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1. EP2691522 - FUNCTIONAL NUCLEIC ACID MOLECULE AND USE THEREOF

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]
Description

Technical Field



[0001]  The present invention relates to (a) a functional nucleic acid molecule having a function of increasing the protein synthesis efficiency, and (b) a use of the functional nucleic acid molecule.

Background Art



[0002]  There are various types of functional nucleic acid molecules, typified by relatively long functional RNA molecules such as antisense RNA, for example. Other typical examples of the functional nucleic acid molecules are relatively short functional RNA molecules such as shRNA (short hairpin RNA), siRNA (small interfering RNA), and miRNA (micro interfering RNA). These functional RNA molecules are generally known to contribute to down-regulation of gene expression. Various examples have been broadly reviewed as "interfering RNA" like in (Non Patent Literature 1).

[0003]  An antisense RNA technique is excellent in target specificity. However, this technique has a disadvantage that an antiviral response in a cell is activated. In contrast, the technique using the relatively short functional RNA molecules such as shRNA does not activate the antiviral response practically. Instead, the relative short functional RNA molecules have the possibility of causing the off-target effects because it is difficult for short sequence length to keep high specificity to the target sequences. Thus, the relatively short functional RNA molecules tend to exhibit poor target specificity as compared with the antisense RNA technique.

[0004]  Patent Literature 1 discloses a functional nucleic acid molecule (DNA molecule) comprising: a pol III type III promoter; a sequence identical or complementary to a target sequence that performs downregulation; and a 7SL small-RNA derived sequence (more specifically, a fragment of an Alu derived sequence) including at least binding domains to srp9 and srp14 proteins. The srp9 and srp14 proteins are members of a family of proteins that bind to a 7SL RNA in gene transcription to form a 7SL RNA complex.

[0005]  It is described that the functional nucleic acid molecule disclosed in Patent Literature 1 is used as a gene expression downregulation technique in which an RNA molecule transcribed from the functional nucleic acid molecule practically causes no antiviral response activation and has excellent target specificity.

[0006]  Alu is classified into one group of SINEs (Short Interspersed Elements). Note that the Alu derived sequence of the functional nucleic acid molecule disclosed in Patent Literature 1 is inserted in a particular orientation and considered to be involved in RNA stability.

[0007]  On the other hand, it has been reported that some small RNA molecules can also enhance the level of transcription.

Citation List



[0008]  Patent Literature 1
International Publication WO 2008/113773 A2 (Publication Date: September 25, 2008)

[0009]  Non Patent Literature 1
He L, Hannon GJ. Nat Rev Genet. 2004 Jul;5(7):522-31. PMID: 15211354

Summary of Invention


Technical Problem



[0010]  As widely reported in Patent Literature 1 and other documents, many types of functional nucleic acid molecules that down-regulate gene expression or the like, are well known. Although there have been some techniques for up-regulating gene expression by increasing the transcription efficiency (like in the case of Nature Chemical Biology 3, 166 - 173 (2007), B. Janowski et al), increasing of the transcription efficiency does not always increase the efficiency of protein synthesis in direct proportion because of the plateau effect. In this regard, the synthesis of translated protein may depend on many other factors, including the ability of a given RNA to interact efficiently with the ribosomes. Additionally, increasing transcription of the natural mRNA is not always possible in the cells or organisms. That is, any functional nucleic acid molecule that increases the protein synthesis efficiency directly has not been reported.

[0011]  The present inventors consider that there are many conditions in which acting only on translation is desired. For instance, enhancement of translation of an animal protein for therapeutic purposes without interfering with its transcription may be highly desired because it does not require reprogramming of mRNA transcription in the nucleus.

[0012]  The present invention is accomplished in view of the above problem. An object of the present invention is to provide a functional nucleic acid molecule having a function of increasing the protein synthesis efficiency, and a use of the functional nucleic acid molecule.

Solution to Problem



[0013]  The present inventors' current knowledge suggested that translation of protein may be regulated by factors including the structure of the regions of the mRNAs that is placed upstream the translated fraction of the mRNAs. This region is known as 5' UTR (5' untranslated region). For the purpose of this invention, the 5' UTR can be a natural one (found in natural RNAs, from the transcription starting site to the protein initiation codon), or can be an artificial sequence, such as the sequence present in a cloning vector or any other recombinant sequence.

[0014]  The present inventors studied diligently to achieve the above object. Consequently, through analyses of functions of RNA molecules known as non-coding RNAs, the inventors found a surprising fact that a particular structure of such an RNA molecule has a function of increasing the protein synthesis efficiency. This functional nucleic acid exhibited the effect against a specific targeted protein through antisense sequence to a target sequence. Based on the findings, the inventors have accomplished the present invention.

[0015]  That is, a functional nucleic acid molecule according to the present invention comprises:
  1. (a) a target determinant sequence comprising antisense sequence to a target sequence in the protein-encoding RNA for which protein synthesis efficiency is to be increased; and
  2. (b) a regulatory sequence having an activity of increasing of the protein synthesis efficiency.


[0016]  In the functional nucleic acid molecule according to the present invention, the regulatory sequence comprises a SINE (Short Interspersed Element) B2 element. SINE B2 sequences are listed in Table 1 in the following.

[0017]  In the functional nucleic acid molecule according to the present invention, the regulatory sequence may be selected from the group consisting of the following (1) through (5):
  1. (1) an RNA, which is encoded by a DNA consisting of the nucleotide sequence shown in SEQ ID No:1 (SINE/B2 in AS Uchll)
  2. (2) an RNA, which is encoded by a DNA consisting of the nucleotide sequence shown in SEQ ID No:2 (SINE/B2 , 39nt spacer indicated as underline and SINE/Alu in AS Uchll)
  3. (3) an RNA, which is encoded by a DNA consisting of the nucleotide sequence shown in SEQ ID No:3 (SINE/B2 in AS Uxt)
  4. (4) an RNA encoded by a DNA consisting of a nucleotide sequence selected from the group consisting of the sequences shown in Table 1; or
  5. (5) an RNA which is at least 95% similar to the RNA of (1), (2), (3) or (4) and has a function of increasing the protein synthesis efficiency.


[0018]  In the functional nucleic acid molecule according to the present invention, the target determinant sequence comprises an antisense sequence to a target sequence in the protein-encoding RNA for which protein synthesis efficiency is to be increased.

[0019]  In the functional nucleic acid molecule according to the present invention, the target determinant sequence may be located between a 5'-terminal and the regulatory sequence in the functional nucleic acid molecule.

[0020]  In the functional nucleic acid molecule according to the present invention, it is preferable that the antisense sequence in the target determinant sequence have a length more than 7 nucleotides but less than 250 nucleotides.

[0021]  In the functional nucleic acid molecule according to the present invention, it is preferable that the antisense sequence in the target determinant sequence be at least 60% similarity to a target sequence in the protein-encoding RNA or to the plasmid sequence upstream of the ATG in the protein-encoding RNA for which protein synthesis efficiency is to be increased.

[0022]  In the functional nucleic acid molecule according to the present invention, it is preferable that the direction of the SINE-derived sequence which is annotated as forward in the regulatory sequence is oriented in a reverse direction relative to the direction (forward direction as defined above), wherein SINE-derived sequence is oriented in the same direction of the consensus sequence of SINE. That is, the regulatory sequence of the functional nucleic acid molecule is oriented in a direct direction relative to the direction of translation and reverse orientation relative to the direction of transcription of the antisense nucleic acid molecule.

[0023]  If the direction from 5' to 3' is defined as the forward direction, the SINE-derived sequence in this invention, wherein its 5' to 3'orientation accords with the SINE consensus sequence, is embedded in the reverse direction of the functional nucleic acid molecule in this invention.

[0024]  In the functional nucleic acid molecule according to the present invention, the antisense sequence in the target determinant sequence may be designed to hybridize with a target sequence in the 5'-UTR of the protein-encoding RNA for which protein synthesis efficiency is to be increased. Alternatively, the target determinant sequence may be designed to hybridize with a target sequence in the coding region of the protein-encoding RNA for which protein synthesis efficiency is to be increased. In addition, the target determinant sequence may overlap to the sequence of plasmid upstream of the starting codon or including the starting codon in the target sequence of the protein encoding RNA. The functional nucleic acid molecule according to the present invention can be targeted to specific splicing variants at the 5- ends of the protein coding RNA or in other parts of the molecule.

[0025]  The functional nucleic acid molecule according to the present invention can be appropriately designed and produced by a skilled person in the related art, as long as the functional nucleic acid molecule includes: a target determinant sequence comprising antisense sequence to a target sequence in the protein-encoding RNA for which protein synthesis efficiency is to be increased; and a regulatory sequence having an activity of increasing of the protein synthesis efficiency. Any modification during or post/synthesis can be applied to the functional nucleic acid molecule by a skilled person in the related art according to the know/how in RNA therapeutics or the like.

[0026]  A DNA molecule according to the present invention encodes any one of the RNA molecules as aforementioned functional nucleic acid molecules.

[0027]  An expression vector according to the present invention includes any one of the RNA molecules or the DNA molecule as aforementioned functional nucleic acid molecules.

[0028]  A composition for increasing protein synthesis efficiency according to the present invention comprises any one of the aforementioned functional nucleic acid molecules and/or the aforementioned expression vector.

[0029]  A method for increasing the protein synthesis efficiency according to the present invention comprises the step of allowing any one of the aforementioned functional nucleic acid molecules or the aforementioned expression vector to coexist with a protein-encoding RNA for which protein synthesis efficiency is to be increased. The protein-encoding RNA is hybridizable with the antisense in the target determinant sequence of the functional nucleic acid molecule.

[0030]  In the protein synthesis efficiency-increasing method of the present invention, the method may comprise the step of transfecting (or transducing) into a cell any one of the aforementioned functional nucleic acid molecules or the aforementioned expression vector.

[0031]  A protein synthesis method according to the present invention is a method for synthesizing a protein, comprising the step of increasing the protein synthesis efficiency by any one of the aforementioned protein synthesis efficiency-increasing methods.

[0032]  The functional nucleic acid molecules of the present invention may be used for treating a disease, wherein the disease is caused by a quantitative decrease in a predetermined normal protein or haploinsufficiency, by increasing the protein synthesis efficiency.

[0033]  The disease may also be caused by a quantitative decrease in a predetermined normal protein or haploinsufficiency. Furthermore, the functional nucleic acid molecule may increase the efficiency of synthesizing of the predetermined normal protein.

[0034]  Further, in case that the disease is caused by a quantitative decrease in a predetermined normal protein or haploinsufficiency, and caused by a quantitative increase of another protein including an abnormal protein like mutant protein, the functional nucleic acid molecules of the present invention can be used in combination with a conventional molecule for suppressing the expression of the another protein by siRNA, shRNA, or the like, and/or a conventional molecule for inactivating a function of a protein by use of an antibody, a low-molecular-weight compound, or the like, as appropriate.

[0035]  Further, the disease may be a neurodegenerative disease or cancer, for example.

Advantageous Effects of Invention



[0036]  According to the present invention, it is successfully possible to provide (a) a functional nucleic acid molecule having a function of increasing the protein synthesis efficiency and (b) a use of the functional nucleic acid molecule.

Brief Description of Drawings



[0037] 

Fig.1
Fig.1, related to the experiment 1, is a schematic diagram of Uchl1/AS Uchl1 genomic organization. Uchl1 exons are in black; 3' and 5' UTRs in white.

Fig.2
Fig.2, related to the experiment 2, is a schematic diagram of AS Uchl1 domain organization. AS Uchl1 exons are in grey; repetitive elements are in red (Alu/SINEBl) and blue (SINEB2). Introns are indicated as lines.

Fig.3
Fig.3, related to the experiment 3, is a view showing AS Uchl1 regulates UchL1 protein levels. AS Uchl1-transfected MN9D cells show increased levels of endogenous UchL1 protein relative to empty vector control, with unchanged mRNA quantity.

Fig.4
Fig.4, related to the experiment 4, is a view showing AS Uchl1 regulates UchL1 protein levels. Increasing doses of transfected AS Uchl1 titrate quantity of UchL1 protein in HEK cells. No changes in Uchl1 mRNA levels.

Fig.5
Fig.5, related to the experiment 5, is a view showing AS Uchl1 regulates UchL1 protein levels. Full length (FL) AS Uchl1 is required for regulating endogenous (MN9D cells, left panel) and overexpressed (HEK cells, right panel) UchL1 protein levels. Scheme of Δ5' or Δ3' deletion mutants is shown.

Fig.6
Fig.6, related to the experiment 6, is a view showing AS Uchl1 regulates UchL1 protein levels via embedded SINEB2. Inverted SINEB2 is sufficient to control UchL1 protein levels. Scheme of mutants is shown.

