WO2014056422A1 - 一种hnRNP A2*蛋白质、编码该蛋白的核酸及用途 - Google Patents

一种hnRNP A2*蛋白质、编码该蛋白的核酸及用途 Download PDF

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WO2014056422A1
WO2014056422A1 PCT/CN2013/084838 CN2013084838W WO2014056422A1 WO 2014056422 A1 WO2014056422 A1 WO 2014056422A1 CN 2013084838 W CN2013084838 W CN 2013084838W WO 2014056422 A1 WO2014056422 A1 WO 2014056422A1
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hnrnp
protein
telomerase
telomere
cells
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PCT/CN2013/084838
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French (fr)
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谭铮
赵勇
王峰
郝玉华
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中国科学院动物研究所
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Priority to EP13845025.9A priority Critical patent/EP2907822B8/en
Publication of WO2014056422A1 publication Critical patent/WO2014056422A1/zh
Priority to US14/683,152 priority patent/US9505815B2/en
Priority to US15/352,507 priority patent/US9617318B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a hnRNP A2* protein, a nucleic acid encoding the same, and use thereof, and belongs to the field of biotechnology. Background technique
  • telomeres consisting of TTAGGG repeat DNA sequences and related proteins. Incomplete terminal replication of telomeric DNA leaves a single-stranded, single-stranded overhang outside the double-stranded region and causes telomere DNA to become shorter as the cell divides.
  • telomere repeats to the 3 ends of telomerase is the primary means of compensating for Telomere erosion.
  • telomere elongation is mediated by Cdcl3.
  • Cdcl3 is a single-stranded telomeric DNA-binding protein that also binds to the telomerase RNA (Tic 1 ) binding protein Estl.
  • Est2 the catalytic subunit of yeast telomerase
  • Est2 is recruited to the telomere by the following interaction: protruding end ⁇ Cdcl3 ⁇ Estl ⁇ Tlcl ⁇ Est2.
  • Tebl establishes an interaction between telomerase and telomeres, allowing telomerase to efficiently extend telomeres.
  • Tebl establishes an interaction between telomerase and telomeres, allowing telomerase to efficiently extend telomeres.
  • telomerase is recruited to the telomere via the cajal body. The mechanism by which telomeres interact with telomerase is unclear, and the factors that mediate this interaction are also Still unknown.
  • the four TTAGGG repeats can form a four-stranded G-quadruplex structure.
  • the intermolecular G-quadruplex can serve as a substrate for ciliate telomerase, the intramolecular G-quadruplex cannot.
  • intramolecular G-quadruplex (hereinafter referred to as G-quadruplex) is preferentially formed at the farthest end of the 3 terminus, making telomerase inaccessible, thereby inhibiting telomere elongation.
  • POT1 is a spherin-binding protein (shelterin) with high affinity for telomere overhangs, which has the effect of unremitting telomere G-quadruplex.
  • shelterin spherin-binding protein
  • the y-terminus of the telomere DNA and the POT1 complex has a tail end of not less than 8 nucleotides, it can be extended by telomerase. Since POT1 preferentially binds to the minimal binding site (MBS) of the 5'-TAGGGTTAG-3', the binding of POT1 to the telomeric DNA leaves the tail of the 3' tail less than 5 nucleotides, thereby inhibiting linearity.
  • MFS minimal binding site
  • telomere extension of the telomere substrate but slightly enhances the extension of the G-quadruplex substrate.
  • a portion of the proteins of the hnRNP family also have the effect of unremitting telomere G-quadruplexes, which interact with single-stranded telomeric DNA and telomerase in vitro, suggesting that they may play a role in telomere function. effect.
  • the hnRNP family plays a major role in RNA metabolism and is one of the most abundant proteins in cells. For example, A2/B1 and A1 of hnRNP have more than 10 7 molecules in a single cell, far more than the dozens of telomere ends and telomerase molecules in a single cell.
  • a HEK-293 human embryonic kidney cell contains only about 20 to 50 telomerase molecules, 92 telomeres, while yeast contains only 29 telomerase molecules and 64 telomeres.
  • the technical problem to be solved by the present invention is to provide a hnRNP A2* protein, a nucleic acid encoding the same, and a use thereof, and hnRNP A2* can efficiently deactivate the telomere G-quadruplex, significantly enhance the catalytic activity of telomerase and carry out Sex, allowing the telomere DNA to be prolonged, will prevent cells from entering replicative senescence and maintaining the cell's cleavage potential. This is important for amplifying useful cells for cell therapy and production.
  • a hnRNP A2* protein comprising a protein, a protein fragment, a protein analog or a neoplasm of the amino acid sequence shown in SEQ ID NO.
  • the present invention also provides the following improvements:
  • amino acid sequence of the protein fragment, analog or derivative has at least 95% identity to the amino acid sequence shown in SEQ ID NO.
  • the protein comprises a protein having the amino acid sequence shown in SEQ ID NO.
  • the present invention solves the above problems and provides a nucleic acid encoding a nucleic acid having a protein, a protein fragment, an analog or a derivative of a protein having the amino acid sequence shown by SEQ ID NO.
  • the present invention also provides the following improvements:
  • nucleic acid comprises a nucleic acid encoding the amino acid sequence shown in SEQ ID NO. Further, the nucleic acid comprises the sequence of position 1-762 of SEQ ID NO.
  • the present invention solves the above technical problems and also provides the use of a hnRNP A2* protein for unwinding a telomere G-quadruplex and promoting telomerase elongation of telomeres.
  • the present invention also provides the following improvements:
  • the hnRNP A2* protein recognizes the nucleic acid sequence of telomere-TAGGGTTAGG-3', and after binding of hnRNP A2* protein to telomeric DNA, the structure of the telomere G-quadruplex is unblocked, and S-GTTAG is exposed.
  • the S-GTTAG-S can be paired with the vertebrate telomerase RNA template S-CUAAC-S to allow telomerase to prolong telomeres and maintain telomere length.
  • the present invention discloses a newly discovered telomere-telomerase-reactive protein (called hnRNP A2*) in mammalian cells, which can colocalize with telomerase in kaja l body (kaja l body) And telomeres, which actively and efficiently unravel the telomere G-quadruplex, bind a minimal binding site at the 3' end of the telomere, and expose a tail of about 5 nucleotides at the 3 end of the telomere.
  • hnRNP A2* telomere-telomerase-reactive protein
  • telomere shortening caused by cell division, and improves and solves cell expansion. problem.
  • the present invention also provides technical insights for controlling cell division life.
  • the telomere length of the cell can be altered, thereby regulating the cell's ability to divide, satisfying the life span and expansion ability of a useful cell population, or inducing the senescence of harmful cells such as cancer cells.
  • the maintenance of cell division potential depends on whether the cell is able to effectively compensate for the shortening of telomere DNA that occurs during the division process and maintain the balance of telomere length.
  • hnRNP A2* recognizes the nucleic acid sequence of 5'-TAGGGTTAGG-3'.
  • this sequence Since the 3' end of this sequence has one more G nucleotide than the 3' end of the native telomeric DNA, it binds to telomeric DNA and unwinds the telomere G-quadruplex structure and exposes its most Five nucleotides of the GTTAG at the end provide telomerase for telomere elongation and maintain telomere length.
  • the hnRNP A2* of the present invention has different functions from the hnRNP protein and is specific for telomere elongation.
  • the low abundance and intracellular location of hnRNP A2* demonstrates a functional distinction between other hnRNP proteins.
  • the abundance of hnRNP A2* transcript is extremely low, and its mRNA can only be detected by RT-PCR after exon 7 of hnRNP A2 and its derivatives are completely excised (Fig. 1C).
  • the hnRNP A2* protein of the present invention and the nucleic acid sequence encoding the same provide a means for establishing a diagnostic tool for diseases involving hnRNP A2* expression, such as cancer, premature aging, including diagnostic nucleic acids for hnRNP A2* nucleic acid sequences Probe hybridization and antibody response to hnRNP A2* protein.
  • Figure 1 Identification of hnRNP A2* of 28 kD size.
  • A (TTAGGG) 3 Affinity Purified Protein SDS-PAGE results (left), 32P-(TTAGGG)3 labeled Southern-western blot (right).
  • B Peptide mapping of 28 kD hnRNP A2* by MALDI-TOF mass spectrometry.
  • C PCR results with or without hnRNP A2 and hnRNP A2* cDNA treated with endonuclease Xho I. The cDNA and other variants of hnRNP A2 were annealed to the complementary DNA at exon 7, and then cleaved using Xho I so that they were not amplified.
  • D mRNA of hnRNP A2* and hnRNP A2.
  • E mRNA of hnRNPA2* detected in rat mice as well as human cells by RT-PCR using junction primer.
  • FIG. 2 hnRNPA2* specifically binds to telomeric DNA in an electrophoretic mobility assay (EMSA).
  • A hnRNP A2* expressed and purified using E. coli.
  • B hnRNP A2* is compared to the hnRNP A2 functional group.
  • C Binding of hnRNP A2* to (TTAGGG) 3 under a concentration gradient in which progressive DNA is gradually increased (indicated by the lower image).
  • D hnRNP A2* binds to the TAGGGTTAGG minimal binding site.
  • E Effect of single nucleotide mutations on the binding of hnRNP A2* to TAGGGTTAGGGTTAG.
  • the DNA used has a wild type sequence (W) and a single mutant sequence with the indicated mutations. The asterisk indicates a mutation that prevents it from binding.
  • hnRNP A2* efficiently cleaves the telomere G-quadruplex, binds to the minimal binding site at the 3' end of telomeric DNA, exposing a 5 nucleotide tail to activate telomerase.
  • A hnRNP A2* mediated efficient unwinding of G-quadruplex versus spontaneous unwinding of G-quadruplex in the presence of a complementary C-rich DNA strand. Real-time monitoring of donor fluorescence during the addition of hnRNP A2* or excess C-rich DNA sequences.
  • B Binding preference of hnRNP A2* to telomeric DNA that does not form a G-quadruplex.
  • FIG. 4 hnRNP A2* interacts with telomerase in both in vivo and in vitro assays.
  • A After fluorescing ffi S6- labeled hnRNP A2* with nickel beads in rat cell lysate, the telomerase activity obtained by TRAP analysis decreased.
  • B Electrophoretic mobility experiments of hnRNP A2* on rat telomerase binding at increasing concentrations (0, 0.25, 0.5, 1 mM).
  • C Three fluorescent assays were used to analyze colocalization of hnRNP A2*, TERT and Rapl/Coilin in cultured rat cells.
  • RAP1 and Coilin are markers of telomeres and caragels, respectively. The rightmost lane represents the number of cells and each co-localization type site.
  • Figure 5 Relationship between hnRNP A2* and telomerase activity and telomere length in an in vivo assay.
  • A hnRNP A2* expression level and hnRNP A2, TERT and telomerase activity in rat tissues at 7 weeks Department. Expression levels of hnRNP A2*, hnRNP A2, and TERT were analyzed using RT-PCR, while telomerase activity was analyzed using TRAP. The relative abundance was first normalized by the intensity of the standard band of actin and then normalized to the intensity of the brain tissue band. IS: Internal standard.
  • B hnRNP A2* overexpression increased telomere length in HeLa cells.
  • hnRNP A2* protein specifically unfolds the tetramer structure of telomere DNA.
  • A Fluorescence intensity changes in fluorescence quadratic structure fluorescence resonance transfer experiments.
  • B Electrophoretic mobility shift after DNA quadruplex structure and free single-stranded random DNA binding to hnRNP A2* protein or complementary DNA.
  • C The POT1 protein binds to the MBS site at the 3' end of the telomeric DNA sequence and closes the end. The 5'-end 32 P-labeled DNA is incubated with the POT1 protein and then digested with T4 polymerase. T4 polymerase digestion removes the exposed nucleotides at the 3' end of the DNA.
  • telomeric DNA sequence was completely digested with T4 polymerase without the addition of hnRNP A2* protein.
  • the first lane is a nucleotide size gradient reference obtained by digesting TTAGGGTTAGGGTTAGGGTTAG with DNasel.
  • the other lanes are exactly the same as in Figure 3B.
  • Figure 7 (A) hnRNP A2* protein enhances telomerase activity of HeLa cells and rat BRL cells.
  • the TRAP assay detects telomerase activity using a non-telomeric TS sequence.
  • the concentration of hnRNP A2* protein is shown in Figure 3D.
  • the last lane indicates a loss of telomerase.
  • (B) hnRNP A2* protein enhances telomerase activity from HeLa cells and rat BRL cells in the TRAP assay.
  • the experiment used a TSG4 sequence which forms a quadruplex structure as a substrate before the extension reaction.
  • the concentration of hnRNP A2* protein is shown in Figure 3E, and the last lane indicates telomerase loss.
  • (C) hnRNP A2* protein enhances the sustained elongation activity of telomerase obtained from HeLa cells and rat BRL cells.
  • the extended extension activity assay was performed using our modified TRAP method with a non-telomeric sequence MTS as a substrate under the addition of 40% (w/v) PEG 200.
  • the last lane indicates telomerase deletion; the IS lane indicates internal reference.
  • Figure 8 (A) Hydrophilic mapping of hnRNP A2 protein. The figure contains fragments deleted from the hnRNP A2* protein. (B) Protein hybridization in different rat cell subcellular fractions detects hnRNP A2* protein. The HA-hnRNP A2* protein was expressed in rat cells, and the HA-hnRNP A2* protein was detected with an anti-HA antibody. The ⁇ -tubulin and p84 proteins are the markers of the cytoplasmic and nuclear matrix, respectively, which are detected by the corresponding antibodies.
  • Figure 9 (A) TRAP method detects telomerase activity of hnRNP A2 protein immobilized on nickel beads from rat cells. (B) Detection of hnRNP A2 eggs by electrophoretic mobility shift assay (EMSA) White binds to rat telomerase RNA components at increasing concentrations (0, 0.25, 0.5, 1 mM). Solid arrows indicate rTR and hnRNP A2* complexes, and open arrows indicate free rTR. The conditions under the experiment were consistent with those of Figs. 4A and 4B, respectively.
  • FIG. 10 (A) Immunofluorescence was used to detect the colocalization of hnRNP A2* and RAP1, TERT, and Coilin in cultured rat cells. RAP1 and Coilin are the hallmarks of telomere and Cajal body, respectively.
  • B Fluorescent immunoassay Cultured HA-hnRNP A2* protein (upper part) and rat cultured cells not expressing HA-TTAP were stained with anti-HA antibody, respectively.
  • FIG 11 (A) Three-color fluorescent staining for the observation of typical colocalization of RAP1, TERT and hnRNP A2* in rat cells.
  • RAP1 is a marker molecule for telomeres.
  • B Three-color fluorescent labeling for the typical colocalization of Coilin, TERT and hnRNP A2* in rat cells.
  • Coilin is a hallmark of Cajalbody.
  • Figure 12 (A) Correlation between hnRNP A2* expression and hnRNP A2 expression, TERT expression, and telomerase activity in 11-month-old rats. Expression of hnRNP A2*, hnRNP A2 and TERT was detected by RT-PCR and telomerase activity was detected by TRAP. Relative abundance is first normalized by normalizing the intensity of actin bands and then normalizing the intensity of brain tissue bands. IS stands for internal reference. (B) The length of telomeres in rat cells increases with increasing hnRNP A2* protein overexpression. The density peaks for each lane are indicated by white arrows. The expression of HA-hnRNP A2* protein was detected by protein hybridization. Other experimental conditions are the same as in Figure 5B. detailed description
  • the present invention illustrates the research ideas of the present invention by the following experiments, reveals the separation and purification process of hnRNP A2* protein, and analyzes the principle of hnRNP A2* unraveling the structure of DNA quadruplex.
  • RNA was used as a template for reverse transcription reaction to obtain cDNA. Then, an oligonucleotide strand complementary to the exon 7 of hnRNP A2 was used to complement the hnRNP A2 cDNA portion, and the pairing portion was cleaved with the endonuclease Xhol.
  • the cDNA obtained by reverse transcription of the hnRNP A2* protein was amplified using the primers 5'-TAGCTAGCATGGAGAGAGAAAAGGAA-3' and 5'-AAGAGCTCTCAATATCGGCTCCTTCCA-3' (Wang F, Zhao Y, Hao YH, Tan Z (2008) Identification of low - abundance alternative spliced mRNA variants by exon exclusive reverse transcriptase polymerase chain reaction. Anal Biochem 383:307-310. ) The exon removal method we established amplifies the hnRNP A2* gene.
  • the amplified fragment was cloned between the Nhel/Sacl digestion of the pET28-b plasmid, and the resulting plasmid was transformed into E. coli BL21 (DE3) strain cells (Beijing Biotek Biotechnology Co., Ltd.).
  • the transformed cells were cultured in LB medium at 37 ° C for 4 hours, and then the recombinant His 6- tagged hnRNP A2* protein was induced with 1 mM of isopropyl- ⁇ -D-thiogalactoside, and induced 2 E.
  • coli cells were collected after an hour, and then buffer A (50 mM sodium dihydrogen phosphate, pH 8.0, 300 mM sodium chloride, 40 mM imidazole, I mM dithiothreitol, 1 mM phenylmethane) in one container.
  • buffer A 50 mM sodium dihydrogen phosphate, pH 8.0, 300 mM sodium chloride, 40 mM imidazole, I mM dithiothreitol, 1 mM phenylmethane
  • the acyl fluoride was sonicated to disrupt the cells at 4 °C.
  • the resulting cell lysate was centrifuged at 20,000 x g for 20 minutes at 4 ° C, and the resulting supernatant was applied to a HisTrap affinity column.
  • buffer A 50 mM sodium dihydrogen phosphate, pH 8.0, 300 mM sodium chloride, 400 mM imidazole. Come down.
  • the rat telomerase RNA sequence was reverse transcribed and amplified by polymerase chain reaction and then cloned into the pMD19-T plasmid.
  • the restriction endonucleases Crf 131, Rsa I, FspB I, BamH I were used to cleave the cleavage site in the rTR sequence, and the plasmid was linearized, and then the linearized plasmid was used as a template for transcription with the T7 transcription kit, followed by T4 multinuclei.
  • the transcription product was labeled with [ ⁇ - 32 ⁇ ] ATP under the action of a glycoside kinase.
  • oligonucleotide strand 50 ⁇ 5'[ ⁇ - 2 ⁇ ] ATP-labeled oligonucleotide strand with 1.5 ⁇ of hnRNP A2* protein or POT1 protein at 4°C 10 (iL binding buffer (10 mM Tris) Incubate for 15 minutes at pH 8.0, 1 mM ethylenediaminetetraacetic acid, 150 mM potassium chloride.
  • telomeres in cell extracts are rich in guanine DNA sequence binding protein with 5' biotin bound to streptomycin agarose-agarose The labeled (TTAGGG) 3 sequence is removed by affinity binding.
  • telomerase substrate was incubated with different concentrations of hnRNP A2* protein or POT1 protein in 5,000 telomerase extracts in 50 ⁇ L. After the addition of 0.25 mM dNTP, the reaction was carried out at 30 ° C (TSG4 as a substrate at 37 ° C) for 10 minutes and then at 75 ° C for 5 minutes to terminate the reaction. Then there is an internal reference standard ⁇ ij
  • 5'-ATCGCTTCTCGGCCTTTT-3' for the TS group, And 5'-CTCGCTTCTCGGCCGCTT-3' used in the TSG4 group) was extracted with phenol chloroform and precipitated with ethanol.
  • the obtained product was collected and amplified by polymerase chain reaction for 33 cycles (94 ° C for 30 s; 59 ° C for 30 s).
  • TS sequences 'J and 5'-GCGCGGCTTACCCTTACCCTTACCCTAACC-3' were used as primers for the TS group reaction; TSG4 and 5'-GTGCCCTTACCCTTACCCTTACCCTAA-3' were used as primers for the TSG4 group reaction.
  • telomerase activity is expressed as a multiple of the control group without hnRNP A2* or POT1 protein, and the applied formula is
  • TP0 and ISO represent the total amount of telomerase catalytic products and the total amount of internal reference products, respectively.
  • TP and IS represent the total obtained under the condition of adding hnRNPA2* or POT1 protein, respectively. The amount of product. Analysis of telomerase sustained extension activity
  • the present invention is specifically designed to analyze the sustained elongation activity of telomerase using an improved telomeric repeat amplification method, such as the literature (Xue Y, Kan ZY, Wang Q, Yao Y, Liu J, et al. (2007). Human telomeric DNA forms parallel-stranded intramolecular G-quadruplex in K+ solution under molecular crowding condition. J Am Chem Soc 129:11185-11191. ) The introduction of TSNT is used as an internal reference. MTS substrate contains 0.25 mM dNTP, 0.5 ⁇ ⁇ hnRNP ⁇ 2* or POT1 protein, 5000 cells of telomerase cell extract, with or without 40% polyethylene glycol 200 and 50 vol.
  • an improved telomeric repeat amplification method such as the literature (Xue Y, Kan ZY, Wang Q, Yao Y, Liu J, et al. (2007). Human telomeric DNA forms parallel-stranded intramolecular
  • reaction was terminated by heating to 75 ° C for 5 minutes, and an internal reference sequence (5'-AGCATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3' and 5'-ATCGCTTCTCGGCCTTTT-3') was added, extracted with phenol chloroform and ethanol precipitated.
  • the collected oligonucleotide strands were amplified by polymerase chain reaction after adding 10 pmol RP, 0.02 pmol RPC 3g and 1 U Taq DNA polymerase, first at 94 ° C for 30 s; 55 ° C for 60 s; 72 ° Two cycles of C 90s, followed by 29 cycles at 94 ° C for 30 s; 63 ° C for 30 s; and 72 ° C for 30 s.
  • the polymerase chain reaction product was electrophoresed on a 12% polyacrylamide gel, then stained with ethidium bromide and photographed with a Chemilmager 5500. Prepare subcellular components and protein hybridization detection
  • the method of obtaining the components of the subtle moon package is according to the literature (Ludems ME, van Steensel B, Chong L, Sibon OC, Cremers FF, et al. (1996) Structure, subnu clear distribution, and nuclear matrix association of the mammalian telomeric complex J Cell Biol 135:867-881. )
  • the method described is improved. It is necessary to say that BRL-3A cells transfected with the hnRNP A2* gene are washed twice with cold Earle's solution and then resuspended in 10 volumes.
  • RSB solution 0.1 M sodium chloride, 1.5 mM magnesium chloride, 10 mM Tris-hydrochloric acid (pH 7.4), 0.1% digitonin
  • RSB solution 0.1 M sodium chloride, 1.5 mM magnesium chloride, 10 mM Tris-hydrochloric acid (pH 7.4), 0.1% digitonin
  • the cells were allowed to flow through a 19.5 gauge needle to lyse
  • the cells were repeated five times.