Fig.7
Fig.7, related to the experiment 7, is a view showing Family of AS transcripts with embedded SINEB2. Family of FANTOM 3 non-coding clones that are AS to protein coding genes and contain embedded SINEB2 in inverted orientation.

Fig.8
Fig.8, related to the experiment 8, is a view showing Family of AS transcripts with embedded SINEB2. Schematic diagram of Uxt/AS Uxt genomic organization. AS Uxt increases endogenous Uxt protein levels in transfected MN9D cells (left), without affecting its transcription (right).

Fig.9
Fig.9, related to the experiment 9, is a view showing expression of AS Uchl1 in the nucleus of dopaminergic neurons. AS Uchl1 (red) and Uchl1 (green) transcripts are expressed in the nucleus and cytoplasm of TH-positive DA neurons of the Substantia Nigra (blue). Details of localization are in zoom images.

Fig.10
Fig.10, related to the experiment 10, is a view showing AS Uchll translocates to the cytoplasm upon rapamycin treatment in MN9D cells. mRNA levels measured with primers spanning 5' overlapping or 3' distal portions of the transcript. Data indicate mean ± s.d., n≥3 (3). **p<0.01; ***p<0.005.

Fig.11
Fig.11, related to the experiment 11, is a view showing Rapamycin treatment induces UchLl protein expression. UchLl protein level is increased in rapamycin-treated MN9D cells.

Fig.12
Fig.12, related to the experiment 12, is a view showing AS Uchll-embedded SINEB2 induces translation of Uchll upon rapamycin treatment. Silencing AS Uchll transcription in MN9D cells (shRNA, encompassing -15/+4 position of target sequence) inhibits rapamycin-induced UchLl protein level. Scramble, shRNA regulatory sequence. Left, mRNA levels; right, protein levels.

Fig.13
Fig.13, related to the experiment 13, is a view showing AS Uchll-embedded SINEB2 induces translation of Uchll upon rapamycin treatment. Deletion of embedded SINEB2 (ΔSINEB2) is sufficient to inhibit rapamycin-induced UchLl protein up-regulation.

Fig.14
Fig.14, related to the experiment 14, is a view showing an artificial AS transcript with embedded SINEB2 (AS Gfp) to an artificial humanized enhanced Green Fluorescent Protein (Gfp) mRNA increases GFP protein synthesis: (a) of Fig. 14 shows Schematic diagram of Gfp/AS Gfp constructs, (b) of Fig. 14 shows how AS Gfp increases Gfp protein levels in transfected HEK cells while a scramble overlapping sequence or an empty plasmid does not increase Gfp protein levels in transfected HEK cells. The overlap is 72nt long and it is centered on the ATG.

Fig.15
Fig.15, related to the experiment 15, is a view showing an artificial AS transcript with embedded SINEB2 (AS Fc clone) to an artificial recombinant antibody in pHYGRO (pHYGRO clones) increases the synthesis of the protein encoded in pHYGRO. a, Schematic diagram of pHYGRO /AS Fc constructs. b, AS Fc increases the encoded protein levels in transfected HEK cells while a scramble overlapping sequence or an empty plasmid do not, detected through the SV5 tag.

Fig.16
Fig.16, related to the experiment 16, is a view showing how an artificial AS transcript with embedded SINEB2 to an artificial Green Fluorescent Protein (EGFP) mRNA increases EGFP protein synthesis.

Fig.17
Fig.17 is a view showing the examples of the potential predicted structures of SINE B2-derived sequence.


Description of Embodiments



[0038]  The following describes an embodiment of the present invention, more specifically.

[1. Functional RNA Molecule]


(Constitution of Functional nucleic acid Molecule)



[0039]  A functional nucleic acid molecule according to the present invention has a feature that it comprises: a target determinant sequence comprising an antisense sequence to a target sequence in the protein-encoding RNA for which protein synthesis efficiency is to be increased; and a regulatory sequence having an activity of increasing the protein synthesis efficiency. The functional nucleic acid molecule according to the present invention can be appropriately designed and produced by a skilled person in the related art based on the description herein.

[0040]  The protein synthesis efficiency is increased according to the present invention. In one embodiment, the protein synthesis efficiency is increased as a result of increase in the translation efficiency, preferably, without changing the transcription efficiency substantially. Thus, the protein synthesis efficiency may be increased by increasing the translation efficiency. In another embodiment, both transcription and translation can be increased by independent means; obviously, it may be the case that an RNA may result lower expression level, and yet the protein results higher expression level.

(Protein-Encoding RNA for Which Protein Synthesis Efficiency is to be Increased)



[0041]  The protein-encoding RNA for which protein synthesis efficiency is to be increased by the functional nucleic acid molecule according to the present invention is not especially limited in regard to its sequence, origin, and the like, provided that the RNA comprises a translation domain (coding region) having a 5'-terminal start codon and a 3'-terminal stop codon. Specifically, the protein-encoding RNA for which protein synthesis efficiency (the target RNA) is to be increased by the functional nucleic acid molecule according to the present invention may further have a 5'-cap structure, a 5' untranslated region (5'-UTR) and/or a 3' untranslated region. These regions may be derived from endogenous sequence in a cell or artificially synthesized sequence. The ORFeome, in which Open Reading Frames (coding sequences) of genes are placed in expression vectors, is one of the examples.

[0042]  Further, it is preferable that the 3' untranslated region includes, at its 3' terminal, a sequence (poly-A addition signal) so that a poly-A sequence can be added. The poly-A addition signal may be, for example, a nucleotide sequence consisting of AAUAAA, a SV40 early poly-A signal having two sequences of AAUAAA, a sequence in which SV40 early poly-A signals are aligned in a tandem manner, or the like. The poly-A addition signal is not limited to them. As examples, various alternative polyadenylation sites have been described in the literature, and some mRNA do not even carry conventional polyadenylation signals ( Carninci et al, Genome Res. 2003 Jun; 13(6B): 1273-89. PMID: 12819125).

[0043]  The protein-encoding RNA for which protein synthesis efficiency is to be increased according to the present invention may have a poly-A sequence at its 3' terminal. An RNA having a poly-A sequence at its 3' terminal has excellent protein synthesis efficiency from a translation domain and excellent stability of the RNA itself. Such an RNA may be, for example, any one of RNAs shown in Uchll, Uxt, GFP or a homologue of any one of these RNAs, although such an RNA is not limited to them.
Mus musculus ubiquitin carboxy-terminal hydrolase L1 (Uchl1) RefSeq: NM_011670.2
The Uchll DNA sequence which encodes the Uchll RNA is shown in SEQ ID NO.4.
Mus musculus ubiquitously expressed transcript (Uxt) RefSeq: NM_013840.3
The Uxt DNA sequence which encodes the Uxt RNA is shown in SEQ ID NO.5.
GFP(Sequence from pEGFP vector)
The GFP DNA sequence which encodes the GFP RNA sequence is shown in SEQ ID NO.6.

[0044]  The protein-encoding RNA for which protein synthesis efficiency is to be increased may be endogenous RNA of biological origin (for example, mRNA) or artificially synthesized. Further, mRNA derived from eucaryotes encompass mature mRNA that has been subjected to what is called processing, and precursor mRNA that has not been subjected to processing.

(Target Determinant Sequence)



[0045]  The target determinant sequence is a sequence that comprises an antisense sequence to a target sequence in the protein-encoding RNA for which protein synthesis efficiency is to be increased. A target sequence is arbitrarily selected from a partial sequence of protein-encoding RNA for which protein synthesis efficiency is to be increased in the present invention. A target sequence may derive from the RNA sequence transcribed from the plasmid DNA in which the protein-encoding cDNA is inserted and around the first 5'-terminal start codon. A length of the antisense sequence is not especially limited. However, from the viewpoint of increasing specificity for a target RNA in a system including different RNAs, the antisense sequence may have a length of preferably more than 7 nucleotides, more preferably 10, more preferably 15. Furthermore, the antisense sequence may have a length of preferably less than 250 nucleotides, more preferably 200, more preferably 150, more preferably 100, more preferably 90, more preferably 80, more preferably 77 nucleotides, more preferably 70 nucleotides, more preferably 60 nucleotides, more preferably 50 nucleotides, more preferably 40 nucleotides, more preferably 30 nucleotides.

[0046]  In one embodiment, more than one different antisense sequences can be included in the target determinant sequence. These multiple antisense sequences can be applied to targeting of multiple proteins, for example. Alternatively, the multiple antisense sequences can be applied to improving the specificity to a protein-encoding RNA wherein the multiple antisense sequences are hybridizable with the same protein-encoding RNA. In one embodiment, the target determinant sequence of the present invention may contain mismatches against the target RNA on purpose to prevent the reaction of gamma interferon that may take place in the cells in presented of long double strand nucleic acids molecule, like long double strand RNAs.

[0047]  Further, from the viewpoint of increasing specificity between the functional nucleic acid molecule of the present invention and the target RNA, the antisense sequence in a target determinant sequence is designed to be preferably at least 60% similarity, more preferably at least 65% similarity, more preferably at least 70% similarity, more preferably at least 75% similarity, more preferably at least 80% similarity, more preferably at least 85% similarity, more preferably at least 90% similarity, more preferably at least 95% similarity to a corresponding target sequence in the protein-encoding RNA for which protein synthesis efficiency is to be increased, as long as the antisense sequence can hybridize to the target sequence in the protein-encoding RNA and/or to the plasmid-derived RNA that contains the target sequence. It is specifically preferable that the antisense sequence in the target determinant sequence be designed to be thoroughly identical with the corresponding sequence of the target sequence.

[0048]  Further, an antisense sequence in a target determinant sequence can be designed to hybridize with a 5'-UTR of the protein-encoding RNA for which protein synthesis efficiency is to be increased. This design can be applied to the synthesizing full length encoded-protein. 5'-UTR may be derived from endogenous sequence in a cell or artificial sequence. The antisense sequence in the target determinant sequence may be designed to be hybridizable with other regions, except the 5'-UTR, of the target RNA, such as a coding region of the target RNA. For example, the antisense sequence in the target determinant sequence can be designed to be hybridizable with a given part of the coding region of the target RNA. This design is useful for a dystrophin gene or the like in which a protein to be encoded by an RNA is very large and has a domain that exhibits bioactivity by itself.

[0049]  Additionally, the antisense sequence in the target determinant sequence can be designed to hybridize both the 5' UTR and a part of the other functional part of the sequence, like the coding sequence or the 3' UTRs of the protein coding mRNAs.

(Regulatory sequence)



[0050]  In the functional nucleic acid molecule according to the present invention, the regulatory sequence has an activity of increasing of the protein synthesis efficiency.

[0051]  In one embodiment, multiple SINE sequences can be included in the regulatory sequence. These multiple SINE sequences may be with combination of different sequences, for example a combination of SINE B2 sequence and Alu-derived sequence or a combination of different SINE B2-derived sequence.

[0052]  The examples of the potential predicted structures of SINE B2 sequences are as shown in (a) and (b) of Fig. 17.

[0053]  The similar sequence indicates a sequence that is at least 25 % similarity, preferably at least 50% similarity, more preferably at least 55% similarity, more preferably at least 60% similarity, more preferably at least 65% similarity, more preferably at least 70% similarity, more preferably at least 75% similarity, more preferably at least 80% similarity, more preferably at least 85% similarity, more preferably at least 90% similarity, more preferably at least 95% similarity to the consensus sequence of the SINE.

[0054]  The sequence of a SINE may deviate from these conservative consensus described above. For instance, the analysis of the consensus similarity between the SINE B2 sequences of three sequences alone, the SINE B2 fraction of SEQ ID NO:1, SEQ ID NO:3, and another SINE B2 randomly taken from the literature (reference: Espinosa et al, http://rnajournal.cshlp.org/content/13/4/583.full), clearly indicates that the SINE B2 fractions alone can share as little as 9 bases out of 36, with only 25% of similarity.

[0055]  Yet, despite diverging, these are still recognizable as a SINE B2 element using programs like RepeatMask as published ( Bioinformatics. 2000 Nov;16(11):1040-1.MaskerAid: a performance enhancement to RepeatMasker.Bedel1 JA, Korf I, Gish W.).

[0056]  In one embodiment, the length can be limited to the shortest sequences with similarity to SINE elements, which are capable to cause increased the efficiency of protein synthesis.

[0057]  In one embodiment, it is possible to synthesize artificial nucleic acid sequences that have partial similarity to the SINE elements described in the present invention and act to increase the level of synthesized protein.

[0058]  Further, the functional nucleic acid molecule according to the present invention may include a plurality of regulatory sequences aligned in a tandem manner, for the purpose of further promoting the protein synthesis efficiency.

[0059]  In the description of the present invention, "SINE" broadly indicates, among non-LTR (long terminal repeat) retrotransposon, an interspersed repetitive sequence (a) which encodes a protein having neither reverse-transcription activity nor endonuclease activity or the like and (b) whose complete or incomplete copy sequences exist abundantly in genomes of living organisms. That is, SINE is a DNA sequence that is inserted into a genome through the reverse transcription from RNA to cDNA, depending on other host factors in these processes. A length of the SINE is not especially limited, but generally, in a range of not less than 20 bp, preferably not less than 30bp, more preferably not less than 50bp, more preferably not less than 50bp, but not more than 700 bp, preferably not more than 600 bp, more preferably not more than 500 bp, more preferably not more than 400 bp. Further, the origin of SINE is not limited, but is generally derived from tRNA and has a sequence having a sequence corresponding to the tRNA on its 5'-terminal side. Further, the SINE may be a 7SL RNA-derived sequence such as Alu, and a 5S rRNA-derived sequence such as SINE 3. In regard to the SINE 3, the document by Kapitonov et al (Vladimir V. Kapitonov and Jerzy Jurka: Molecular Biology AND Evolution 20(5): p694-702, 2003) and the like document can be referred to.

[0060]  The regulatory sequence may be, for example, selected from the following (1) to (5):
  1. (1) an RNA, which is encoded by a DNA consisting of the nucleotide sequence shown in SEQ ID No:1
  2. (2) an RNA, which is encoded by a DNA consisting of the nucleotide sequence shown in SEQ ID No:2
  3. (3) an RNA, which is encoded by a DNA consisting of the nucleotide sequence shown in SEQ ID No:3
  4. (4) an RNA encoded by a DNA consisting of a nucleotide sequence selected from the group consisting of the sequences shown in Table 1; or
  5. (5) an RNA which is at least 95% similar to the RNA of (1), (2), (3) or (4) and has a function of increasing the protein synthesis efficiency.


[0061]  The number of nucleotides to be deleted, substituted, added, and/or inserted is preferably not less than 1 but not more than 175, more preferably not less than 1 but not more than 150, more preferably not less than 1 but not more than 125, more preferably not less than 1 but not more than 100, more preferably not less than 1 but not more than 75, more preferably not less than 1 but not more than 50, more preferably not less than 1 but not more than 30, more preferably not less than 1 but not more than 20.

(Positional Relationship between Target Determinant Sequence and Regulatory sequence)



[0062]  In the present invention, a direction (a sense-strand direction) along which a target protein is translated is defined as "forward direction", and the direction opposite to the forward direction is defined as "reverse direction". In the functional nucleic acid molecule according to the present invention, a positional relationship between the target determinant sequence and the regulatory sequence is not especially limited. However, it is preferable that the target determinant sequence be located closer to a forward-direction side in the functional nucleic acid molecule than the regulatory sequence. The target determinant sequence may be directly linked to the regulatory sequence. Alternatively, a linker sequence and/or the like sequence for connecting the target determinant sequence and the regulatory sequence may be inserted there between.

[0063]  In the functional nucleic acid molecule according to the present invention, it is preferable that the direction of the SINE sequence which is annotated as forward in the regulatory sequence is oriented in a reverse direction relative to the direction (forward direction as defined above), wherein SINE sequence is oriented in the same direction of the consensus sequence of SINE. That is, the regulatory sequence of the functional nucleic acid molecule is oriented in a forward direction relative to the direction of translation.

[0064]  If the direction from 5' to 3' is defined as the forward direction, the SINE sequence in this invention, wherein its 5' to 3'orientation accords with the SINE consensus sequence, is embedded in the reverse direction of the functional nucleic acid molecule in this invention. For example, in case of one of SINE B2 sequence, Abox site of SINE B2-derived sequence is located on the 3' side of the functional nucleic acid molecule compared to Bbox site of SINE B2 sequence.

(Production of Functional nucleic acid Molecule)



[0065]  A method according to the present invention for producing a functional nucleic acid molecule comprises the step of preparing the aforementioned RNA molecule. The functional nucleic acid molecules may be prepared by a well-known nucleic acid biosynthesis method, or such a method that (i) a DNA molecule encoding the functional RNA molecule is produced and (ii) the DNA molecule is transcribed into the functional RNA molecule, for example. A size of the functional nucleic acid molecule is not especially limited, but the functional nucleic acid molecule has a size of preferably not more than 2000 nucleotides, more preferably not more than 250 nucleotides, for example, from the viewpoint of producing the functional nucleic acid molecule by the nucleic acid biosynthesis method.

[2. DNA Molecule, Expression Vector, Composition for Increasing Protein Synthesis Efficiency]



[0066]  A DNA molecule according to the present invention encodes any one of the aforementioned functional nucleic acid molecules according to the present invention. Further, an expression vector according to the present invention is an RNA vector comprising any one of the aforementioned functional RNA molecules of the present invention or a DNA vector comprising the DNA molecule according to the present invention. Further, a composition for increasing protein synthesis efficiency according to the present invention comprises any one of the aforementioned functional nucleic acid molecules or the aforementioned expression vector.

[0067]  In one embodiment, the composition according to the present invention may comprise a translation agent based on in vitro system like reticulocyte extract to produce protein in vitro; or to produce protein in vivo in mammalian cells expressing a protein for industrial use, for research purpose, or for any other screening, for example.

[0068]  The backbone of the expression vector according to the present invention is not especially limited to any particular type, and may be appropriately selected from a plasmid vector. The plasmid vector may be a mammalian, yeast, insect expression vector, a virus vector (for example a lentiviral or retroviral expression vector, adenovirus or adeno-associated virus vectors), a phage vector, a cosmid vector, and the like, depending on types of host cells to be used and the purpose of use. For example, in a case where the present invention is used for gene treatment of mammals including a human, the present invention may be prepared in a form of a virus vector, such as an adenovirus or adeno-associated vector or a lentivirus vector.

[0069]  Alternatively, the expression vector may ultimately be integrated in the genome of the expressing cells or organism to be targeted.

[3. Method for Increasing Protein Synthesis Efficiency]


(Method for Increasing Protein Synthesis Efficiency)



[0070]  A method for increasing protein synthesis efficiency according to the present invention comprises the step of allowing a functional nucleic acid molecule according to the present invention or the aforementioned expression vector to coexist with a protein-encoding RNA for which protein synthesis efficiency is to be increased. This step can be carried out in vivo or in vitro using, for example, cell-free protein synthesis system. In the present invention, "in vivo" means a system of using either cell culture or whole animal specifically, and "in vitro" means a system using cell-free assay specifically. In a case where the step is carried out in vivo, the functional nucleic acid molecule or the aforementioned expression vector may be allowed to coexist, in an isolated cell or tissue, with a protein-encoding RNA for which protein synthesis efficiency is to be increased. Alternatively, the functional nucleic acid molecule or the aforementioned expression vector may be allowed to coexist, in a living organism, with an RNA for which protein synthesis efficiency is to be increased.

[0071]  The method for increasing protein synthesis efficiency according to the present invention may comprise transfecting into a cell an aforementioned expression vector encoding the functional nucleic acid molecule or the functional nucleic acid molecule itself so as to allow the functional nucleic acid molecule to coexist with the protein-encoding RNA. The "cell" indicates not only an isolated cell but also cells constituting an individual. The RNA for which protein synthesis efficiency is to be increased may be derived from an endogenous sequence in a cell or an RNA encoding a protein synthesized artificially. The transfection (or gene induction) of a nucleic acid molecule into a cell may be carried out appropriately by conventional methods, for example, self-infection by a vector, a microinjection technique, a lipofection technique, an electroporation technique, a calcium phosphate method, transduction of a virus and the like. The vector may or may not be permanently integrated in the host genome.

[0072]  The cells may be derived from any one of cells from any organism including animals and plants, or any one of cells selected from the established cell lines.

[0073]  In the present invention, animals include vertebrate, preferably mammals including a human, but are not limited to these examples.

[0074]  In the present invention, plants include both monocotyledons and dicotyledons. In one embodiment, the plants are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, or pea), or other legumes. In another embodiment, the plants may be vegetables or ornamental plants. The plants of the invention may be: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), flax (Linum usitatissimum), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolour, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato, (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Lopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango, (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, or barley, but are not limited to these examples.

[0075]  In the present invention, the established cell lines including mammalian derived cell such as COS-1 (ATCC No.CRL 1650), COS-7 (ATCC CRL 1651), human embryonic kidney line 293 (ATCC NO.CRL 1573), PerC6 (Crucell), baby hamster kidney cell (BHK) (ATCC CRL.1632), BHK570 (ATCC NO: CRL 10314), Chinese hamster ovary cells CHO (e.g. CHO-K1, ATCC NO: CCL 61 , DHFR minus CHO cell line such as DG44, particularly those CHO cell lines adapted for suspension culture, mouse Sertoli cell, monkey kidney cell, African green monkey kidney cell (ATCC CRL-1587), HeLa cell, SH-Y5Y cell, canine kidney cell (ATCC CCL 34), human lung cell (ATCC CCL 75), Hep G2 and myeloma or lymphoma cells e.g. NSO (see US 5,807,715), Sp2/0, YO, other animals derived cells such as Sf9 cell,DT40, but are not limited to these examples. Furthermore, the established cell lines can be hybridoma or a cell given a particular feature by gene transfer, nuclear transfer and/or treatment of chemical compound, for example a nuclear transfer embryonic stem cell or an iPS cell( WO2007/069666, JP-A 2010-273680, JP-A 2010-284088, JP-A 2011-50379, JP-A 2011-4674, etc), a neuronal cell differentiated from neural stem cell, iPS cell or the like, or neuronal cells derived from re-programmed fibroblasts or the like, but are not limited to these examples.

[0076]  The protein synthesis efficiency increasing method according to the present invention optimally contributes to increase the efficiency of the translation from RNA. The increase of the protein synthesis efficiency indicates that the protein synthesis efficiency is increased as compared with a case where the functional nucleic acid molecule according to the present invention or aforementioned expression vector is not allowed to coexist with the target RNA in a system. How much the protein synthesis efficiency is to be increased is not limited especially. However, it is preferable that an amount of a protein to be synthesized by the protein synthesis efficiency-increasing method be at least 1.5 times, more preferably at least 2 times more than an amount of a protein to be produced in the case where the functional RNA molecule is not allowed to coexist with the target RNA in the system.

[0077]  In a case where the functional nucleic acid molecule or aforementioned expression vector is allowed to coexist with the target RNA in the system, a quantitative ratio between the functional nucleic acid molecule (or the expression vector) and the target RNA is not especially limited. The quantitative ratio between them may be, for example, 1:1 to 1:10.

(Method for synthesizing Protein)



[0078]  The aforementioned protein synthesis efficiency-increasing method according to the present invention can be used as a protein synthesis method. That is, a protein synthesis method according to the present invention is a method for producing a target protein, comprising the step of increasing the protein synthesis efficiency by any one of the aforementioned protein synthesis efficiency-increasing methods. It is preferable that the protein synthesis method allow for efficient synthesis of the target protein through the increasing the efficiency of protein translation from the target RNA.

[0079]  In the protein synthesis method of the present invention, one example of the protein to be synthesized is an antibody, particularly synthesizing a light or a heavy chain or both of the antibody or single chain recombinant version of an antibody. The synthesis of the antibody is carried out preferably in an in vitro system or in an isolated cell system so that a functional nucleic acid molecule according to the present invention or aforementioned expression vector can be allowed to coexist, in the system, with a target RNA for which protein synthesis efficiency is to be increased.

(Method for Treating Disease)



[0080]  The functional nucleic acid molecule according to the present invention can be used for treatment of a disease caused by a quantitative decrease in a predetermined normal protein, for example. The disease is not especially limited, but may be, for example; myodegeneration such as muscular dystrophy; neurodegenerative disease such as Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis, and triplet repeat diseases both at the protein encoding level (i.e. Huntington's disease) or at DNA/RNA level (i.e. Fragile X).

[0081]  Furthermore, a tumor can be also applied to the present invention through the increase of expression of a pro-apoptotic protein or a tumor suppression protein for tumor treatment as for instance for p53 family members.

[0082]  Such a tumor, including a cancer, may be any one of tumors including without limitation carcinoma, melanoma and sarcoma, bladder carcinoma, brain tumor, breast tumor, cervical tumor, colorectal tumor, esophageal tumor, endometrial tumor, hepatocellular carcinoma, gastrointestinal stromal tumor, laryngeal tumor, lung tumor, osteosarcoma, ovarian tumor, pancreatic tumor, prostate tumor, renal cell carcinoma, skin tumor, or thyroid tumor.

[0083]  In order to obtain the increase of protein synthesis efficiency, the functional nucleic acid molecule according to the present invention or aforementioned expression vector is allowed to coexist, in a body of a subject, with a target RNA. The functional nucleic acid molecule itself or an aforementioned expression vector is transfected into a cell of the subject. The target RNA may be an endogenous RNA (mRNA or the like) in the cell of the subject or artificially synthesized RNA. Alternatively, the target RNA or a DNA molecule encoding the RNA may be transfected into the cell of the subject.