  • the cell lysate was then centrifuged at 23,000 X g for 30 minutes in RSB solution containing 10% glycerol. The supernatant after centrifugation was collected as a cytoplasmic extract.
  • the nuclear components in the pellet were collected and incubated in RSB solution at 37 ° C for 20 minutes, and then nucleated with LIS solution at room temperature for 10 minutes at a concentration of 2 X 10 6 cells per ml of LIS solution.
  • the LIS solution consists of 10 mM LIS, 100 mM lithium acetate, 1 mM ethylenediaminetetraacetic acid, 0.1% digitonin, 0.05 mM spermine, 0.125 mM spermidine, 0.25 mM phenylmethylsulfonyl fluoride, and 20 mM Hepes-potassium hydroxide (pH 7.4).
  • the mixture was centrifuged at 20000 x g for 20 minutes, and the obtained supernatant component and the precipitate component were collected as a core component and a core matrix component, respectively.
  • the proteins in these three cellular fractions were electrophoresed on a 10% sodium dodecylsulfate polyacrylamide gel and transferred to a nitrocellulose membrane.
  • the HA-hnRNPA2* protein was detected with a monoclonal antibody against mouse anti-HA, and the hnRNPA2/B1 protein was detected with a polyclonal antibody against goat hnRNP A2/B 1 .
  • telomerase activity analysis Male mice were sacrificed after sacrifice and immediately used for telomerase activity analysis and RNA extraction.
  • tissue to be tested is added with lysate in an amount of 0.5 mg tissue per ml of telomerase extraction lysate, and the tissue is ground with a sterilized tissue grinder on ice. Incubate for 30 minutes. The cell lysate was then centrifuged at 12000 x g for 30 minutes at 4 °C. The obtained supernatant was quickly frozen with liquid nitrogen and stored in a -80 °C refrigerator until use. The protein content of the resulting supernatant was determined by the Bradford method, and the amount of the extract containing 200 ng of protein was used for each telomerase extension experiment. Detection of RNA content Using RT-PCT method, total RNA of 0.05 was reverse transcribed with ⁇ LV reverse transcriptase and then amplified by polymerase chain reaction.
  • Cell culture method
  • Rat BRL-3A cells and HeLa (HeLa) cells were supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 0.1 mg/ml streptomycin in DMEM medium at 5% carbon dioxide concentration of 37 °C. to cultivate. Retroviral vector and virus particle production
  • cDNA of hnRNP A2* protein was amplified by HM6 forward primer 5'-GATGGTACCATGGAGAGAAAAGGAACAGTTC-3' and HM6 reverse primer 5'-GACGAATTCATTCTCAATATCGGCTCCTTCCAC-3' polymerase chain reaction, and the amplified product was inserted into Kpnl/EcoRI of pHM6 expression vector. At the site of the enzyme cleavage. After cloning The HA-hnRNP A2* protein gene fragment on the plasmid was amplified with the T7 promoter forward primer 5'-TAATACGAGTCACTATAGGGA-3' and the HM6 reverse primer.
  • the amplified product was then cloned into the pGEM-T vector.
  • the fragment was then cloned into the Notl-EcoRI cleavage site of the pQCXIN retroviral expression vector (for BRL-3A cells), or cloned into the EcoRV-Kpnl cleavage site of the pMSCV-IThyl-1 retroviral vector. (for HeLa cells).
  • the obtained recombinant pGEM-T HA-hnRNP A2* vector was transfected into EcoPack 2-293 cells, and the pMSCV-IThyl-1 HA-hnRNP A2* vector was transfected together with pGag-pol and pMD.G-VSVG-env plasmids. Used to produce retroviruses in 293T cells. The medium containing the virus particles was collected 48 hours after the cells were transfected. Stable expression of HA-hnRNP A2* protein in rat BRL-3A cells
  • Rat BRL-3A cells grown to a density of about 70% were exposed to a mixture of virus-containing medium and fresh medium in a 1:1 ratio and cultured for 12 hours.
  • the old medium of BRL-3A cells was then replaced with fresh medium containing 800 g/ml G418 antibiotic, and the old medium of HeLa cells was replaced with fresh medium containing 300 g/ml G418 antibiotic.
  • BRL-3A cells were cultured in this medium for two weeks, while HeLa cells were cultured at a concentration of 100 ⁇ g/ml G418 antibiotic.
  • the clone used in BRL-3A, HeLa cells are non-clonal cells.
  • Rat BRL-3A cells grown on the surface of glass coverslips were fixed in PBS (pH 7.4) buffer containing 2% paraformaldehyde for 8 minutes at room temperature, and then buffered with PBS containing 0.5% Triton X-100. The liquid is permeabilized. The cells were then pre-blocked with 10% normal serum from the same species as the secondary antibody used in the experiment.
  • the primary antibodies used in the experiments were as follows, goat or rabbit anti-TERT polyclonal antibody; rabbit anti-Coilin polyclonal antibody; mouse anti-HA monoclonal antibody; rabbit anti-hRAP1 polyclonal antibody.
  • the secondary antibodies used are the following, Cy3-labeled scorpion anti-goat antibodies; fluorescein-labeled goat or scorpion anti-rabbit antibodies, DyLight 649-labeled scorpion anti-rabbit antibodies. These secondary antibodies are used according to the dilution recommended by the manufacturer. Primary antibody treatment was performed for 1 hour at room temperature, followed by secondary antibody treatment for 45 minutes, followed by 4 washes with PBS buffer for 5 minutes each wash. Finally, the coverslips were covered with VECTASHIELD embedding medium containing 0.5 ⁇ g/ml DAPI, and the immunofluorescence staining results were observed under a 100-fold oil microscope using a ZEISS 510 META confocal microscope.
  • telomeres 1 (POT1) is a negative regulator of telomerase activity in vitro. Mol Cell Biol 25: 808-818. Plasmid pET-14-POTl was a gift from Joachim Lingner. The pET-14-POT1 plasmid was transformed into E. coli BL21 (DE3) strain cells, and then the cells were cultured in TB medium supplemented with 1% glucose and 0.05 mg/ml carbenicillin for 5 hours at 37 °C.
  • Protein expression was induced with 5 ⁇ M concentration of isopropyl- ⁇ -D-thiogalactoside and induced at 25 °C for 2 hours. After collecting the induced cells, the cells were resuspended in a container with a solution C containing 1 mg/ml lysozyme.
  • the composition of the solution C was 20 mM sodium dihydrogen phosphate, pH 8.0, 200 mM sodium chloride, 0.2% poly Oxyethylene sorbitan monolaurate, 10 mM imidazole, 20% glycerol, 5 mM ⁇ -mercaptoethanol. It was then placed at 4 ° C for 20 minutes and the cells were disrupted by sonication.
  • the cell lysate was centrifuged at 20000 x g for 20 minutes at 4 ° C, and the resulting supernatant was applied to a HisTrap HP affinity column, followed by rinsing with 10 column volumes of solution A, followed by 400 mM imidazole solution C.
  • the His-POT1 protein bound to the column was eluted, followed by dialysis treatment with solution D.
  • Solution D (20 mM sodium dihydrogen phosphate, pH 8.0, 50 mM sodium chloride, 0.2% polyoxyethylene sorbitan) Monolaurate, 20% glycerol, 5 mM ⁇ -mercaptoethanol), and the resulting protein product was stored at -70 °C.
  • the A2* protein or the ⁇ complementary paired nucleic acid sequence CCC(TAACCC) 3 was mixed in TE buffer pH 8.0 containing 150 mM potassium chloride.
  • the polymerase chain reaction was carried out in a 25 ⁇ l system, and poly(T) was used as a primer to reverse transcribe 0.2 ⁇ g of rat BRL-3A cells, human HeLa cells, and mouse liver cell RNA to obtain cDNA.
  • the amplification reaction was first pre-denaturing at 94 °C for 2 minutes, then at 94 °C for 30 s; at 62 °C for 30 s; at 72 °C for 30 s.
  • Rat cells were expanded for 29 cycles, and human and mouse cells were expanded for 32 cycles.
  • telomere pull-down experiment was performed according to the literature ( Eversole A, Maizels N (2000) In vitro properties of the conserved mammalian protein hnRNP D suggest a role In telomere maintenance. Mol Cell Biol 20: 5425-5432. ) The method described is improved. 10 6 RL-3A cell pellets were lysed in ⁇ CHAPS solution. The composition of the CHAPS solution was 0.5% 3-[(3-cholesterosylpropyl)dimethylamino]propanesulfonic acid, 10 mM trishydroxymethylamino Methane, pH 8.0, 1 mM magnesium chloride, 0.1 mM phenylmethylsulfonyl fluoride.
  • RNA sequences Two short RNA sequences, GCCAGCATGTTAGGAAGAA, which form a double-stranded pair with the rat TERT sequence, were used as interfering RNA (provided by RiboBio Co., Ltd (Guangzhou, China)).
  • a non-targeted RNA short chain was also used as a control.
  • the cells grown on the coverslips in a 6-well cell culture plate were incubated with 2 mL of Lipofectamine 2000-containing medium containing 50 nM of interfering RNA, and then the expression level of TERT protein was detected by immunofluorescence, and the level used in the experiment was used.
  • the antibody anti-TERT (D-16) antibody was supplied by Santa Cmz Co., Ltd.
  • the FITC-labeled scorpion anti-goat secondary antibody was supplied by Protein Tech Groupjnc.
  • hnRNP A2* an identification of single-stranded telomeric DNA-binding proteins from nuclear Jjf.
  • the present invention prepares a protein of a nuclear matrix, and proteins which specifically bind to single-stranded telomeric DNA are isolated by affinity purification, and are subjected to SDS-PAGE electrophoresis (Fig. 1A, left). Use 32 P marked single
  • the present invention cut out the most abundant band with a molecular weight of 28 kd, and identified nine tryptic peptides by MALDI-TOF mass spectrometry (Table 1). These nine peptide chains are paired with hnRNP, with eight at the N-terminal half and the other at the front C-end ( Figure 1B). Since the span of these peptide chains is greater than 28 kD, the present inventors speculate that this protein may be a splice variant of hnRNP A2 (36 kD) lacking several exons.
  • the present invention uses a newly established "exon row" of the inventor Exon exclusive RT-PCR (Wang F, Zhao Y, Hao YH, Tan Z (2008) Identification of low-abundance alternative spliced mRNA variants by exon exclusive reverse transcriptase polymerase chain reaction. Anal Biochem 383:307-310) method to selectively amplify this variant (Fig. 1C). Subsequent sequencing identified this to be a new isoform of exon 7-9 of exon of hnRNP A2 (Fig. 1D) It is a conserved protein present in rat, mouse, human cells (Fig. 1E).
  • hnRNP A2* recognizes by unique sequences and binds to single-stranded telomeric DNA.
  • the present invention cloned and expressed hnRNP A2* in E. coli, and its molecular weight was as expected (Fig. 2A).
  • Exon 7-9 encodes a glycine rich region (GRD).
  • RRMs RNA recognition sites
  • this region provides an additional DNA/RNA binding site (Fig. 2B).
  • the Electrophoretic Mobility Assay (EMSA) revealed that deletion of exon 7-9 in hnRNP A2* resulted in its high specificity for telomeric DNA.
  • hnRNPA2* binds only to single-stranded (TTAGGG)3, but not to (TAAGGG)3, (TTGGGG)3, (TTAGAG)3 or double-stranded (TTAGGG)3 (Fig. 2C). This is in sharp contrast to the wide sequence of hnRNP that can be represented by N(A, C, T)(C, T)(A, G)G(C, G, T)(A, T)NNN. .
  • EMSA electrophoretic mobility experiments
  • this sequence is one more nucleotide than the smallest binding site of the human P0T1 protein at the 3' end.
  • the present invention mutated 10 nucleotides in its minimal binding site to cytosine, respectively. In addition to mutations in the thymidine immediately adjacent to GGG, these mutations abolished binding (Fig. 2E), further illustrating the high specificity of hnRNP A2* for telomeric DNA. Mutations outside the minimum binding site do not affect binding.
  • hnRNP A2* efficiently cleaves the G-quadruplex and preferentially binds to the 3' end of the telomeric DNA, exposing it to a 5 nucleotide 3' end.
  • FRET fluorescence energy resonance transfer
  • FAM Fluorescein
  • TAMRA rhodamine tetramethyl isothiocyanate
  • telomere protrudes at the 3' end mainly ending with TTAG-3'.
  • the present invention uses T4 polymerase to digest linear telomeric DNA in the presence of hnRNP A2* or POT1, respectively.
  • both proteins preferentially bind to the closest binding site closest to the 3 ends, so the end of hnRNP A2* exposure is GTTAG-3 (Fig. 3B) and POT1 completely covers 3 End and completely prevent the occurrence of elimination ( Figure 6C). More importantly, the two proteins still have a preference for the 3' end when using the telomere G-quadruplex (Fig. 3C).
  • hnRNP A2* enhances the catalytic activity and progression of telomerase in vitro.
  • the exposed 5 -GTTAG-3' end of hnRNP A2* is perfectly paired with the vertebrate telomerase RNA template 5, -CUAAC-3'. It is thus conceivable that the combination of hnRNP A2* and telomere ends can promote telomerase action. Indeed, traditional TRAP analysis found that hnRNP A2* in both human and rat cells significantly enhanced telomerase activity (Fig. 3D, 7A).
  • TRAP analysis uses a substrate for non-telomeric sequences.
  • the formation of the G-quadruplex and the binding of hnRNP A2* may only occur if the substrate has been added with several telomere repeats. Therefore, this result suggests that hnRNP A2* can play a role in telomere elongation, possibly by blocking the formation of G-quadruplexes, providing telomerase A usable y-end is used to achieve this.
  • POT1 has no effect. This result is consistent with previous reports.
  • the reported experiments used non-PCR direct extension analysis of non-telomeric sequence primers, demonstrating that POT1 does not affect telomerase activity.
  • the results of the present invention support the statement that "POT1 may be involved in the regulation of telomerase activity by affecting the proximity of telomerase to primers.”
  • the present invention uses the modified TRAP method to examine the effect of hnRNP A2* on the G-quadruplex substrate.
  • the substrate TSG4 was obtained by mutating the T in the native telomere sequence near the GGG fragment to C, which can form a G-quadruplex and still be recognized by hnRNP A2* (Fig. 2E). This mutation distinguishes this substrate from the extension sequence, allowing for PCR amplification.
  • the results showed that hnRNP A2* significantly increased telomerase activity against TSG4 compared to the TS substrate of the non-telomeric sequence (Fig. 3D, 7A) (Fig.
  • telomere has the ability to add multiple telomere repeats to a particular primer at a time. However, the formation of a G-quadruplex will disrupt the elongation process by interfering with the translocation of telomerase. Since hnRNP A2* is capable of efficiently dissociating G-quadruplexes, the present invention uses a TRAP method specifically designed to detect telomerase progression to analyze how hnRNP A2* affects the progression of telomerase. In dilute solutions, telomerase showed a strong progression (Fig. 3F, 7C, lane 1).
  • telomere G-quadruplex a reagent PEG200, which is widely used to mimic the crowded environment of intracellular molecules, can significantly enhance the thermal stability of the telomere G-quadruplex and inhibit the progression of human telomerase.
  • the extension was carried out in the presence of PEG 200, and the present invention observed a significant decrease in progress (second lane) as previously reported.
  • hnRNP A2* can restore this progressive extension (third lane), while POT1 does not (fourth lane). Since both proteins can unravel the G-quadruplex, this difference can be explained by the fact that when bound to telomere DNA, hnRNP A2* can expose the free 3 ends and POT1 will cover the end of the sputum.
  • Intracellular hnRNP A2* is located in the nuclear matrix and interacts with telomerase in telomeres and kadarian bodies.
  • the hnRNP A2* deleted exon 7-9 sequences are all hydrophilic (Fig. 8A), which makes hnRNP A2* more hydrophobic than hnRNP A2.
  • Western blots demonstrated that hnRNP A2* only appeared in the nuclear matrix compared to hnRNP A2, which is predominantly present in the nucleoplasm (Fig. 8B). This difference indicates that hnRNP A2* may have a different function than hnRNP A2.
  • hnRNP family proteins can bind not only to telomeric DNA, but also directly to telomerase.
  • the present inventors have found that hnRNP A2* can drag down telomerase activity in cell lysates like hnRNP A2 (Fig. 9A), indicating that hnRNP A2* is also physics It can bind to telomerase (Fig. 4A).
  • hnRNP A2* also binds to RNA components in telomerase (rTR) (Fig. 4B). This binding is dependent on the size of the RNA fragment, indicating that this interaction is affected by the rTR structure.
  • hnRNP A2 also interacts with rat telomerase, but in a different manner than hnRNP A2*, since hnRNP A2 binds to the 0-269 nucleotide fragment (Fig. 9B), whereas hnRNP A2* does not bind (Fig. 4B) .
  • the present invention expresses HA-tagged hnRNP A2* in rat cells and detects its location using immunofluorescence. Consistent with the in vitro results, the present invention observed that hnRNP A2* colocalizes with RAP 1 and TERT (Fig. 10A, top and middle row images).
  • hnRNP A2* Fluorescence elimination of TERT by siRNA (Fig. 10B), cell-selective staining of HA-tagged hnRNP A2* (Fig. 10C) confirmed the specificity of the antibody for HA-tagged hnRNP A2* and TERT, respectively.
  • the present invention also observed colocalization of hnRNP A2* with the Cargill body (Fig. 10A, bottom line image). Because Cargill is involved in the delivery of telomerase to the telomeres, this result suggests that hnRNP A2* may be involved in the regulation of telomerase activity by the Cargill.
  • telomere colocalization of most hnRNP A2* with telomeres was accompanied by the presence of telomerase (Fig. 4C, top line image and Fig. 11A).
  • Fig. 4C top line image and Fig. 11A.
  • 37 were associated with the presence of telomerase.
  • hnRNPA2*/RAPl/TERT co-localization The colocalization of hnRNP A2*, TERT, and Cargill also reveals a close relationship between hnRNP A2* and telomerase.
  • 21 colocalizations observed for the 21 hnRNP A2* and the Cargill 20 were in the form of hnRNP A2*, Cargill, TERT complex (Fig. 4C, bottom row image and Fig. 11B).
  • Fig. 4C bottom row image and Fig. 11B
  • hnRNP A2* (Cajal body Box, CAB) is responsible for its accumulation in the Cargill body.
  • the expression level of hnRNP A2* in vivo is related to telomerase activity, and t ⁇ J ⁇ of hnRNP A2* is longer than telomere length.
  • the present inventors investigated the correlation between hnRNP A2* expression levels and telomerase activity in vivo.
  • the present invention detects the contents of hnRNP A2, hnRNP A2* and TERT in rat tissues by RT-PCR, respectively, and detects by TRAP. Telomerase activity (Fig. 5A, 12A).
  • telomerase activity is not always positively correlated with the mRNA level of TERT.
  • hepatocytes have very high mRNA levels of TERT, while TRAP detects very low telomerase activity.
  • Telomerase activity is highly correlated with the expression level of hnRNP A2*. This correlation demonstrates that hnRNP A2* plays an important role in telomerase activity in vivo. Conversely, this correlation does not occur between hnRNP A2 and telomerase.
  • the present invention presupposes overexpression of hnRNP A2* Will lead to the elongation of telomeres.
  • the present invention utilizes retrovirus transfection of cultured HeLa (HeLa) and rat cells to overexpress hnRNP A2*. Telomeres were prolonged in the hnRNP A2* overexpressing experimental group cells compared to the empty vector transfected control group (Fig. 5B, 12B).
  • telomere extension is a positive factor in telomerase-regulated telomere extension.