[0084]  Further, the functional nucleic acid molecule according to the present invention is applicable to treatment of a disease by amplifying in a body of a subject a protein factor (e.g., interferon, an apoptosis-inducing factor, or the like) that ameliorates the disease. For example, an apoptosis-inducing factor amplified in accordance with the present invention can be effectively used for treatment of tumors or the like.

[0085]  When the functional nucleic acid molecule according to the present invention or aforementioned expression vector is allowed to coexist with a target RNA encoding a protein that ameliorates a disease in a body of a subject, the target RNA hybridizes the antisense in the target determinant sequence of the functional nucleic acid molecule. The functional nucleic acid molecule itself or the aforementioned expression vector is transfected into a cell of the subject. The RNA encoding the protein that ameliorates the disease may be an endogenous RNA in the cell of the subject. Alternatively, the RNA encoding the protein that ameliorates the disease or a DNA molecule encoding the RNA may be transfected into the cell of the subject.

[0086]  The functional nucleic acid molecule may be used as a pretreatment to an isolated cell to be transplanted into a body of the subject. The cell may be isolated from the body of the subject before treatment and transplantation. The functional nucleic acid molecule according to the present invention may be allowed to coexist in an isolated cell with a target RNA; and the cell can then be transplanted into a body of a subject. An isolated cell may be a cell which has the ability of differentiation, for example an Embryonic Stem cell (ES cell), an Embryonic Germ cell (EG cell), a somatic stem cell, especially a multi potent adult progenitor cell, stem cell, a hematopoietic stem cell, a vascular endothelial stem cell, a mesenchymal stem cell, a hepatic stem cell, a neural stem cell, an endothelial stem cell, a pancreatic stem cell, a primordial germ cell, or a multilineage-differentiating cell like a Muse cell ( Kuroda et al., 2010, PNAS). Additionally, an isolated cell may be also an artificial undifferentiated cell, for example a nuclear transfer embryonic stem cell or a cell acquired the pluripotent ability by gene transfer and/or treatment of chemical compound, like an induced pluripotent stem cell (iPS cell ( WO2007/069666, JP-A 2010-273680, JP-A 2010- 284088, JP-A 2011-50379, JP-A 2011-4674, etc)). Additionally, an isolated cell may also be a fibroblast or an adult somatic cell that acquired the ability to become another somatic cell upon reprogramming. Additionally, after the step (a) but before the step (b), the isolated cell may be grown undifferentiated. Alternatively, after the step (a) but before the step (b), the isolated cell may be differentiated to obtain a differentiated cell or a group of differentiated cells (cell sheet or the like), and the differentiated cell or the group of differentiated cells may be transplanted into the body.

[0087]  The subject indicates animals, wherein the animals include a human, preferably mammals including a human, more preferably a human. Further, the subject generally encompasses (a) one who has already shown the symptoms of a disease and (b) one who has a genetic predisposing cause but has not shown the symptoms of a disease yet. In view of this, the concept of "treatment" in the present invention includes therapeutic treatment and preventive treatment of a disease.

[Example]



[0088]  The present invention will be described below more specifically based on Examples, Comparative Examples, and the like.Antisense transcription in synthenic PARKS/Uchl1 locus.

[0089]  The FANTOM2 clone Rik6430596G22 was identified as a putative spliced AntiSense (AS) noncoding RNA (ncRNA9 of the Ubiquitin Carboxy Terminal Hydrolase 1 (UCHL1) gene 7 (Experiment 1). UchLl is a neuron-restricted protein that acts as deubiquinating enzyme, ubiquitin ligase or monoubiquitin stabilizer (Reference 1; Liu, Y., Fallon, L., Lashuel, H.A., Liu, Z. & Lansbury, P.T., Jr. The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility. Cell 111, 209-218 (2002)., Reference 2; Osaka, H., et al. Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron. Hum Mol Genet 12, 1945-1958 (2003).). It is mutated in an autosomal dominant form of PD (PARK5) (Reference 3; Leroy, E., et al. The ubiquitin pathway in Parkinson's disease. Nature 395, 451-452 (1998).). In Substantia Nigra (SN) of sporadic post-mortem brains UchLl expression was found reduced and correlated to the formation of aSYN aggregates. Loss of Uchll activity has been also associated to Dementia with Lewy bodies (DLB) and Alzheimer's disease (AD). Increasing Uchll expression has been proposed as therapeutic strategy for AD since its ectopic expression rescued beta-amyloid-induced loss of synaptic function and contextual memory in a mouse model (Reference 1, Reference 4; Butterfield, D.A., et al. Redox proteomics identification of oxidatively modified hippocampal proteins in mild cognitive impairment: insights into the development of Alzheimer's disease. Neurobiol Dis 22, 223-232 (2006)., Reference 5; Castegna, A., et al. Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71. J Neurochem 82, 1524-1532 (2002)., Reference 6; Choi, J., et al. Oxidative modifications and down-regulation of ubiquitin carboxyl-terminal hydrolase L1 associated with idiopathic Parkinson's and Alzheimer's diseases. J Biol Chem 279, 13256-13264 (2004).).

[0090]  Rik6430596G22 is a typical 5' head to head transcript that initiates within the second intron of Uchll and overlaps the first 72 nts of the Sense (S) mRNA including the AUG codon (Experiment 1). The non overlapping part of the transcript also contains two embedded repetitive sequences, SINEB2 and Alu, identified by Repeatmasker. The FANTOM2 cDNA clone spans a genomic region of 70kb and is a spliced transcript composed of four exons whose intron-junctions follow the traditional GT-AG rule (Experiment 2). In the remaining part of the disclosure Rik6430596G22 is referred as a natural AntiSense transcript to Uchll (AS Uchll).

[0091]  AS Uchll increases UchLl protein levels with a mechanism that requires an embedded inverted SINEB2 repeat.

[0092]  The interplay between S and AS transcripts was then examined. After cloning the full length cDNA for AS Uchll from MN9D cells with 5' rapid amplification of cDNA ends (RACE), a CMV-driven AS Uchll was transiently overexpressed in MN9D dopaminergic cells and endogenous Uchll mRNA and protein levels were monitored by qRT-PCR and western blotting. While no significant change in Uchll mRNA endogenous levels was observed, a strong and reproducible upregulation of UchLl protein product was detected within 24 hours (Experiment 3). The inventors tested whether co-transfection of both cDNAs into HEK cells which do not express either transcript could recapitulate what was seen in MN9D cells. When increasing amounts of AS Uchll were co-transfected with murine Uchll, a dose-dependent UchLl protein upregulation was recorded in absence of any significant change in exogenous Uchll mRNA level (Experiment 4). This specific effect was not observed for unrelated controls such as GFP. To identify sequences and/or structural elements of AS Uchll mRNA that elicit its functional activity on UchLl protein, deletion mutants were produced and tested in MN9D cells as well as in co-transfection in HEK cells. AS Uchll deletion constructs lacking the 5' first exon (AS Uchll Δ 5'), or the last three exons (AS Uchll Δ 3') failed to induce Uchll protein levels in both MN9D and HEK cell models, suggesting both 5' and 3' components were important to AS Uchll function (Experiment 5).

[0093]  Additional deletion mutants were thus synthesized to assess the role of the embedded repetitive sequences, Alu and SINEB2, in UchLl protein upregulation.

[0094]  Targeted deletion of the region containing both the SINEB2 and Alu repeat elements (AS Uchll Δ SINEB2+ALU) prevented Uchll protein induction. Deletion of a single repetitive element, (A-SINE B2 (764-934) and Δ-ALU (1000-1045), revealed the SINEB2 was the functional region of the transcript required for UchLl protein increase (Experiment 6). In all cases no change in Uchll mRNA level was detected by transfection of AS Uchll wild type and deletion constructs.

[0095]  Additionally the Δ-SINEB2 mutant has a dominant negative activity on the full-length AS Uchll.

[0096]  Since the deletion mutant Δ-SINEB2 lacks 170 nucleotides potentially impairing AS Uchll RNA secondary structure, a mutant was produced with the SINEB2 sequence flipped in between nucleotide 764-934. Interestingly, SINEB2 flip was unable to increase UchLl protein levels thus proving the orientation-dependent activity of the SINEB2 domain embedded within AS Uchll (Experiment 6).

[0097]  S/AS pairs with an embedded inverted SINEB2 repeat in the AS transcript identify a new functional class of ncRNA. :
The FANTOM3 collection of non-coding cDNAs was bioinformatically screened for other examples of natural AS transcripts that contain SINEB2 elements (B3 subclass) in the correct orientation and 5' head to head overlapping to a protein coding gene. This identified 31 S/AS pairs similar to the Uchl1/AS Uchll structure (Experiment 7).

[0098]  To test whether the observation for Uchl1/AS Uchll generalizes to other examples, the AS overlapping transcript of Uxt (ubiquitously-expressed transcript), AS Uxt (Rik4833404H03) were cloned and over-expressed. Transfection of AS Uxt in MN9D dopaminergic cells showed up-regulation of Uxt protein product with no change in the total mRNA levels confirming a more general mechanism is at work (Experiment 8).

[0099]  AS Uchll is a nuclear-enriched transcript expressed in dopaminergic neurons. :
Multiplex RT-PCR on a panel of mouse adult tissues, macroscopically dissected brain regions and neuronal cell lines found that AS Uchll expression was restricted to ventral midbrain, cortex and MN9D dopaminergic cells but absent in non-neuronal tissues, and cell lines. Double in situ hybridization with riboprobes targeting the non-overlapping region of Uchll showed that the mRNA was prevalent in the cytoplasm of cells of the hippocampus, cortex and subcortical regions as well as of the dorsal and ventral midbrain. AS Uchll riboprobe decorated similar structures. A combination of double in situ hybridization with anti-tyrosine hydroxylase immunohystofluorescence showed that mRNAs for Uchll and AS were expressed in the same DA neurons of the SN. Intriguingly, transcripts for the S/AS pair were prevalently localized in two different subcellular compartments: mature Uchll mRNA was mainly observed in the cytoplasm, while the AS RNA was nuclear, accumulating in specific subnuclear regions (Experiment 9). 50% of cellular transcripts have been recently found enriched in the nucleus representing mainly ncRNAs with unknown function. Nuclear-retained RNAs tend to accumulate in areas, called paraspeckles, that strongly resemble sites of AS Uchll localization and which association is regulated by embedded SINEs (Reference 7; Chen, L.L., DeCerbo, J.N. & Carmichael, G.G. Alu element-mediated gene silencing. Embo J 27, 1694-1705 (2008).).

[0100]  By taking advantage of RACE, the precise transcriptional start site (TSS) of the AS Uchll gene was mapped in MN9D cells. As shown in Experiment 2, the TSS lies 250 bps upstream the previously annotated sequence and is localized in the second intron of Uchll.

[0101]  AS Uchll is down-regulated in PD neurochemical models and human post-mortem brains. :
A 70-kb region of the mouse genome encompassing the AS UchLl locus was then compared to the corresponding human genomic sequence using Genome Vista alignment (http://genome.lbl.gov/cgi-bin/GenomeVista). By the use of primers designed on the human sequence in correspondence to CST peaks, a 1.6 kb non-coding transcript, 5' head to head AS to human UCHL1 gene, was cloned from human brain RNA. The anatomical organization of hAS UCHL1 gene was very similar to its mouse counterpart including the extension of the S/AS pair overlapping region as well as the presence of embedded repetitive elements. hAS UCHL1 expression was highly restricted to neuronal tissues as found for mouse.

[0102]  UCH-L1 protein synthesis is increased upon rapamycin treatment through nucleus-cytoplasmic shuttling of AS Uchll RNA and AS-dependent recruitment of Uchll mRNA to active polysomes.

[0103]  So far, AS ncRNA is able to increase S protein levels with no change in the quantity of S mRNA. Since in physiological conditions S mRNA and AS ncRNA seem to be localized in different subcellular compartments, several stressors that have been implicated in PD pathogenesis for their ability to redistribute the nuclear AS ncRNA into the cytoplasm, where translation takes place, were assayed. MN9D cells were exposed to hydrogen peroxide 1mM, serum starvation, rapamycin 1 ug/ml, tunycamycin 20nM and TNFalpha 20 nM for 45 minutes and AS Uchll mRNA content was independently measured in the cytoplasm and nucleus by qRT-PCR. The majority of treatments had no effect, however rapamycin strongly up-regulated the amount of AS Uchll cytoplasmic mRNA (Experiment10).

[0104]  Rapamycin is a well known inhibitor of CAP-dependent translation through its effect on mTORC1 and subsequent repression of S6K and 4E-BP1 activities. It is currently tested as anti-cancer drug and proposed for clinical trials for neurodegenerative diseases. Block of translation initiation mediated by rapamycin is able to rescue DA cell loss observed in knock-out flies for parkin and pink1 as well as in those over-expressing the dominant PD-associated mutation of LRRK2 (Reference 8; Tain, L.S., et al. Rapamycin activation of 4E-BP prevents parkinsonian dopaminergic neuron loss. Nat Neurosci 12, 1129-1135 (2009).). Furthermore, it protects mammalian DA cells from neurochemical intoxication in vitro and in mice (Reference 9; Malagelada, C., Jin, Z.H., Jackson-Lewis, V., Przedborski, S. & Greene, L.A. Rapamycin protects against neuron death in in vitro and in vivo models of Parkinson's disease. J Neurosci 30, 1166-1175 (2010)., Reference 10; Malagelada, C., Ryu, E.J., Biswas, S.C., Jackson-Lewis, V. & Greene, L.A. RTP801 is elevated in Parkinson brain substantia nigral neurons and mediates death in cellular models of Parkinson's disease by a mechanism involving mammalian target of rapamycin inactivation. J Neurosci 26, 9996-10005 (2006).). Recently, rapamycin was shown to prevent L-DOPA-induced dyskinesia, a common severe motor side effect of the symptomatic treatment for PD (reference 11; Santini, E., Heiman, M., Greengard, P., Valjent, E. & Fisone, G. Inhibition of mTOR signaling in Parkinson's disease prevents L-DOPA-induced dyskinesia. Sci Signal 2, ra36 (2009).).

[0105]  The effects of rapamycin on the cytoplasmic content of AS Uchll were confirmed by the presence of a concomitant decrease in its nuclear steady state levels, and by the absence of any de-novo transcription of AS Uchll (Experiment 10). Total cellular content of these transcripts remained constant. Uchll mRNA showed no changes in subcellular distribution, de novo transcription or total cellular content.

[0106]  Despite the block in CAP-dependent translation, upon rapamycin treatment UchLl protein level increased several fold (Experiment 11).

[0107]  The inventors assessed whether AS Uchll was required for UchLl induction. Stable MN9D cell lines were then established expressing constitutively shRNA for AS Uchll, targeting the AS Uchll promoter region from -4 to + 15 nt around the RACE-validated TSS (Reference 12; Hawkins, P.G., Santoso, S., Adams, C., Anest, V. & Morris, K.V. Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells. Nucleic Acids Res 37, 2984-2995 (2009)). As expected, scrambled cells showed UchLl protein up-regulation as in MN9D parental line while cells expressing shRNA for AS Uchll lacked any changes in UchLl protein levels proving a causal link between rapamycin induction of Uchll protein and AS Uchll ncRNA expression (Experiment 12). As independent model, stable cell lines with expression of a dominant negative mutant of AS Uchl1(Δ-SINEB2) were established. When control MN9D cells stable for empty vector were treated with rapamycin, UchLl protein was found increased. In presence of the dominant negative form of AS Uchll this upregulation was no longer visible (Experiment 13).

[0108]  A model for AS-dependent increase in S-encoded protein levels upon rapamycin treatment. :
In growing cells mTORC1 signaling is required for proliferation and controls CAP-dependent translation machinery through the phosphorylation of its downstream substrates 4E-BPs and S6K. The cytostatic drug rapamycin inhibits mTORC1 activity leading to block of CAP-dependent translation. Here, the inventors show that in these conditions AS transcription is required for protein synthesis of selected mRNAs. Upon rapamycin addition, the nuclear-enriched ncRNA AS Uchll is transported into the cytoplasm where it recruits mRNAs of its S protein-encoding partner to polysomes for translation.

[0109]  AS Uchll is thus the representative member of a new functional class of ncRNAs that are associated to S/AS pairs in the mammalian genome and appears to be composed by two domains. The overlapping region at the 5' provides specificity to a protein-encoding mRNA partner transcribed from the complementary strand. An inverted SINEB2 element at 3' is required for translational activation, representing a new function for embedded SINEB2 in the cytoplasm.

[0110]  The manipulation of Uchll expression in vivo has been proposed for therapeutic intervention in neurodegenerative diseases, including PD and AD. Natural AS transcripts with embedded repetitive elements may thus represent endogenous molecular tools to increase protein synthesis of selected mRNAs defining a potential new class of RNA therapeutics.

[0111]  Rapamycin is currently under intense scrutiny in biomedical research both as neuroprotective agent for neurodegenerative diseases and as anti-cancer drug. In mice rapamycin prevents L-DOPA-induced dyskinesia, a common severe motor side effect of the symptomatic treatment for PD. Furthermore, it protects neurons from apoptosis both in Drosophila genetic models as well as upon neurochemical intoxication in mammals making it an attractive molecule for anti-parkinsonian therapies (Reference 11; Santini, E et al., (2009)). It is thus important to better understand its modes of action in vivo and its interplay with pathways involved in familiar cases of PD, as shown here for Uchll. The role of AS transcription in rapamycin-induced protein synthesis adds an unexpected switch to its activities.

[0112]  Artificially synthesized AS for increasing EGFP expression or antibody expression up-regulates the expression of each target in the cell. :
The inventors tested whether or not increased protein synthesis can be achieved on a synthetic RNA by inserting a 72 nt long target determinant sequence antisense to the enhanced green fluorescent protein (EGFP) into the appropriate sequence in AS Uchll. As in Experiment 14, the expression of the artificial AS for up-regulating EGFP expression was designed. The fragments encoding the artificial AS was cloned into pcDNA3.1- vector (Invitrogen). This proves that a functional nucleic acid molecule with a target determinant sequence and a regulatory sequence can increase protein synthesis efficiency of any gene of interest.

[0113]  In the same manner, the antibody can be applied as shown in Experiment 15. An artificial AS for up-regulating a recombinant antibody was designed to target the leader sequence of the recombinant antibody (overlap is 72bp around the ATG) and embedded into the vector (AS Fc). An artificial AS comprising a scramble sequence as the target determinant sequence (Scrambled) was also produced as control (Fig. 15). The target sequence of the antibody was included in pHYGRO vectors (pHYGRO). HEK cell line was co-transfected with pHYGRO and AS Fc or control. Transient transfection in HEK cells produced an AS Fc specific upregulation of the recombinant antibody in cell lysates. AS Fc increases the encoded protein levels in transfected HEK cells while a scramble overlapping sequence or an empty plasmid does not, detected through the SV5 tag.

[0114]  For further examination (Experiment 16), HEK cell line was transfected with EGFP expression vector pEGFP-C2 (Clontech) with Lipofectamine 2000 (Invitrogen) in accordance with manufacturer's instructions. Then, the transfectants which stably express low EGFP expression were selected. AS GFP comprises target determinant sequence against EGFP and it was artificially designed to increase the efficiency of EGFP protein synthesis (Fig. 16). The stable transfectants were further transfected with the vector encoding AS GFP or control vector. After 24 hours or 48 hours from the transfection of AS GFP construct or control vector, the cells were collected and lysed. The GFP level in the cell lysate was monitored by western blotting. The low expression of EGFP in the transfectant was increased by transfection of the AS GFP construct (Fig. 16).

[0115]  In an additional experiment, a construct with a target determinant sequence that overlaps for 44nt to the plasmid backbone till the ATG codon for EGFP has been synthesized. This contruct (SINEup005) increases protein synthesis of the target gene very efficiently (more than 10 fold).

[0116]  Therefore, this is one of evidences that the new type of AS which the inventors found can be artificially designed as long as the functional nucleic acid molecule includes a target determinant sequence comprising an antisense sequence to a target sequence in the protein-encoding RNA for which protein synthesis efficiency is to be increased and a regulatory sequence having an activity of increasing of the protein synthesis efficiency.

Methods


Plasmids



[0117]  RACE fragment : The 5' UTR of AS Uchll was amplified by RACE PCR (GeneRacer, Invitrogen) by MN9D total RNA and cloned into pGEM®-T Easy vector (Promega).
Full length AS Uchl1 : Full length DNA sequence of AS was amplified via fusion PCR starting from RACE fragment and FANTOM clone Rik 6430596G22 with following primers For mAS Uchll fl 5'-ACAAAGCTCAGCCCACACGT- 3' (SEQ ID No:13) and Rev mAS Uchllfl 5'-CATAGGGTTCATT -3'(SEQ ID No:14).
Uchll : Mouse Uchll mRNA was cloned from FANTOM 2900059022 with following primers: For mUchl1 5'-ATGCAGCTGAAGCCGATG-3'(SEQ ID No:15) and Rev mUchl1 5'-TTAAGCTGCTTTGCAGAGAGC-3' (SEQ ID No:16) AS Uchll shRNA : Oligo containing the sequence -14/+4 around the TSS of AS Uchll CGCGCAGTGACACAGCACAAA (SEQ ID No:17) are cloned into pSUPER.retro.puro vector (OligoEngine, Seattle, WA), scrambled sequence was used as control.

Deletional mutants :



[0118]  Δ5': For mAS Uchll fl and Rev Δ 5'AS Uchl15' TACCATTCTGTGCGGTGCA-3' (SEQ ID No:18).
Δ3': For mAS Uchll GACCTCCTCTAGCACTGCACA-3' (SEQ ID No:19) and Rev mAS Uchll fl.
For fine deletional mutants, PCR fragment I is cloned NheI-EcoRI site in PcDNA3.1- and PCR fragment II into following EcoRI-HindII site.

AS Uchll Δ (Alu+SINEB2) :



[0119]  PCR fragment I: For mAS Uchll fl and Rev pre-SINE B2 5'-CAATGGATTCCATGT-3' (SEQ ID No:20). PCR fragment II: For post-ALU 5'-GATATAAGGAGAATCTG-3' (SEQ ID No:21) and Rev mAS fl.

AS Uchll Δ(Alu) :



[0120]  PCR fragment I: mAS Uchll fl and Rev pre-Alu 5'-TTATAG TATGTGTTGTC-3' (SEQ ID No:22). PCR fragment II: For post-ALU 5'-GATATAAGGAGAATCTG-3' (SEQ ID No:23) and Rev mAS fl cloned into EcoRI-HindII site.

AS Uchll Δ (SINEB2) :



[0121]  PCR fragment I: For mAS Uchll fl and and Rev pre-SINE B2 5'-CAATGGATTCCATGT-3' (SEQ ID No:24). PCR fragment II: For post-SINE B2 5'- GAATTCCTCCAGTCTCTTA -3' (SEQ ID No:25) and Rev mAS fl.
AS uchl1 (Alu+SINEB2) flip : PCR fragment I: obtained with For SINE B2 inside 5'-TGCTAGAGGAGG-3' (SEQ ID No:26) and Rev Alu flip 5'- GTCAGGCAATCC -3' (SEQ ID No:27) are cloned in the unique EcoRI site of AS Uchll Δ (Alu+SINEB2). AS uchl1 SINEB2 flip: PCR fragment obtained with For SINE B2 inside 5'-TGCTAGAGGAGG-3' (SEQ ID No:28) and Rev SINE flip 5'-AAAGAGATGGC-3' (SEQ ID No:29) are cloned in the unique EcoRI site of AS Uchll Δ (SINEB2).

Cells



[0122]  MN9D cells were seeded in 10 mm petri-dishes in Dulbecco's modified Eagle's medium containing 10% of fetal bovine serum and, penicillin (50 units/ml), streptomycin (50 units/ml). Treatments were done by adding Rapamycin (R0395, Sigma) at final concentration of 1ug/ml in fresh medium for 45 minutes.

[0123]  For the establishment of MN9D stable cells (siRNA -15/+4, siRNA scrambled, pcDNA 3.1- and Δ SINE B2) MN9D cells were seeded in 100mm petri-dishes and transfected with Lipofectamine 2000 (Invitrogen) according to manufacturer's instruction, the day after cells seeded for selection with 500uM of Neomycin (#N1142, Sigma). HEK-293T cells were grown in DMEM (GIBCO) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 units/mL penicillin, and 100 µm/mL streptomycin (Sigma) at 37 °C in a humidified CO 2 incubator.

PCR



[0124]  PCR analysis: Total RNA was extracted using Trizol reagent (Invitrogen) according to manufacturers instruction. It was subjected to DNAse I treatment (Ambion) and 1 ug was retrotranscribed using iScript cDNA Synthesis Kit (BioRad). Real Time qRT-PCR was carried out using Sybr green fluorescence dye (2X iQ5 SYBR Green supermix, BioRad). Actin and GAPDH were used as internal standard. Relative quantification was performed with the comparative Ct method.

Actin: sense 5'- CACACCCGCCACCAGTTC-3' (SEQ ID No:30), antisense 5'-CCCATTCCCACCATCACACC-3' (SEQ ID No:31).

Gapdh: sense 5'-GCAGTGGCAAAGTGGAGATT-3' (SEQ ID No:32), antisense 5'-GCAGAAGGGGCGGAGATGAT-3' (SEQ ID No:33).