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Abstract

本发明提供了一种hnRNP A2*蛋白质及为其编码的核酸及用途。该蛋白质为包含SEQ ID NO.1所示的氨基酸序列的蛋白质及其片段、类似物或衍生物。所述蛋白质能主动解开端粒G-四链体，在端粒3'末端结合最小结合位点，并暴露出一个可以与端粒酶RNA模板配对的5个核苷酸尾巴，促进端粒酶对端粒的延长，进而维持细胞的分裂潜能。

Description

技术领域
本发明涉及一种 hnRNP A2*蛋白质、 编码该蛋白的核酸及用途， 属于生 物技术领域。 背景技术
人染色体末端由 TTAGGG重复 DNA序列和相关蛋白组成的端粒所保 护。 端粒 DNA的不完全末端复制在双链区外留下了一个 3 端的单链突出末 端， 并使得端粒 DNA随着细胞分裂而变短。 端粒酶向 3 端添加端粒重复序 列是补偿端粒损耗（ Telomere erosion ) 的主要方式。
在出芽酵母中， 端粒延长是由 Cdcl3 介导的。 Cdcl3 是一个单链端粒 DNA结合蛋白， 同时也与端粒酶 RNA ( Tic 1 ) 结合蛋白 Estl结合。 这样， Est2(酵母端粒酶的催化亚基)通过以下相互作用被募集至端粒处： 突出末端 →Cdcl3→Estl→Tlcl→Est2。类似的情况也发生在四膜虫中， Tebl建立起端 粒酶与端粒之间的相互作用， 使得端粒酶能够高效地延长端粒。 但是在哺乳 动物细胞中， 端粒酶经由卡佳尔体（cajal body )被募集至端粒处， 端粒与端 粒酶相互作用的机制尚不清楚， 介导这一相互作用过程的因子也还是未知 的。
四个 TTAGGG重复序列可以形成一种四链的 G-四链体结构。 虽然分子 间的 G-四链体可以作为纤毛虫端粒酶的底物， 但是分子内的 G-四链体却不 能。 对于脊推动物端粒 DNA, 分子内 G-四链体 (以下均称为 G-四链体）会 优先在 3 端最远处形成， 使得端粒酶无法靠近， 从而抑制端粒的延伸。 到现 在为止， 已经发现了一些可以解开端粒 G-四链体结构的蛋白。 POT1是一种 对端粒突出末端具有高亲和力的端粒结合蛋白 （shelterin ), 具有解开端粒 G-四链体的作用。 当端粒 DNA和 POT1的复合体 y端存在不小于 8个核苷 酸尾端时可以被端粒酶延伸。 由于 POT1优先与 ? 端 5'-TAGGGTTAG-3'的 最小结合位点（MBS )结合， POT1与端粒 DNA的结合会留下的 3' 尾端小 于 5个核苷酸的尾巴， 从而抑制线性端粒底物的延伸， 但是略微增强对 G- 四链体底物的延伸。 hnRNP家族的一部分蛋白质也具有解开端粒 G-四链体 的作用， 它们在体外实验中能与单链端粒 DNA和端粒酶的相互作用， 说明 它们可能在端粒功能中起一定的作用。 hnRNP家族主要在 RNA代谢中起作用， 是细胞中最丰富的蛋白之一。 例 如， hnRNP的 A2/B1和 A1在单个细胞中分子数超过 107, 远超过一个细胞中 仅有的数十个端粒末端和端粒酶分子。一个 HEK-293人胚肾细胞中只含有约 20 ~ 50个端粒酶分子， 92个端粒， 而酵母中则只含有 29个端粒酶分子， 64 个端粒。 这些事实质疑这些蛋白会与端粒与端粒酶直接发生相互作用， 因为 它们在理论上只会饱和结合端粒与端粒酶，从而阻止端粒与端粒酶之间的相 互作用。 发明内容
本发明所要解决的技术问题是提供一种 hnRNP A2*蛋白质、 编码该蛋白 的核酸及用途， hnRNP A2*能高效地解开端粒 G-四链体， 显著增强端粒酶的 催化活性和进行性， 使端粒 DNA得以延长， 将会防止细胞进入复制性衰老， 维持细胞的分裂潜能。 这一点对于扩增有用细胞， 用于细胞治疗和生产有重 要的价值。
本发明解决上述技术问题的技术方案如下： 一种 hnRNP A2*蛋白质， 所述蛋 白质包含有 SEQ ID NO. 1所示的氨基酸序列的蛋白质、 蛋白质片段、 蛋白质 的类似物或 †生物。
在上述技术方案的基础上本发明还做了如下改进：
进一步， 所述蛋白质片段、 类似物或衍生物的氨基酸序列具有与 SEQ ID NO. 1所示的氨基酸序列至少 95%的相同性。
进一步，所述蛋白质包含具有 SEQ ID NO. 1所示的氨基酸序列的蛋白质。 本发明解决上述技术问题还提供了一种核酸，所述核酸编码具有 SEQ ID NO. 1 所示的氨基酸序列的蛋白质、蛋白质片段、蛋白质的类似物或衍生物的核酸。
在上述技术方案的基础上本发明还做了如下改进：
进一步，所述核酸包含编码具有 SEQ ID NO. 1所示的氨基酸序列的核酸。 进一步， 所述核酸包含有 SEQ ID NO. 2中的 1-762位的序列。
本发明解决上述技术问题还提供了一种 hnRNP A2*蛋白质的用途， 所述 hnRNP A2*蛋白质用于解开端粒 G-四链体，促进端粒酶对端粒的延长。
在上述技术方案的基础上本发明还做了如下改进：
进一步， 所述 hnRNP A2*蛋白质能够识别端粒 -TAGGGTTAGG-3'的核酸 序列， hnRNP A2*蛋白质与端粒 DNA结合后， 解开端粒 G -四链体的结构， 并暴露出 S -GTTAG-S 的末端， 所述 S -GTTAG-S 能和脊推动物端粒酶 RNA 模板 S -CUAAC-S 配对， 使端粒酶对端粒进行延长， 维持端粒长度。 本发明的有益效果是：
1 )本发明公开了一个在哺乳动物细胞中新发现的端粒与端粒酶相互作 用的蛋白（称为 hnRNP A2* ),它可以与端粒酶共定位于卡佳尔体（kaja l body) 和端粒， 它通过主动高效地解开端粒 G-四链体， 在端粒 3'末端结合最小结合 位点，将端粒 3 端的一个长约 5个核苷酸的尾巴暴露出来，从而使得它可以 和端粒酶的 RNA模板配对， 显著增强了端粒酶的催化活性和进行性， 延长了 细胞的端粒， 有效补偿细胞分裂导致的端粒缩短， 改善和解决细胞扩增的问 题。
2 )本发明也为控制细胞分裂寿命提供了技术启示。通过控制 hnRNP A2* 蛋白的表达可以改变细胞的端粒长度， 进而调节细胞的分裂能力， 满足增加 有用细胞群体的寿命和扩增能力， 或者诱导有害细胞如癌细胞的衰老。 细胞 分裂潜能的维持取决于细胞是否能够有效地补偿它在分裂过程中所发生的 端粒 DNA 缩短， 维持端粒长度的平衡。 hnRNP A2*能够识别 5'-TAGGGTTAGG-3'的核酸序列。由于这一序列的 3'末端比天然的端粒 DNA 的 3'末端多出一个 G核苷酸，所以它结合端粒 DNA后会解开端粒 G-四链体 结构， 并暴露出其最末端的 GTTAG五个核苷酸， 提供给端粒酶进行端粒延 长， 维持端粒长度。
3 )本发明 hnRNP A2*与 hnRNP蛋白质具有不同的功能， 并对端粒延长 具有特异性。 hnRNP A2*的低丰度和细胞内位置证明它与其它 hnRNP蛋白有 功能上的区分。相较于 hnRNP的高丰度， hnRNP A2*转录本的丰度极低， 它 的 mRNA只有在 hnRNP A2和它的衍生物的外显子 7被完全切除后才能用 RT-PCR检测到 （图 1C )。
4 ) 本发明的 hnRNP A2*蛋白质和编码该蛋白质的核酸序列对涉及 hnRNP A2*表达改变的疾病如癌症， 早老症提供了建立诊断工具的手段， 这 些诊断工具包括针对 hnRNP A2*核酸序列的核酸探针杂交和针对 hnRNP A2*蛋白的抗体反应。 附图说明
图 1 : 28 kD大小的 hnRNP A2*的鉴定。 （A) ( TTAGGG ) 3亲和纯化蛋白 SDS-PAGE结果（左）， 32P-(TTAGGG)3标记的 Southern-western印迹（右）。 ( B ) 28 kD的 hnRNP A2*的 MALDI-TOF质谱分析肽段图谱。 （ C )使用或 未使用使用核酸内切酶 Xho I处理的 hnRNP A2和 hnRNP A2*cDNA的 PCR 结果。 hnRNP A2的 cDNA和其他变体与外显子 7处的互补 DNA退火后使 用 Xho I切割， 使其不被扩增。 （ D ) hnRNP A2*和 hnRNP A2的 mRNA。 （E) 使用接点引物（junction primer )的 RT-PCR在大鼠小鼠以及人细胞中检测到 的 hnRNPA2*的 mRNA。
图 2:在电泳迁移率实验（EMSA )中 hnRNPA2*特异性结合于端粒 DNA。 （A ) 使用大肠杆菌表达并纯化后的 hnRNP A2*。 （ B ) hnRNP A2*与 hnRNP A2功 能基团对比。 （C )在竟争性 DNA逐渐提高的浓度梯度下 （下方图像表示）， hnRNP A2*对（ TTAGGG ) 3的结合。 （D ) hnRNP A2*与 TAGGGTTAGG最小 结合位点结合。 （ E )单核苷酸突变对 hnRNP A2*与 TAGGGTTAGGGTTAG结 合的影响。 使用的 DNA有野生型序列 (W)和具有图示突变的单突变序列。 星 号表示使其无法结合的突变。
图 3: hnRNP A2*高效解开端粒 G-四联体，与端粒 DNA的 3'端的最小结合位点 结合， 暴露出一个 5核苷酸的尾部而激活端粒酶。 （A ) hnRNP A2*介导的 G- 四联体的高效解开与在互补富 C的 DNA链存在下 G-四联体的自发解开的对 比。 hnRNP A2*或过量富 C的 DNA序列加入过程中供体荧光的实时监测。 ( B ) hnRNP A2*对不形成 G-四联体的端粒 DNA的结合偏好性。 5'端使用 32P 标记的 DNA与 hnRNP A2*共孵育后，使用可以从 3'末端切除核苷酸的 T4聚合 酶消化。 小写字母表示用于控制最小结合位点（由方括号表示）的数量和位 置的突变。 （ C ) hnRNPA2*和 POT1对形成 G-四联体的端粒 DNA的结合偏好 性。 实线方括号表示 hnRNP A2*的最小结合位点， 而虚线方括号则表示 POT1 的最小结合位点。 其他条件与（B )中相同。 （ D,E )使用 TRAP分析在 hnRNP A2*或 POTl 存在的情况下， 大鼠细胞或海拉（HeLa ) 细胞裂解液中端粒酶 分别对（D ) 中非端粒的端粒酶底物以及（E ) 中 G-四联体的 TSG4底物的催 化活性。 （E ) 中插入的图像表示自由的 TSG4 (左）和 TSG4/hnRNPA2*复合 物（右） 的电泳迁移实验结果。 （F )在 40 % (质量体积比） 的 PEG200存在 下， 使用改进 TRAP分析海拉（HeLa ) 细胞裂解液中端粒酶对非端粒的 MTS 底物的持续合成能力的结果。
图 4: hnRNP A2*在体内和体外试验中均可以与端粒酶相互作用。 （ A ) 大鼠 细胞裂解液中使用镍珠免疫固化 ffiS6标记的 hnRNP A2*后， TRAP分析得到的 端粒酶活性下降。 （ B )在逐渐提高的浓度 ( 0, 0.25, 0.5, 1 mM )下 hnRNP A2* 对大鼠端粒酶结合的电泳迁移率实验结果。 （C )培养的大鼠细胞中使用三种 荧光分析 hnRNP A2*,TERT和 Rapl/Coilin的共定位。 RAP1和 Coilin分别作为 端粒和卡佳尔体的标志。 最右边的泳道表示了细胞数量和每一种共定位类型 位点。
图 5:体内试验中 hnRNP A2*与端粒酶活性和端粒长度的关系。 （ A )七周大 鼠组织中 hnRNP A2*表达水平和与 hnRNP A2, TERT以及端粒酶活性的关 系。 hnRNP A2*,hnRNP A2,和 TERT的表达水平使用 RT-PCR分析， 而端粒 酶活性则使用 TRAP分析。相对丰度先通过对肌动蛋白的标准条带强度作归 一化， 然后在对脑组织条带强度作归一化获得. IS: 内标。 （B ) hnRNP A2* 过表达提高了海拉（HeLa ) 细胞中端粒长度。 使用 HA标记的 hnRNP A2* 表达载体或对照的空载体转染细胞后，使用药物筛选出克隆并培养至所示的 细胞群体倍增数（ PDs )。分别使用 Southern印迹（左）和泳道数字化分析（右） 分析了端粒限制酶切片段。 M:DNA分子量标记。
图 6: hnRNP A2*蛋白专一性解开端粒 DNA四链体结构。 （ A ) DNA四链体结 构荧光共振转移实验中的荧光强度变化。 （B ) DNA四链体结构和游离单链 随机 DNA结合 hnRNP A2*蛋白或互补 DNA后电泳迁移率改变。 （ C ) POT1蛋 白结合端粒 DNA序列 3 末端的 MBS位点并封闭末端。 5'端 32P标记的 DNA与 POT1 蛋白孵育， 之后再用 T4 多聚酶消化处理， T4 多聚酶消化可以除去 DNA3'末端暴露的核苷酸。小写字母表示引入的突变以改变 MBS的数量和位 置。方括号状表示 MBS位置。 （ D )在不添加 hnRNP A2*蛋白情况下端粒 DNA 序 列 被 T4 多 聚酶完全消化。 第 一个泳道是用 DNasel消化 TTAGGGTTAGGGTTAGGGTTAG所得到的 1个核苷酸大小梯度参照。 其它 泳道情况与图 3B完全一致。
图 7: ( A ) hnRNP A2*蛋白增强海拉（HeLa ) 细胞和大鼠 BRL细胞的端粒 酶催化活性。 TRAP实验用非端粒的 TS序列检测端粒酶活性。 hnRNP A2* 蛋白的浓度如图 3D所示。 最后一个泳道表示端粒酶缺失。 （B ) TRAP实验 中 hnRNP A2*蛋白增强从海拉（HeLa ) 细胞和大鼠 BRL细胞获得的端粒酶 催化活性。 实验使用在延伸反应前就能形成四链体结构的 TSG4序列作为底 物。 hnRNP A2*蛋白的浓度如图 3E所示， 最后一个泳道表示端粒酶缺失。 ( C ) hnRNP A2*蛋白增强从海拉（ HeLa )细胞和大鼠 BRL细胞获得的端粒 酶的持续延伸活性。 用我们改进的 TRAP方法在添加 40% (w/v) PEG 200条 件下以非端粒序列 MTS为底物进行持续延伸活性检测实验。 最后一个泳道 表示端粒酶缺失； IS泳道表示内部参照。
图 8: ( A ) hnRNP A2蛋白的亲水性作图。 图中含有 hnRNP A2*蛋白所缺失 的片段。 （B )在不同的大鼠细胞亚细胞成分中蛋白质杂交检测 hnRNP A2* 蛋白。 HA-hnRNP A2*蛋白在大鼠细胞中表达， 用抗 HA 的抗体检测 HA-hnRNP A2*蛋白。 γ-tubulin和 p84蛋白分别是细胞质胞浆和细胞核基质 的标志分子， 它们分别用相应的抗体检测。
图 9: ( A ) TRAP方法检测固定于镍珠的 hnRNP A2蛋白从大鼠细胞拖拽下 来的端粒酶活性。 （ B )用电泳迁移率实验 ( EMSA )方法检测 hnRNP A2蛋 白在增加浓度 (0, 0.25, 0.5, 1 mM)条件下结合大鼠端粒酶 RNA成分的情况。 实心箭头表示 rTR和 hnRNP A2*复合物， 空心箭头表示游离的 rTR。 实验进 行的条件分别和图 4A、 图 4B相一致。
图 10: ( A )用免疫荧光法检测 hnRNP A2*和 RAP1、 TERT, Coilin在培养 的大鼠细胞中的共定位。 RAP1和 Coilin分别是端粒和卡佳尔体（ Cajal body ) 的标志分子。 （B ) 荧光免疫法用抗 HA 的抗体分别染色培养的表达 HA-hnRNP A2*蛋白 （上部分）和不表达 HA-TTAP的大鼠培养细胞。 （C ) 荧光免疫法检测表明大鼠 TERT蛋白的表达受到了 TERT干扰 RNA的抑制。 所示的实验结果图片都是从三次独立的实验中获得， 所有的图片都是在相同 的曝光时间下拍摄。
图 11: ( A )三色荧光标记染色观察大鼠细胞内 RAP1、 TERT和 hnRNP A2* 典型共定位。 RAP1是端粒的标志分子。 （B )三色荧光标记观察在大鼠细胞 内 Coilin、 TERT和 hnRNP A2*的典型共定位。 Coilin是卡佳尔体（ Cajalbody ) 的标志分子。
图 12: ( A ) 11个月年龄大鼠组织中 hnRNP A2*表达量和 hnRNP A2表达量、 TERT表达量、 端粒酶活性的相关性。 hnRNP A2*、 hnRNP A2和 TERT的表 达量用 RT-PCR方法检测， 端粒酶的活性用 TRAP检测。 相对丰度先通过对 肌动蛋白的标准条带强度作归一化， 然后在对脑组织条带强度作归一化获 得。 IS表示内部参照。 （B ) 大鼠细胞端粒的长度随 hnRNP A2*蛋白过表达 量增加而增加。 每个泳道的密度峰用白色箭头标示。 HA-hnRNP A2*蛋白的 表达用蛋白质杂交的方法检测。 其它实验条件和图 5B—样。 具体实施方式
以下结合本发明的原理和特征进行描述， 所举实例只用于解释本发明， 并非用于限定本发明的范围。
本发明通过以下实验阐述本发明的研究思路，揭示 hnRNP A2*蛋白的分 离纯化过程， 分析 hnRNP A2*解开 DNA四链体结构的原理等等。
亲和纯化端粒 DNA结合蛋白的实验
用文献 ( Ma H, Siegel AJ, Berezney R (1999) Association of chromosome territories with the nuclear matrix. Disruption of human chromosome territories correlates with the release of a subset of nuclear matrix proteins. J Cell Biol 146:531-542. ) 中的方法从大鼠的肝脏中分离出与维持染色体相关的核基质 蛋白。这些核基质蛋白用来与生物素标记的并且结合在链霉素亲和素包被的 琼脂糖珠子上的端粒 DNA寡聚核苷酸序列 （TTAGGG ) 3做亲和纯化分离 实验。 纯化分离实验是在有竟争性酵母 tRNA和超声破碎的鮭精 DNA存在 的条件下进行。 最后用 2M的氯化钠溶液将结合在珠子上的蛋白洗脱下来。 亲和分离的蛋白质产物上样到 12% 十二烷基磺酸钠聚丙烯酰胺凝胶电泳， 切取 28kD分子量大小的蛋白， 用胰蛋白酶消化处理。 再用 MALDI-TOF质 谱来分析胰蛋白酶消化所得到的片段， 之后将得到的数据提交到 MS-Fit 网 站 (http://prospector.ucsf.edU/ucsfhtml4.0/msfit.htm), 返还的 9个片段信息表 明它们与 hnRNPA2蛋白上的片段匹配。 hnRNP A2*蛋白 cDNA的克隆和蛋白质的分离纯化
用 poly-T做引物以 DNase处理过的大鼠肝脏 mRNA做模板做逆转录反应 得到 cDNA。然后用一条与 hnRNP A2的 7号外显子互补配对的寡聚核苷酸链 使之与 hnRNP A2 cDNA部分互补配对， 在用核酸内切酶 Xhol切断配对部分。 hnRNP A2* 蛋 白 的 mRNA 逆 转 录 得 到 的 cDNA 用 引 物 5'-TAGCTAGCATGGAGAGAGAAAAGGAA-3' 和 5'-AAGAGCTCTCAATATCGGCTCCTTCCA-3'才艮据文献 ( Wang F, Zhao Y, Hao YH, Tan Z (2008) Identification of low- abundance alternatively spliced mRNA variants by exon exclusive reverse transcriptase polymerase chain reaction. Anal Biochem 383:307-310. ) 中我们所建立的外显子去除方法扩增 hnRNP A2*基因。 扩增出来的片段被克隆到 pET28-b质粒的 Nhel/Sacl酶切之 间， 然后将得到的质粒转化到大肠杆菌 BL21 (DE3)菌株细胞（北京百泰克生 物技术有限公司） 内。 转化的细胞在 37°C条件下 LB培养基中培养 4小时， 然后用 ImM的异丙基- β -D-硫代半乳糖苷诱导重组的带 His6标签的 hnRNP A2*蛋白表达， 诱导 2小时后收集大肠杆菌细胞， 再在一个容器里用緩沖液 A ( 50 mM磷酸二氢钠，pH 8.0, 300 mM 氯化钠， 40 mM咪唑， I mM 二巯 基苏糖醇， 1 mM苯甲磺酰氟）在 4°C条件下超声破碎细胞。 所得细胞裂解液 在 4°C20000x g离心力下离心 20分钟， 所得到的上清液上柱到 HisTrap亲和 柱上。再用 10倍柱子体积的緩沖液 A漂洗后，结合在柱子上的 His-hnRNP A2* 用緩沖液 B (50 mM磷酸二氢钠， pH 8.0, 300 mM氯化钠， 400 mM咪唑)洗脱下 来。
制备大鼠端粒酶 RNA序列
大鼠端粒酶 RNA序列逆转录后用聚合酶链式反应扩增， 然后克隆到 pMD19-T质粒中。再用限制性内切酶 Crf 131, Rsa I, FspB I, BamH I切割 rTR序 列中存在酶切位点， 线性化质粒， 然后把线性化质粒作为模板用 T7 转录试 剂盒转录， 再在 T4多核苷酸激酶作用下用 [γ-32Ρ] ATP标记转录产物。 电泳迁移率改变实验
80 nM 5'端被 [γ-32Ρ] ATP标记的寡聚核苷酸链与 400 nM的重组 hnRNP A2*蛋白在 4°C结合緩沖液 (10 mM 三羟甲基氨基甲烷， pH 8.0, 1 mM 乙二胺 四乙酸， 150 mM 氯化钾)条件下孵育 30分钟， 然后上样在 8%非变性聚丙烯 酰胺胶中电泳， 再在 Typhoon phosphor imager上放射性自显影。
T4聚合酶水解实验
50 ηΜ 5'[γ- 2Ρ] ATP标记的寡聚核苷酸链与 1.5 μ Μ的 hnRNP A2*蛋白 或 POT1蛋白在 4°C 10(iL结合緩沖液 (10 mM 三羟甲基氨基甲烷， pH 8.0, 1 mM 乙二胺四乙酸， 150 mM 氯化钾）中孵育 15分钟。 然后加入 0.5 U T4聚 合酶， 1 μ L l0 x内切酶緩沖液 (330 mM三（羟甲基）氨基甲烷醋酸盐，ρΗ 7.8, 100 mM Mg(CH3CO2)2, 5 mM二巯基苏糖醇）， 4°C孵育 1分钟进行切割。 最 后用酚氯仿抽提终止反应， 产物再用乙醇沉淀， 上样到含 7 M尿素 19%聚 丙烯酰胺凝胶进行电泳。
准备端粒酶提取物
用文献（ Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, et al. (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011-2015. ) 所描述的方法提取海拉（HeLa ) 细胞或是 RRL-3A大鼠细胞提取物， 细胞提取物中的端粒富含鸟嘌呤 DNA序列结合蛋 白用结合在链霉素亲和素 -琼脂糖上的 5'端生物素标记的 (TTAGGG)3序列亲 和结合而除去。 端粒 化活性分析
用端粒重复序列扩增 （TRAP ) 实验方法以 TS
(5'-AATCCGTCGAGCAGAGTT-3') 或是 TSG4
(5'-GGGCTAGGGCTAGGGCTAGGGAGTT-3')作为底物来分析端粒酶催化活 性。 端粒酶底物与不同浓度的 hnRNP A2*蛋白或是 POT1蛋白在 5000个细 胞的端粒酶提取物在 50 μ L中孵育。在添加 0.25 mM dNTP后在 30 °C( TSG4 为底物在 37°C )进行延伸反应 10分钟后 75°C加热 5分钟终止反应。 接着在 有内参标准序歹 ij