AS Uchll overlap: sense 5'-GCACCTGCAGACACAAACC-3' (SEQ ID No:34), antisense 5'-TCTCTCAGCTGCTGGAATCA-3' (SEQ ID No:35).

AS Uchll : 5'CTGGTGTGTATCTCTTATGC (SEQ ID No:36) antisense 5'CTCCCGAGTCTCTGTAGC (SEQ ID No:37).

Uchll : sense 5'- CCCGCCGATAGAGCCAAG (SEQ ID No:38), antisense 5'-ATGGTTCACTGGAAAGGG-3' (SEQ ID No:39).

ASUchll pre RNA : 5'-CCATGCACCGCACAGAATG-3' (SEQ ID No:40), antisense 5'-GAAAGCTCCCTCAAATAGGC-3' (SEQ ID No:41).

Pre_A0ribosomal RNA sense 5'-TGTGGTGTCCAAGTGTTCATGC-3' (SEQ ID No:42), antisense5'-CGGAGCACCACATCGATCTAAG-3 (SEQ ID No:43).

AS_Uxt: sense5'-CAACGTTGGGGATGACTTCT (SEQ ID No:44), antisense5'-TCGATTCCCATTACCCACAT (SEQ ID No:45);

Uxt: sense5'-TTGAGCGACTCCAGGAAACT-3' (SEQ ID No:46), antisense 5'-GAGTCCTGGTGAGGCTGTC-3' (SEQ ID No:47).

Multiplex RT PCR was performed with SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Invitrogen). 500 mg of total DNAse traeated RNA was incubated with reverse primers for Gapdh, Uchll, AS Uchll. The reaction was performed for 60 minutes at 60 degrees.

[0125]  Each volume was then splitted in three and forward primers were added at final concentration of 200nM to the reaction. The PCR reaction comprised 40 cycles of 95 degrees for 15 seconds, followed by 60 degrees for 45 seconds, and final 68 degrees for 30 seconds.

Western blot



[0126]  Cells were lysed in SDS sample buffer 2X. Proteins were separated in 15% SDS-polyacrilamide gel and transferred to nitrocellulose membrane. Immunoblotting was performed with the primary antbodies: anti-Uchll (#3524 Cell Signalling) 1:300 and anti-b actin (A5441, Sigma) 1:5000. Signals were revealed after incubation with recommended secondary antibodies conjugated with horseradish peroxidase by using enhanced chemioluminescence for Uchll (#WBKLS0500 Immobilion Western Chemioluminescent HRP substrate) and ECL detection reagent (RPN2105, GE Healthcare).

Cellular fractionation



[0127]  Nucleo cytoplasmic fractionation was performed using Nucleo Cytoplasmic separation kit (Norgen) according to manufacturer's instruction. RNA was eluted and DNAseI treated. The purity of the cytoplasmic fraction was confirmed by Real Time qRT-PCR on Pre-ribosomal RNA.

Two colour In situ hybridization (ISH)



[0128]  Reagents: anti DIG antibody D8156 (Sigma); streptavidin HRP RPN1231-100UL (Amersham Bioscience); DIG labeling mix #11 277 073 910 (Roche) BIO-labelling Mix # 11 685 597 910 (Roche), Ribonucleic acid, transfer from baker's yeast R8759 (Sigma), Deoxyribonucleic acid, single stranded from salmon testes D7656 (Sigma), Blocking reagent #11 096 176 001 (Roche), TSA Cy3 system (Perkin Elmer, Heidelberg, Germany).

[0129]  After perfusion with 4% formaldehyde, the mouse brain was cryoprotected overnight in 30% sucrose. In situ hybridization was performed on cryostat slices (16 um). Sense and antisense probes were generated by in vitro transcription from the cDNA encoding the distal 600 bps of mouse Uchll cDNA and the last 1000 bps of mouse AS Uchll. The probes for Uchll were labeled with digoxigenin, probes for AS Uchll were labeled with biotin. Incorporation of both bioting and digoxigenin was checked via Northern Blot. Slices were pretreated with hydrogenum peroxide 3% for 30 minutes. Hybridization was performed with probes at a concentration of 1 mg/ml (Uchll) and 3 mg/ml for AS Uchll at 60°C for 16 h. For biotinilated RNA detection, streptavidin-HRP was used 1:250 for 2 hours in TNB buffer (Tris HCl PH 7,5 100mM, NaCl 150 mM, 0,5% Blocking Reagent), and signals are visualized using the TSA Cy3 system after washing in TNT buffer (Tris HCl PH 7,5 100mM, NaCl 150 mM, 0,05% tween).

[0130]  ISH on DIG-labeled probe was performed by incubating slices with monoclonal anti-DIG antibody after TSA reaction. To combine RNA ISH with immunofluorescence, slice were incubated with the antibody anti TH (#AB152, Chemicon) 1:1000. Signals are then detected with fluorescent dye-conjugated secondary antibody goat anti-rabbit 405 and goat anti-mouse 488. Sections were then washed, mounted with Vectashield (Vector lab) mounting medium and observed at confocal microscope (Leica).

Post-mortem human brain samples



[0131]  Brain samples were obtained from the brain bank at the Institute of Neuropathology, Bellvitge Hospital (University of Barcelona, Spain). Samples were dissected at autopsy with the informed consent of patients or their relatives and the institutional approval of the Ethics Committee of the University of Barcelona. Brains were obtained from Caucasian, pathologically confirmed PD cases and age-matched controls (Navarro et al., 2009). Briefly, all cases of PD had suffered from classical PD, none of them had cognitive impairment and their neuropathological characterization was made according to established criteria. Control healthy subjects showed absence of neurological symptoms and of metabolic and vascular diseases, and the neuropathological study disclosed no abnormalities, including lack of Alzheimer disease and related pathology. The time between death and tissue preparation was in the range of 3 to 5 hours.

Bioinformatic analysis



[0132]  For the identification of a cadidate human hortologue of AS Uchll, conservation between human and mouse in the orthologus region of AS uchl1 was performed using VISTA genome browser. The inventors selected parameters for conserved sequence tags (CTS) that have a minimum of 75% identity between the mouse and human genome. For each conserved element a primer on the homologus human region was designed.

[0133]  For the identification of additional translational activator candidates, the inventors searched for FANTOM3 full-length cDNAs that were non-coding RNAs and overlap the 5' end of coding transcripts in a head to head configuration [PMID: 16141072]. The filtered set of 8535 FANTOM3 ncRNA transcripts described in the Nordstrom et al 2009 ( Nordstrom, K.J., et al. Critical evaluation of the FANTOM3 non-coding RNA transcripts. Genomics 94, 169-176 (2009).) was used as our starting point. Genomic locations of these ncRNA transcripts and REFSEQ ( Maglott, D.R., Katz, K.S., Sicotte, H. & Pruitt, K.D. NCBI's LocusLink and RefSeq. Nucleic Acids Res 28, 126-128 (2000)) coding transcripts were extracted from the alignments in the UCSC Genome browser ( Kent, W.J., et al. The human genome browser at UCSC. Genome Res 12, 996-1006 (2002)) to identify a set of 788 coding-sense: non-coding-antisense pairs. The ncRNAs were then checked by repeat masker to identify SINEB2 related sequences (Smit, AFA, Hubley, R & Green, P. RepeatMasker Open-3.0.1996-2010 <http://www.repeatmasker.org>). This reduced the number of pairs to 127 protein coding transcripts with overlap at the 5' end (60 with a sense strand version of the repeat, 53 with an antisense version and 14 with both sense and antisense versions).

[0134]  Alignment of the SINEB2 related elements was then carried out using Clustalw (http://www.ebi.ac.uk/Tools/clustalw2/index.html. From this analysis the antisense overlapping transcripts with a repeat most like that of Uchll-as and in the same orientation were chosen for experimental testing (Uxt1-AS).

Sequences



[0135]  As example, the following is a list of sequences that are complementary to protein coding mRNAs. They contain a fraction that provides examples of a target determinant sequence [now in light blue highlight] and a regulatory sequence [how highlighted in red]. The regulatory sequence in this list of natural antisense is as short at 89 nucleotides in this example. The length of the adaptor sequences in this partial list of antisense RNAs is as short as 44 nt.

[0136]  Below each sequence there is a summary of the alignments with the retrotransposon elements as determined by Repeatmask program.





[0137]  Other sequences in the study:









[0138]  The examples of SINE B2 embedded in cDNA clones sequence are as follows.
Table 1
Gene with AS_refseq start B2 end B2   strand subtype         Legend; cDNA cloneID_alignment Start position_Alignent end_type of Repeat element
NM_177182 973 1013 -2766 + B2_M m2 SINE/ B2 2 42 -153 >AK032380_973_10 13_+_B2_Mm2
NM_009351 859 1044 -1470 + B2_M m1t SINE/ B2 2 185 -8 >AK033525_859_10 44_+_B2_Mm1t
NM_144515 1564 1718 -761 + B2_M m2 SINE/ B2 1 158 -37 >AK033993_1564_1 718_+_B2_Mm2
NM_198300 520 712 -1160 + B2_M m2 SINE/ B2 1 188 -7 >AK039361_520_71 2_+_B2_Mm2
NM_0284 28 1801 1997 -245 + B2_M m1t SINE/ B2 1 193 0 >AK042841_1801_1 997_+_B2_Mm1t
NM_0106 61 946 1111 -1515 + B2_M m2 SINE/ B2 1 195 0 >AK043817 _946_11 11_+_B2_Mm2
NM_0074 85 722 912 -417 + B2_M m2 SINE/ B2 1 194 -1 >AK044205_722_91 2_+_B2_Mm2
NM_0106 33 971 1164 -67 + B2_M m2 SINE/ B2 1 195 0 >AK047213_971_11 64_+_B2_Mm2
NM_0302 07 714 787 -446 + B2_M m2 SINE/ B2 1 88 -107 >AK079217_714_78 7_+_B2_Mm2
NM_1454 70 1498 1697 -1057 + B2_M m2 SINE/ B2 1 193 -2 >AK081722_1498_1 697_+_B2_Mm2
NM_0242 82 1379 1555 -2 + B2_M m2 SINE/ B2 2 187 -8 >AK132990_1379_1 555_+_B2_Mm2
NM_1339 94 1001 1057 -86 + B2_M m2 SINE/ B2 2 58 -137 >AK133457_1001_1 057_+_B2_Mm2
NM_1339 94 1057 1137 -6 + B2_M m2 SINE/ B2 91 171 -24 >AK133457_1057_1 137_+_B2_Mm2
NM_1782 44 2323 2513 -45 + B2_M m2 SINE/ B2 1 195 0 >AK133632_2323_2 513_+_B2_Mm2
NM_0089 97 734 923 -3 + B2_M m2 SINE/ B2 1 186 -9 >AK133808_734_92 3_+_B2_Mm2
NM_0805 55 286 382 -4 + B2_M m1t SINE/ B2 75 171 -22 >AK134674_286_38 2_+_B2_Mm1t
NM_0103 32 2428 2606 -1 + B2_M m2 SINE/ B2 1 183 -12 >AK135599_2428_2 606_+_B2_Mm2
NM_1724 67 2948 3108 -3 + B2_M m2 SINE/ B2 1 162 -33 >AK137583_2948_3 108_+_B2_Mm2
NM_1751 15 81 274 -2006 + B2_M m2 SINE/ B2 2 195 0 >AK138675_81_274 _+_B2_Mm2
NM_0100 71 3150 3339 -54 + B2_M m2 SINE/ B2 1 195 0 >AK155102_3150_3 339_+_B2_Mm2
NM_1830 14 288 442 -20 + B3A SINE/ B2 48 198 0 >AK015655_288_44 2_+_B3A
NM_0265 55 277 435 -24 + B3A SINE/ B2 40 198 0 >AK021299_277_43 5_+_B3A
NM_0330 77 1109 1295 -1732 + B3A SINE/ B2 5 189 -9 >AK029689_1109_1 295_+_B3A
NM_0197 89 1052 1211 -1195 + B3 SINE/ B2 33 196 -20 >AK030353_1052_1 211_+_B3
NM_1788 91 2403 2558 -3 + B3 SINE/ B2 20 176 -40 >AK030551_2403_2 558_+_B3
NM_1455 79 2546 2719 -346 + B3 SINE/ B2 2 206 -10 >AK031007_2546_2 719_+_B3
NM_0085 10 2255 2404 -429 + B3A SINE/ B2 57 198 0 >AK034030_2255_2 404_+_B3A
NM_1535 79 1071 1262 -798 + B3 SINE/ B2 1 214 -2 >AK035406_1071_1 262_+_B3
NM_1459 42 1006 1219 -681 + B3 SINE/ B2 2 216 0 >AK037188_1006_1 219_+_B3
NM_0257 88 1871 2045 -757 + B3 SINE/ B2 1 215 -1 >AK039409_1871_2 045_+_B3
NM_0080 19 877 1044 -1057 + B3 SINE/ B2 2 211 -5 >AK040162_877_10 44_+_B3
NM_0010 81475 1431 1582 -6 + B3 SINE/ B2 69 216 0 >AK040401_1431_1 582_+_B3
NM_0010 81475 1586 1745 -2 + B3 SINE/ B2 3 206 -10 >AK040401_1586_1 745_+_B3
NM_0074 85 722 810 -519 + B3 SINE/ B2 1 89 -127 >AK044205_722_81 0_+_B3
NM_0074 85 835 912 -417 + B3 SINE/ B2 138 215 -1 >AK044205_835_91 2_+_B3
NM_1787 94 826 959 -986 + B3A SINE/ B2 2 129 -69 >AK045196_826_95 9_+_B3A
NM_0089 15 737 940 -298 + B3 SINE/ B2 8 201 -15 >AK046652_737_94 0_+_B3
NM_0106 33 971 1096 -135 + B3 SINE/ B2 1 129 -87 >AK047213_971_10 96_+_B3
NM_1994 76 3267 3405 -1039 + B3 SINE/ B2 2 149 -67 >AK047540_3267_3 405_+_B3
NM_1447 95 2052 2248 -546 + B3 SINE/ B2 2 210 -6 >AK048854_2052_2 248_+_B3
#N/A 1858 2028 -5 + B3A SINE/ B2 1 176 -22 >AK049524_1858_2 028_+_B3A
NM_0010 38621 1207 1363 -359 + B3A SINE/ B2 2 160 -38 >AK053130_1207_1 363_+_B3A
NM_1814 23 1666 1733 -71 + B3A SINE/ B2 2 72 -126 >AK054359_1666_1 733_+_B3A
NM_1535 15 1403 1520 -470 + B3A SINE/ B2 63 183 -15 >AK078013_1403_1 520_+_B3A
NM_1984 15 403 623 -1212 + B3 SINE/ B2 1 216 0 >AK078328_403_62 3_+_B3
NM_1522 20 1794 1979 -561 + B3 SINE/ B2 3 213 -3 >AK078537_1794_1 979_+_B3
NM_0257 29 1 165 -203 + B3 SINE/ B2 15 177 -39 >AK079403_1_165_ +_B3
NM_0166 93 1307 1517 -13 + B3 SINE/ B2 2 216 0 >AK080235_1307_1 517_+_B3
NM_0101 51 814 1024 -451 + B3 SINE/ B2 2 216 0 >AK082108_814_10 24_+_B3
NM_0097 13 1690 1882 -5 + B3 SINE/ B2 1 199 -17 >AK082325_1690_1 882_+_B3
NM_0010 29985 221 280 -1179 + B3 SINE/ B2 108 168 -48 >AK084376_221_28 0_+_B3
NM_0097 37 1805 2005 -406 + B3 SINE/ B2 1 207 -9 >AK085337_1805_2 005_+_B3
NM_0270 81 984 1130 -1155 + B3 SINE/ B2 1 162 -54 >AK086470_984_11 30_+_B3
NM_0187 79 876 1062 -530 + B3A SINE/ B2 2 188 -10 >AK089148_876_10 62_+_B3A
NM_0279 19 1468 1667 -185 + B3 SINE/ B2 1 213 -3 >AK090182_1468_1 667_+_B3
NM_0110 34 434 608 -12 + B3 SINE/ B2 2 216 0 >AK131819_434_60 8_+_B3
NM_1755 51 1802 1895 -513 + B3A SINE/ B2 64 176 -22 >AK132737_1802_1 895_+_B3A
NM_1755 51 1829 1928 -480 + B3 SINE/ B2 101 213 -3 >AK132737_1829_1 928_+_B3
NM_0079 21 951 1134 -1650 + B3A SINE/ B2 3 187 -11 >AK133325_951_11 34_+_B3A
NM_0010 39042 1220 1315 -292 + B3A SINE/ B2 48 142 -56 >AK134755_1220_1 315_+_B3A
NM_1773 28 1378 1504 -285 + B3 SINE/ B2 85 216 0 >AK134874_1378_1 504_+_B3
NM_0218 99 1172 1259 -140 + B3 SINE/ B2 1 95 -121 >AK135206_1172_1 259_+_B3
NM_0087 05 1521 1717 -572 + B3A SINE/ B2 1 197 -1 >AK136279_1521_1 717_+_B3A
NM_0010 79932 2116 2299 -702 + B3 SINE/ B2 6 208 -8 >AK137643_2116_2 299_+_B3
NM_0010 79932 3059 3253 -2 + B3 SINE/ B2 4 205 -11 >AK137643_3059_3 253_+_B3
NM_0010 33286 2524 2698 -63 + B3A SINE/ B2 4 181 -17 >AK138296_2524_2 698_+_B3A
NM_0089 62 928 1095 -375 + B3 SINE/ B2 1 153 -63 >AK138521_928_10 95_+_B3
NM_1753 49 2151 2347 -181 + B3A SINE/ B2 11 196 -2 >AK139254_2151_2 347_+_B3A
NM_1770 03 1277 1466 0 + B3 SINE/ B2 1 200 -16 >AK139647_1277_1 466_+_B3
NM_0079 65 1338 1488 -5 + B3 SINE/ B2 51 216 0 >AK140072_1338_1 488_+_B3
NM_0101 92 1808 1933 -45 + B3 SINE/ B2 1 126 -90 >AK140346_1808_1 933_+_B3
NM_0274 46 1871 1983 -2794 + B3A SINE/ B2 1 130 -68 >AK140616_1871_1 983_+_B3A
NM_0274 46 2318 2342 -2435 + B3A SINE/ B2 131 156 -42 >AK140616_2318_2 342_+_B3A
NM_1771 86 2061 2142 -351 + B3 SINE/ B2 130 211 -5 >AK142359_2061_2 142_+_B3
NM_1451 34 2963 3148 -1292 + B3 SINE/ B2 28 210 -6 >AK142507_2963_3 148_+_B3
NM_0098 90 2740 2968 -135 + B3 SINE/ B2 1 216 0 >AK142879_2740_2 968_+_B3
NM_1460 55 5202 5272 -94 + B3 SINE/ B2 60 141 -75 >AK143143_5202_5 272_+_B3
NM_0089 77 1344 1513 -645 + B3 SINE/ B2 11 205 -11 >AK143279_1344_1 513_+_B3
NM_0260 36 2001 2167 -272 + B3A SINE/ B2 1 169 -29 >AK149843_2001_2 167_+_B3A
NM_00 11 59519 1440 1548 -1422 + B3A SINE/ B2 55 163 -35 >AK157402_1440_1 548_+_B3A
NM_00 11 59519 2503 2556 -558 + B3 SINE/ B2 1 56 -160 >AK157402_2503_2 556_+_B3
NM_00 11 59519 2544 2616 -498 + B3 SINE/ B2 109 177 -39 >AK157402_2544_2 616_+_B3
NM_00 11 59519 2702 2896 -412 + B3A SINE/ B2 1 198 0 >AK157402_2702_2 896_+_B3A
NM_00 11 10504 1778 1909 -25 + B3 SINE/ B2 58 210 -6 >AK160921_1778_1 909_+_B3
NM_0307 14 2153 2335 -62 + B3 SINE/ B2 40 216 0 >AK165234_2153_2 335_+_B3
NM_0258 25 945 1052 -383 R C B2_M m1a SINE/ B2 -85 108 1 >AK014613_945_10 52_RC_B2_Mm1a
NM_1337 56 1102 1289 -246 R C B2_M m2 SINE/ B2 -4 191 1 >AK016234_1102_1 289_RC_B2_Mm2
NM_1832 94 1565 1750 -1384 R C B2_M m1a SINE/ B2 -8 185 4 >AK029702_1565_1 750_RC_B2_Mm1a
NM_0094 46 531 710 -2730 R C B2_M m2 SINE/ B2 -6 189 2 >AK030803_531_71 0_RC_B2_Mm2
NM_0257 88 1687 1839 -963 R C B2_M m2 SINE/ B2 -11 184 1 >AK039409_1687_1 839_RC_B2_Mm2
NM_1777 85 1626 1682 -1130 R C B2_M m2 SINE/ B2 -4 191 137 >AK040275_1626_1 682_RC_B2_Mm2
NM_1777 85 1682 1739 -1073 R C B2_M m2 SINE/ B2 -94 101 46 >AK040275_1682_1 739_RC_B2_Mm2
NM_1777 85 1776 1952 -1036 R C B2_M m1t SINE/ B2 -13 180 2 >AK040275_1776_1 952_RC_B2_Mm1t
NM_0074 85 1137 1341 -192 R C B2_M m2 SINE/ B2 0 195 2 >AK044205_1137_1 341_RC_B2_Mm2
NM_1752 73 537 714 -1793 R C B2_M m2 SINE/ B2 -5 190 1 >AK048762_537_71 4_RC_B2_Mm2
NM_0294 09 1005 1172 -2204 R C B2_M m1t SINE/ B2 -14 179 1 >AK049449_1005_1 172_RC_B2_Mm1t
NM_0078 36 282 407 -2067 R C B2_M m2 SINE/ B2 -67 128 2 >AK054076_282_40 7_RC_B2_Mm2
NM_0078 36 1764 1860 -614 R C B2_M m2 SINE/ B2 -97 98 2 >AK054076_1764_1 860_RC_B2_Mm2
NR_0028 91 1 181 -2344 R C B2_M m1a SINE/ B2 -10 183 2 >AK076350_1_181_ RC_B2_Mm1a
NM_0265 00 984 1157 -1501 R C B2_M m2 SINE/ B2 -14 181 1 >AK076438_984_11 57_RC_B2_Mm2
NM_0529 94 2158 2323 -1225 R C B2_M m2 SINE/ B2 -4 191 8 >AK079094_2158_2 323_RC_B2_Mm2
NM_1531 00 1870 2057 -1774 R C B2_M m2 SINE/ B2 0 195 1 >AK086953_1870_2 057_RC_B2_Mm2
NM_1753 13 2088 2261 -2958 R C B2_M m2 SINE/ B2 -9 186 14 >AK132441_2088_2 261_RC_B2_Mm2
NM_00 11 14140 5305 5461 -1145 R C B2_M m2 SINE/ B2 -38 157 2 >AK133162_5305_5 461_RC_B2_Mm2
NM_0095 79 1059 1251 -2941 R C B2_M m2 SINE/ B2 0 195 1 >AK137370_1059_1 251_RC_B2_Mm2
NM_0105 67 1613 1794 -1882 R C B2_M m2 SINE/ B2 -2 193 1 >AK138181_1613_1 794_RC_B2_Mm2
NM_0010 08423 2088 2222 -57 R C B2_M m2 SINE/ B2 -60 135 1 >AK141165_2088_2 222_RC_B2_Mm2
NM_1990 27 935 1064 -63 R C B2_M m2 SINE/ B2 -65 130 1 >AK141411_935_10 64_RC_B2_Mm2
NM_0010 33316 3103 3283 -440 R C B2_M m2 SINE/ B2 -5 190 2 >AK145736_3103_3 283_RC_B2_Mm2
NR_0034 92 748 810 -977 R C B2_M m1a SINE/ B2 -6 187 123 >AK147092_748_81 0_RC_B2_Mm1a
NM_0105 67 935 1129 -78 R C B2_M m2 SINE/ B2 0 195 2 >AK148373_935_11 29_RC_B2_Mm2
NM_0261 15 3593 3791 -204 R C B2_M m2 SINE/ B2 -1 194 1 >AK155374_3593_3 791_RC_B2_Mm2
NM_0103 98 1080 1265 -909 R C B2_M m2 SINE/ B2 -8 187 1 >AK157261_1080_1 265_RC_B2_Mm2
NM_0078 93 764 820 -1012 R C B2_M m2 SINE/ B2 -94 101 47 >AK163105_764_82 0_RC_B2_Mm2
NM_0078 93 894 1062 -938 R C B2_M m2 SINE/ B2 -13 182 1 >AK163105_894_10 62_RC_B2_Mm2
NM_0078 93 1390 1436 -564 R C B2_M m2 SINE/ B2 -5 190 137 >AK163105_1390_1 436_RC_B2_Mm2
NM_0078 93 1436 1490 -510 R C B2_M m2 SINE/ B2 -94 101 47 >AK163105_1436_1 490_RC_B2_Mm2
NM_0078 93 1543 1701 -299 R C B2_M m1t SINE/ B2 -24 169 1 >AK163105_1543_1 701_RC_B2_Mm1t
NM_1771 86 1783 1964 -258 R C B2_M m1t SINE/ B2 -7 186 2 >AK163831_1783_1 964_RC_B2_Mm1t
NM_0307 14 1401 1588 -809 R C B2_M m2 SINE/ B2 0 195 3 >AK165234_1401_1 588_RC_B2_Mm2
NM_0011 10101 1253 1420 -1139 R C B2_M m2 SINE/ B2 -22 173 2 >AK169421_1253_1 420_RC_B2_Mm2
NM_0076 01 387 544 -1715 R C B3A SINE/ B2 -22 176 1 >AK016423_387_54 4_RC_B3A
NM_0273 46 1459 1690 -74 R C B3 SINE/ B2 -2 214 1 >AK019925_1459_1 690_RC_B3
NM_1386 64 1382 1411 -1334 R C B3A SINE/ B2 -14 8 50 21 >AK028982_1382_1 411_RC_B3A
NM_0138 40 160 290 -556 R C B3 SINE/ B2 -79 137 9 >AK029359_160_29 0_RC_B3
NM_0138 40 774 960 -72 R C B3 SINE/ B2 -7 209 1 >AK029359_774_96 0_RC_B3
NM_1535 91 769 982 -1569 R C B3 SINE/ B2 -6 210 1 >AK032194_769_98 2_RC_B3
NM_0287 94 1926 2130 -654 R C B3 SINE/ B2 -12 204 2 >AK032215_1926_2 130_RC_B3
NM_0010 12311 1317 1437 -1013 R C B3 SINE/ B2 -44 172 38 >AK034331_1317_1 437_RC_B3
NM_0010 12311 1335 1437 -1013 R C B3A SINE/ B2 -54 144 38 >AK034331_1335_1 437_RC_B3A
NM_1341 22 1094 1205 -719 R C B3 SINE/ B2 -88 128 1 >AK035015_1094_1 205_RC_B3
NM_1535 79 1443 1617 -577 R C B3 SINE/ B2 -2 214 45 >AK035406_1443_1 617_RC_B3
NM_1535 79 1483 1633 -561 R C B3A SINE/ B2 -18 180 27 >AK035406_1483_1 633_RC_B3A
NM_0010 81014 1615 1715 -218 R C B3A SINE/ B2 -88 110 1 >AK039704_1615_1 715_RC_B3A
NM_0187 47 432 531 -1672 R C B3A SINE/ B2 -31 167 49 >AK040672_432_53 1_RC_B3A
NM_1768 41 1677 1886 -64 R C B3 SINE/ B2 0 216 2 >AK041236_1677_1 886_RC_B3
NM_1338 78 1676 1882 -314 R C B3 SINE/ B2 -7 209 1 >AK041654_1676_1 882_RC_B3
NM_1452 15 2334 2533 -484 R C B3 SINE/ B2 0 216 3 >AK041742_2334_2 533_RC_B3
NM_1453 69 1132 1336 -593 R C B3 SINE/ B2 -7 209 5 >AK042861_1132_1 336_RC_B3
NM_1726 91 248 442 -151 R C B3A SINE/ B2 -18 180 10 >AK043958_248_44 2_RC_B3A
NM_0074 85 938 1127 -202 R C B3 SINE/ B2 -37 179 2 >AK044205_938_11 27_RC_B3
NM_0074 85 989 1127 -202 R C B3A SINE/ B2 -57 141 2 >AK044205_989_11 27_RC_B3A
NM_0079 25 592 713 -719 R C B3 SINE/ B2 -54 162 39 >AK045677_592_71 3_RC_B3
NM_1770 06 3566 3620 -1104 R C B3 SINE/ B2 -16 0 56 2 >AK046828_3566_3 620_RC_B3
NM_0106 33 431 640 -591 R C B3 SINE/ B2 -4 212 8 >AK047213_431_64 0_RC_B3
NM_0106 33 457 640 -591 R C B3A SINE/ B2 -7 191 8 >AK047213_457_64 0_RC_B3A
NM_0088 42 1460 1636 -53 R C B3A SINE/ B2 -4 194 10 >AK047301_1460_1 636_RC_B3A
NM_1994 76 2716 2780 -1664 R C B3A SINE/ B2 -36 162 98 >AK047540_2716_2 780_RC_B3A
NM_0135 14 1203 1416 -311 R C B3 SINE/ B2 0 216 1 >AK048309_1203_1 416_RC_B3
NM_0807 93 2342 2396 -1333 R C B3A SINE/ B2 -5 193 144 >AK048747_2342_2 396_RC_B3A
NM_0807 93 2430 2530 -1199 R C B3A SINE/ B2 -55 143 47 >AK048747_2430_2 530_RC_B3A
NM_0010 38621 159 275 -1447 R C B3 SINE/ B2 -56 160 34 >AK053130_159_27 5_RC_B3
NM_0078 36 1747 1860 -614 R C B3 SINE/ B2 -97 119 2 >AK054076_1747_1 860_RC_B3
NM_0108 78 1134 1337 -1192 R C B3 SINE/ B2 0 216 1 >AK078161_1134_1 337_RC_B3
NM_0116 70 521 690 -995 R C B3 SINE/ B2 -60 156 1 >AK078321_521_69 0_RC_B3
NM_0260 86 882 1046 -355 R C B3A SINE/ B2 -18 180 1 >AK079515_882_10 46_RC_B3A
NM_0253 96 1081 1179 -370 R C B3 SINE/ B2 -27 189 75 >AK080749_1081_1 179_RC_B3
NM_0079 23 2420 2567 -582 R C B3A SINE/ B2 -48 150 4 >AK086589_2420_2 567_RC_B3A
NM_0284 27 1420 1597 -495 R C B3 SINE/ B2 -5 211 2 >AK090347_1420_1 597_RC_B3
NM_0261 57 1871 2066 -1227 R C B3A SINE/ B2 0 198 5 >AK132393_1871_2 066_RC_B3A
NM_0261 57 2342 2408 -885 R C B3A SINE/ B2 -13 1 67 1 >AK132393_2342_2 408_RC_B3A
NM_0261 57 2473 2606 -687 R C B3A SINE/ B2 -25 173 28 >AK132393_2473_2 606_RC_B3A
NM_1753 13 2113 2261 -2958 R C B3 SINE/ B2 -40 176 14 >AK132441_2113_2 261_RC_B3
NM_1753 13 4127 4308 -948 R C B3 SINE/ B2 -6 210 28 >AK132441_4127_4 308_RC_B3
NM_0242 82 1137 1290 -91 R C B3A SINE/ B2 -39 159 1 >AK132990_1137_1 290_RC_B3A
NM_1773 28 277 358 -1283 R C B3A SINE/ B2 -56 142 66 >AK134874_277_35 8_RC_B3A
NM_1773 28 405 484 -1157 R C B3A SINE/ B2 -13 3 65 1 >AK134874_405_48 4_RC_B3A
NM_1773 28 545 713 -928 R C B3A SINE/ B2 -4 194 14 >AK134874_545_71 3_RC_B3A
NM_1773 28 787 971 -670 R C B3A SINE/ B2 -4 194 7 >AK134874_787_97 1_RC_B3A
NM_0218 99 161 307 -1092 R C B3A SINE/ B2 -46 152 1 >AK135206_161_30 7_RC_B3A
NM_0103 32 228 409 -1967 R C B3 SINE/ B2 -28 188 2 >AK135599_228_40 9_RC_B3
NM_1724 07 1470 1683 -74 R C B3 SINE/ B2 -5 211 2 >AK143014_1470_1 683_RC_B3
NM_0010 09935 1389 1495 -157 R C B3 SINE/ B2 -96 120 1 >AK143784_1389_1 495_RC_B3
NM_0198 27 2953 3143 -1064 R C B3 SINE/ B2 -1 215 1 >AK145079_2953_3 143_RC_B3
NM_0112 12 2131 2283 -1263 R C B3A SINE/ B2 -47 151 2 >AK148045_2131_2 283_RC_B3A
NM_0105 67 984 1129 -78 R C B3A SINE/ B2 -60 138 2 >AK148373_984_11 29_RC_B3A
NM_00 11 10504 1406 1594 -400 R C B3A SINE/ B2 -10 188 2 >AK149403_1406_1 594_RC_B3A
NM_0260 36 2270 2439 0 R C B3 SINE/ B2 -4 212 27 >AK149843_2270_2 439_RC_B3
NM_0103 98 1292 1476 -698 R C B3A SINE/ B2 -11 187 3 >AK157261_1292_1 476_RC_B3A
NM_00 11 10504 1292 1362 -572 R C B3A SINE/ B2 -11 2 86 11 >AK160921_1292_1 362_RC_B3A
NM_0078 93 1080 1254 -746 R C B3 SINE/ B2 0 216 21 >AK163105_1080_1 254_RC_B3
NM_0078 93 1427 1491 -509 R C B3A SINE/ B2 -82 116 46 >AK163105_1427_1 491_RC_B3A
NM_0307 14 1954 2064 -333 R C B3 SINE/ B2 -95 121 2 >AK165234_1954_2 064_RC_B3
NM_0011 10101 853 1043 -1516 R C B3 SINE/ B2 -5 211 6 >AK169421_853_10 43_RC_B3
NM_0011 10101 1742 1936 -623 R C B3 SINE/ B2 -2 214 8 >AK169421_1742_1 936_RC_B3