(5'-AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3' 和
5'-ATCGCTTCTCGGCCTTTT-3' 用于 TS组， 和 5'-CTCGCTTCTCGGCCGCTT-3' 用于 TSG4组)存在下用酚氯仿抽提，再 用乙醇沉淀。 收集获得的产物用聚合酶链式反应 33个循环扩增（94 °C 30s; 59 °C 30s )。 TS序歹 'J和 5'-GCGCGGCTTACCCTTACCCTTACCCTAACC-3'作 为 TS组反应的引物； TSG4和 5'-GTGCCCTTACCCTTACCCTTACCCTAA-3' 作为 TSG4组反应的引物。 聚合酶链式反应产物在 12%聚丙烯酰胺凝胶上电 泳， 然后用溴化乙锭染色， 用 Chemilmager 5500照相。 端粒酶的活性用未添 加 hnRNP A2*或是 POT1蛋白的对照组的倍数来表示， 应用的公式为
(TP/TP0 ) (ISO/IS), TP0 和 ISO分别代表端粒酶催化产物总量和内参的产 物总量， TP和 IS分别代表在添加了 hnRNPA2*或是 POT1蛋白条件下所获 得的总产物量。 分析端粒酶持续延伸活性
本发明用一个改进的端粒重复序列扩增方法专门用来分析端粒酶的持 续延伸活性， 这个方法如文献（ Xue Y, Kan ZY, Wang Q, Yao Y, Liu J, et al. (2007) Human telomeric DNA forms parallel- stranded intramolecular G-quadruplex in K+ solution under molecular crowding condition. J Am Chem Soc 129:11185-11191. )介绍的采用 TSNT作为内部参照。 MTS底物在含有 0.25 mM dNTP, 0.5 μ Μ hnRNP Α2*或 POT1蛋白， 5000个细胞的端粒酶细 胞提取物， 含有或不含 40% 聚乙二醇 200和的 50 体积中 37°C反应 10 分钟， 然后加热到 75 °C 5 分钟终止反应， 再加入内部参照序列 (5'-AGCATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3' 和 5'-ATCGCTTCTCGGCCTTTT-3') , 用酚氯仿抽提和乙醇沉淀。 收集得到的寡 聚核苷酸链在添加 10 pmol RP, 0.02 pmol RPC 3g和 1 U Taq DNA聚合酶后进 行聚合酶链式反应扩增， 先在 94°C 30s; 55 °C 60s; 72 °C 90s条件下 2个循 环， 再在 94°C 30s; 63 °C 30s; 72 °C 30s条件下进行 29个循环。 聚合酶链 式反应产物在 12%聚丙烯酰胺凝胶上电泳， 然后用溴化乙锭染色， 用 Chemilmager 5500照像。 准备亚细胞成分和蛋白质杂交检测
亚细月包成分的获取的方法是按照文献 （Ludems ME, van Steensel B, Chong L, Sibon OC, Cremers FF, et al. (1996) Structure, subnu clear distribution, and nuclear matrix association of the mammalian telomeric complex. J Cell Biol 135:867-881. )所描述的方法改进而来。 筒要的说， 转染了 hnRNP A2*基因的 BRL-3A细胞用冷的 Earle's溶液洗涤两次， 然后把细胞重悬于 10倍体积的 RSB溶液 ( 0.1 M 氯化钠， 1.5 mM 氯化镁， 10 mM Tris-盐酸（pH 7.4), 0.1% 洋地黄皂苷 ) , 4°C放置 10分钟； 然后把让细胞流过一个 19.5 gauge的针头来 裂解细胞，重复五次。之后细胞裂解液在含 10%的甘油的 RSB溶液中 23000X g离心 30分钟。 离心后的上清液收集作为细胞质提取物。 收集沉淀中的细胞 核成分后在 RSB溶液中 37°C孵育 20分钟， 然后用 LIS溶液在室温下作用 10 分钟裂解细胞核成分， 浓度为 2 X 106细胞每毫升 LIS溶液。 LIS溶液成分为 10 mM LIS, 100 mM 醋酸锂， 1 mM 乙二胺四乙酸， 0.1% 洋地黄皂苷， 0.05 mM 精胺， 0.125 mM 亚精胺， 0.25 mM 苯甲基磺酰氟化物， 和 20 mM Hepes-氢氧化钾 （pH 7.4)。 LIS溶液处理后， 20000x g离心力下离心 20分钟， 所得的上清液成分和沉淀成分分别收集作为核质成分和核基质成分。这三种 细胞组分中的蛋白质分别在 10%十二烷基磺酸钠聚丙烯酰胺凝胶上电泳，然 后转移到硝化纤维素膜上。 HA-hnRNPA2*蛋白用小鼠抗 HA的单克隆抗体来 检测， hnRNPA2/B 1蛋白用山羊抗 hnRNP A2/B 1的多克隆抗体来检测。 不同大鼠组织中端粒酶活性和 mRNA含量水平
雄性小鼠处死后分离器官， 立即用来做端粒酶活性分析和 RNA提取。 用 于端粒酶活性分析时，待检测的组织按 0. 5毫克组织每毫升端粒酶提取裂解 液的量添加裂解液， 用一个灭菌处理过的组织研磨器磨碎组织， 在冰上孵育 30分钟。 然后将细胞裂解物在 4 °C 12000x g离心力下离心 30分钟。 得到 的上清液用液氮迅速冻存， 保存在 -80 °C冰箱直至使用。 用 Bradford方法来 测定所得上清液中蛋白质含量， 每次端粒酶延伸实验用含 200 ng蛋白质的 提取物的量。 RNA含量的检测用 RT-PCT的方法， 0. 05 的总 RNA用匪 LV 逆转录酶做逆转录， 然后用聚合酶链式反应扩增。 细胞培养方法
大鼠 BRL-3A细胞和海拉（HeLa )细胞在添加了 10%胎牛血清， 100单 位 /毫升青霉素和 0.1毫克 /毫升链霉素的 DMEM培养基中， 5%二氧化碳浓度 37°C条件下培养。 逆转录病毒载体和病毒颗粒产生
hnRNP A2* 蛋 白 的 cDNA 用 HM6 正 向 引 物 5'-GATGGTACCATGGAGAGAGAAAAGGAACAGTTC-3'和 HM6反向引物 5'-GACGAATTCATTCTCAATATCGGCTCCTTCCAC-3'聚合酶链式反应扩 增， 扩增产物插入到 pHM6表达载体的 Kpnl/EcoRI酶切位点处。 克隆之后 质粒上的 HA-hnRNP A2*蛋白基因片段用 T7 启动子正向引物 5'-TAATACGAGTCACTATAGGGA-3' 和 HM6反向引物扩增。然后把扩增产 物克隆到 pGEM-T载体上。 然后再将片段克隆到 pQCXIN逆转录病毒表达 载体的 Notl-EcoRI 酶切位点处 （用于 BRL-3A 细胞）， 或者克隆到 pMSCV-IThyl-1 逆转录病毒载体的 EcoRV-Kpnl 酶切位点处 （用于海拉 ( HeLa ) 细胞）。 获得的重组 pGEM-T HA-hnRNP A2*载体转染到 EcoPack 2-293 细胞中 ， pMSCV-IThyl-1 HA-hnRNP A2*载体和 pGag-pol 、 pMD.G-VSVG-env质粒一起转染到 293T细胞中用来产生逆转录病毒。 转染 细胞 48小时后收集含有病毒颗粒的培养基。 在大鼠 BRL- 3A细胞中稳定表达 HA- hnRNP A2*蛋白
将大约生长到 70%密度的大鼠 BRL-3A细胞暴露到含病毒颗粒培养基和 新鲜培养基按 1:1比例混合液中， 培养 12个小时。 然后用含有 800 g/ml G418 抗生素的新鲜培养基替换 BRL-3A细胞的旧培养基， 用含 300 g/ml G418抗 生素的新鲜培养基替换海拉（HeLa ) 细胞的旧培养基。 BRL-3A细胞在这个 培养基中培养两个星期， 而海拉（HeLa ) 细胞则保持 100 μ g/ml G418抗生 素浓度条件下培养。 BRL-3A使用的克隆， 海拉（HeLa ) 细胞为非克隆的细 胞。
荧光免疫显微法
在玻璃盖玻片表面生长的大鼠 BRL-3A细胞用含 2%的多聚甲醛的 PBS ( pH7.4 )緩沖液中室温下固定 8分钟， 然后用含 0.5% Triton X-100的 PBS 緩沖液做透化处理。 之后细胞用同实验所使用二级抗体来源相同的物种的 10%浓度正常血清做预封闭处理。 实验中用到的一级抗体有以下一些， 山羊 或兔抗 TERT多克隆抗体； 兔抗 Coilin多克隆抗体； 鼠抗 HA单克隆抗体； 兔抗 hRAPl 多克隆抗体。 用到的二级抗体有以下几种， Cy3标记的驴子抗 山羊抗体； fluorescein标记的山羊或驴子抗兔抗体， DyLight649标记的驴子 抗兔抗体。 这些二级抗体都是根据生产商所推荐的稀释浓度使用的。 在室温 下一级抗体处理 1个小时， 再二级抗体处理 45分钟， 之后接着用 PBS緩沖 液洗涤 4 次， 每次洗涤 5 分钟。 最后， 盖玻片用含 0.5 μ g/ml DAPI 的 VECTASHIELD包埋介质盖好， 再用 ZEISS 510 META共聚焦显微镜的 100 倍油镜下观察免疫荧光染色结果。
P0T1蛋白的制备
重组人源 POT1蛋白是根据文献（Kelleher C, Kurth I, Lingner J (2005) Human protection of telomeres 1 (POTl) is a negative regulator of telomerase activity in vitro. Mol Cell Biol 25:808-818. ) 描述的方法准备。 质粒 pET-14-POTl由 Joachim Lingner惠赠。 把 pET-14-POTl质粒转化到大肠杆菌 BL21 (DE3)菌株细胞内， 然后让细胞在添加了 1%葡萄糖和 0.05 mg/ml浓度 的羧苄青霉素的 TB培养基中 37°C培养 5小时， 再用 5 μ Μ浓度的异丙基 - β -D-硫代半乳糖苷诱导蛋白表达， 在 25 °C条件下诱导 2小时。 收集诱导菌体 细胞后用含 1 mg/ml浓度溶菌酶的溶液 C在一个容器中重悬细胞， 溶液 C的成 分是 20 mM磷酸二氢钠， pH 8.0, 200 mM 氯化钠， 0.2% 聚氧乙烯山梨糖醇 酐单月桂酸酯， 10 mM咪唑， 20%甘油， 5 mM β -巯基乙醇。 然后在 4°C放置 20分钟， 再用超声破碎细胞。 细胞裂解液在 4°C 20000x g离心力下离心 20 分钟，所得到的上清液上样到 HisTrap HP亲和柱上，接着用 10倍柱子体积的 溶液 A漂洗， 然后用 400 mM咪唑溶液 C把结合在柱子上的 His-POTl蛋白洗 脱下来， 接着用溶液 D做透析处理， 溶液 D成分 (20 mM磷酸二氢钠， pH 8.0, 50 mM 氯化钠， 0.2% 聚氧乙烯山梨糖醇酐单月桂酸酯， 20%甘油， 5 mM β - 巯基乙醇)， 最后所得蛋白产物保存于 -70°C。
分析 hnRNP A2*解开 DNA四链体结构
20 nM浓度的 5'端标记了作为供体的荧光素和 3' 端标记了作为受体的四 甲基罗丹明荧光染料的 GGG(TTAGGG)3寡聚核苷酸链分别与 200 nM的 hnRNP A2*蛋白或是 ΙμΜ互补配对的核酸序列 CCC(TAACCC)3在含 150mM 浓度氯化钾的 pH8.0的 TE緩沖液中混合。 之后在 25 °C , 5nM狭缝下以 480nm 激发记录 515nm处荧光发射，检测在加入 hnRNP A2*蛋白或是互补配对 DNA 链后 DNA四链体结构解开的动态变化。 在大鼠， 小鼠和人细胞中聚合 式反应扩增 hnRNP A2*基因