Industrial Applicability



[0139]  According to the present invention, it is possible to provide a functional nucleic acid molecule having a function of improving efficiency of translation from an RNA, and a use thereof.

[Sequence Listing]



[0140]  RK23223PCT Sequence Listing

SEQUENCE LISTING



[0141] 

<110> RIKEN, INTERNATIONAL SCHOOL FOR ADVANCED STUDIES

<120> FUNCTIONAL NUCLEIC ACID MOLECULE AND USE THEREOF

<130> RK23223PCT

<150> US 61/469,399
<151> 2011-03-30

<160> 47

<170> PatentIn version 3.5

<210> 1
<211> 250
<212> DNA
<213> Mus musculus

<400> 1

<210> 2
<211> 363
<212> DNA
<213> Mus musculus

<400> 2

<210> 3
<211> 187
<212> DNA
<213> Mus musculus

<400> 3

<210> 4
<211> 1146
<212> DNA
<213> Mus musculus

<400> 4



<210> 5
<211> 875
<212> DNA
<213> Mus musculus

<400> 5



<210> 6
<211> 748
<212> DNA
<213> Mus musculus

<400> 6



<210> 7
<211> 1685
<212> DNA
<213> Mus musculus

<400> 7



<210> 8
<211> 1145
<212> DNA
<213> Mus musculus

<400> 8



<210> 9
<211> 209
<212> DNA
<213> Mus musculus

<400> 9

<210> 10
<211> 228
<212> DNA
<213> Mus musculus

<220>
<221> misc_feature
<222> (125)..(125)
<223> n is a, c, g, or t

<400> 10

<210> 11
<211> 1013
<212> DNA
<213> Mus musculus

<400> 11



<210> 12
<211> 1929
<212> DNA
<213> Mus musculus

<400> 12





<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 13
acaaagctca gcccacacgt   20

<210> 14
<211> 13
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 14
catagggttc att   13

<210> 15
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 15
atgcagctga agccgatg   18

<210> 16
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 16
ttaagctgct ttgcagagag c   21

<210> 17
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 17
cgcgcagtga cacagcacaa a   21

<210> 18
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 18
taccattctg tgcggtgca   19

<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 19
gacctcctct agcactgcac a   21

<210> 20
<211> 15
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 20
caatggattc catgt   15

<210> 21
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 21
gatataagga gaatctg   17

<210> 22
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 22
ttatagtatg tgttgtc   17

<210> 23
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 23
gatataagga gaatctg   17

<210> 24
<211> 15
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 24
caatggattc catgt   15

<210> 25
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 25
gaattcctcc agtctctta   19

<210> 26
<211> 12
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 26
tgctagagga gg   12

<210> 27
<211> 12
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 27
gtcaggcaat cc   12

<210> 28
<211> 12
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 28
tgctagagga gg   12

<210> 29
<211> 11
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 29
aaagagatgg c   11

<210> 30
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 30
cacacccgcc accagttc   18

<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 31
cccattccca ccatcacacc   20

<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 32
gcagtggcaa agtggagatt   20

<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 33
gcagaagggg cggagatgat   20

<210> 34
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 34
gcacctgcag acacaaacc   19

<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 35
tctctcagct gctggaatca   20

<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 36
ctggtgtgta tctcttatgc   20

<210> 37
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 37
ctcccgagtc tctgtagc   18

<210> 38
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 38
cccgccgata gagccaag   18

<210> 39
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 39
atggttcact ggaaaggg   18

<210> 40
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 40
ccatgcaccg cacagaatg   19

<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 41
gaaagctccc tcaaataggc   20

<210> 42
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 42
tgtggtgtcc aagtgttcat gc   22

<210> 43
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 43
cggagcacca catcgatcta ag   22

<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 44
caacgttggg gatgacttct   20

<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 45
tcgattccca ttacccacat   20

<210> 46
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 46
ttgagcgact ccaggaaact   20

<210> 47
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> primer

<400> 47
gagtcctggt gaggctgtc   19