聚合酶链式反应是在一个 25μ1的体系中进行， 以 poly(T)为引物逆转录 0.2μg大鼠 BRL-3A细胞， 人海拉（HeLa ) 细胞， 小鼠肝脏细胞的 RNA得 到 cDNA。 扩增反应先是在 94 °C预变性处理 2分钟， 然后进行 94 °C 30s; 62 °C 30s; 72 °C 30s循环。 大鼠细胞扩增 29个循环， 人和小鼠细胞扩增 32个 循环。 端粒酶被 hnRNP A2*蛋白拖拽 ( pul l-down )实验
端粒酶拖拽（ pull-down )实验是按照文献 ( Eversole A, Maizels N (2000) In vitro properties of the conserved mammalian protein hnRNP D suggest a role in telomere maintenance. Mol Cell Biol 20:5425-5432. )所描述方法改进后进 行。 106个 RL-3A细胞沉淀在 ΙΟΟμΙ CHAPS溶液中裂解， CHAPS溶液的成分 为 0.5% 3-[ ( 3-胆固醇氨丙基）二甲基氨基]小丙磺酸， 10 mM 三羟甲基氨基 甲烷， pH 8.0, 1 mM 氯化镁， 0.1 mM 苯甲基磺酰氟化物。 3(^g的重组含 His6 标签的 hnRNP A2*蛋白被固定在镍珠上， 然后用 1% 牛血清蛋白在 4°C做封 闭处理，再用冷的 CHAPS溶液洗涤两次，最后与 ΙΟΟμΙ的细胞裂解物和 ΙΟΟμΙ 的 CHAPS溶液在 4°C下孵育 2小时。孵育结束之后， 用冷的 CHAPS溶液洗涤 镍珠三次， 再用来检测端粒酶活性。 对照实验采用未包被的镍珠拖拽的端粒 酶或用 20(^g/ml RNase A室温下处理 30分钟的细胞裂解物。 端粒的限制性片段长度测量
按照文献（ Zhao Y, Sfeir AJ, Zou Y, Buseman CM, Chow TT, et al. (2009) Telomere extension occurs at most chromosome ends and is uncoupled from fill-in in human cancer cells. Cell 138:463-475. )介绍的方法进行端粒的限制性 片段平均长度测量。 用干扰 RNA敲除大鼠细胞中的 TERT
两个能与 大鼠 TERT 序歹 ll形成双链配对的短 RNA 序列 GCCAGCATGTTAGGAAGAA 作为干扰 RNA ( 由 RiboBio Co., Ltd (Guangzhou, China)公司提供）。另外用非靶向的 RNA短链作对照。在 6孔细 胞培养板中盖玻片上生长的细胞用含 50nM 的干扰 RNA 的 2mL 含 Lipofectamine 2000的培养基与之孵育，之后用免疫荧光方法检测 TERT蛋白 的表达水平，实验中用到的一级抗体抗 TERT (D-16)抗体是由 Santa Cmz Co., Ltd提供， FITC标记的驴子抗山羊二级抗体是由 Protein Tech Groupjnc提供。 实验结果
hnRNP A2*, 一种来自细胞核 Jjf的单链端粒 DNA结合蛋白的鉴定。 本发明制 备了细胞核基质的蛋白，通过亲和纯化分离出与单链端粒 DNA特异性结合的 蛋白， 并将它们进行了 SDS-PAGE电泳分离 （图 1A, 左）。 使用 32P标记的单
1A , 右）。 本发明切出了丰度最高的?分子量为 28kd处的条带， 使用 MALDI-TOF质谱分析鉴定出了九条胰蛋白酶肽（表 1 )。这九条肽链与 hnRNP 配对， 八条位于 N端半部， 另一条则更靠前 C端 （图 1B )。 由于这些肽链的 跨度大于 28 kD, 本发明推测这一蛋白可能是 hnRNP A2 ( 36 kD ) 的一个缺 失了几个外显子的剪接变体。 本发明使用了一种发明人新建立的 "外显子排 除逆转录 -聚合酶链式反应" ( exon exclusive RT-PCR ) ( Wang F, Zhao Y, Hao YH, Tan Z (2008) Identification of low-abundance alternatively spliced mRNA variants by exon exclusive reverse transcriptase polymerase chain reaction. Anal Biochem 383:307-310 )方法来选择性扩增这种变体（图 1C )。 随后的测序鉴 定出这是 hnRNP A2的一种外显子 7-9被剪切掉的新异构体（图 1D )。 它是 存在于大鼠， 小鼠， 人细胞中的一种保守性蛋白 （图 1E )。
表 1. MALDI-TOF mass spectrometry of TTIP. m/z MH+ Delta
start end Peptide Sequence Modifications submitted matched Da
727.3752 727.4466 -0.0714 78 83 (R)VVEPKR(A)
781.4059 781.3593 0.0466 298 305 (K)SGNFGGSR(N)
1087.4952 1087.4849 0.0103 27 34 (R)NYYEQWGK(L)
1087.4952 1087.4681 0.0271 35 42 (K)LTDCVVMR(D) 1P04 ICys-am
1188.6464 1188.6476 -0.0012 126 135 (K)IDTIEIITDR(Q)
1338.6715 1338.7018 -0.0302 88 100 (R)EESGKPGAHVTVK(K)
1695.7682 1695.7655 0.0028 142 156 (R)GFGFVTFDDHDPVDK(I)
1798.9305 1798.9227 0.0077 11 26 (K)LFIGGLSFETTEESLR(N)
1879.9572 1879.9666 -0.0094 102 117 (K)LFVGGIKEDTEEHHLR(D)
2220.0923 2220.0712 0.0211 118 135 (R)DYFEEYGKIDTIEIITDR(Q)
hnRNP A2*通过独特的序列识别并与单链端粒 DNA结合。 本发明在大肠杆 菌中克隆表达了 hnRNP A2*, 其分子量与预期相符（图 2A )。 外显子 7-9编 码了富甘氨酸区域（GRD )。 除了两个 RNA识别位点 （RRM )夕卜， 这一区 域提供了一个额外的 DNA/RNA结合位点（图 2B )。电泳迁移率实验（ EMSA ) 揭示了 hnRNP A2*中外显子 7-9的缺失导致了它对端粒 DNA的高特异性。 hnRNPA2*仅与单链 (TTAGGG)3结合， 但不与（TAAGGG)3, (TTGGGG)3, (TTAGAG)3或双链 (TTAGGG)3结合（图 2C )。这与 hnRNP可以与 N(A, C, T)(C, T)(A, G)G(C, G, T)(A, T)NNN所代表的一个广泛的序列结合形成 了强烈的对比。 通过在电泳迁移率实验（EMSA ) 中使用不同大小的端粒 DNA, 本发明鉴定出了 hnRNP A2*的最小结合位点为 5 -TAGGGTTAGG-3 (图 2D ),这一序列比人 P0T1蛋白的最小结合位点在 3'端的多一个核苷酸。 为了进一步检验 hnRNP A2*的结合特异性， 本发明将其最小结合位点中的 10个核苷酸分别突变为了胞嘧啶。除了紧靠着 GGG的胸腺嘧啶的突变以外， 这些突变都消除了结合（图 2E ), 进一步说明了 hnRNP A2*对端粒 DNA的 高特异性。 在最小结合位点以外的突变并不影响结合。
hnRNP A2*高效地解开 G-四链体， 优先结合于端粒 DNA的 3'端， 使其暴露 出一个 5个核苷酸的 3'端尾巴。 为了检测 hnRNP A2*是如何处理端粒 G-四 链体的， 本发明使用了荧光能量共振转移 （FRET )进行了分析。 用荧光素 ( FAM )在 5 -(GGGTTA)3GGG-3/的 5 端标记作为供体， 在 端使用四甲 基异硫氰酸罗丹明 (TAMRA)标记作为受体（图 3A )。 这个 DNA形成 G-四链 体后将使得两个荧光基团靠近而导致发生荧光能量共振转移，从而使得供体 荧光淬灭。 hnRNP A2*的加入导致了供体荧光量的迅速增长， 说明 G-四链体 被解开了，从而使得两个荧光基团分离，导致从供体到受体的能量转移减弱。 而 G-四链体被 hnRNP A2*快速解开与它在过量的互补 DNA S -CCC ATTCCC^^ 链的存在下的緩慢自发解链形成对比， 说明 hnRNP A2*可以主动解开 G-四链体。 而一个无关随机的发卡结构 DNA 则不受 hnRNP A2*的影响 （图 6A-B )。
天然的端粒突出 3'末端主要以 TTAG-3'结尾。 为了对比 hnRNP A2*和 POT1在 3 端的结合， 本发明使用 T4聚合酶在 hnRNP A2*或 POT1分别存 在的情况下对线性端粒 DNA进行了消化。 当有多个最小结合位点存在时， 两种蛋白均会优先与最靠近 3 端的最小结合位点结合， 因此 hnRNP A2*暴 露的末端是 GTTAG-3 (图 3B ) , POT1则完全覆盖了 3 端并且完全阻止了 消 4匕的发生（图 6C ) 。 更重要的是使用端粒 G-四链体时两个蛋白仍然具有对 3'末端 的偏好性（图 3C )。 由于这些 DNA在没有蛋白存在的情况下均被完全消化， 这说明了这种消化带型并不是由潜在的二级结构的形成所导致的（图 6D ) 。 体外实验中 hnRNP A2*增强了端粒酶的催化活性和进行性。 hnRNP A2*暴露 出的 5 -GTTAG-3'末端完美地和脊推动物端粒酶 RNA模板 5， -CUAAC-3' 形成配对。 由此可以想到， hnRNP A2*和端粒末端的结合可以促进端粒酶作 用。 确实，传统的 TRAP分析发现人和大鼠细胞的 hnRNP A2*均显著地增强 了端粒酶活性（图 3D, 7A )。 TRAP分析使用了非端粒序列的底物。 这样， G-四链体的形成和 hnRNP A2*的结合只有可能在底物已经添加了数段端粒 重复序列的情况下发生。 所以， 这个结果说明， hnRNP A2*可以在端粒延伸 过程中起作用， 这一作用可能是通过阻止 G-四链体的形成， 为端粒酶提供 了一个可使用的 y末端来实现的。 相反， POT1没有效果。 这个结果与之前 的报道是一致的， 这个报道的实验中使用了非 PCR 的直接延伸分析了非端 粒序列引物， 证明 POT1不影响端粒酶的活性。 本发明的结果支持了 "POT1 可能是通过调控端粒酶与引物的接近， 而不是在延伸过程中影响端粒酶活 性" 的说法。
随后本发明使用改进过的 TRAP方法检测了 hnRNP A2*对 G-四链体底 物的作用。 所用底物 TSG4由天然端粒序列中靠近 GGG片段的 T突变为 C 而成， 它既可以形成 G-四链体， 而又仍然可以被 hnRNP A2*识别 （图 2E )。 这一突变使得这一底物与延伸序列区别开来， 从而允许 PCR扩增。 实验结 果表明与非端粒序列的 TS底物相比（图 3D , 7A ), hnRNP A2*更显著地提 高了端粒酶对 TSG4的活性（图 3E, 7B ), 这一点说明了 hnRNP A2*解开 G-四链体在端粒酶延伸过程中的贡献。 相反， POT1对 TSG4的延长几乎没 有影响 （图 3E )。
人端粒酶具有一次往一个特定引物上加上多个端粒重复序列的能力。但 是 G-四链体的形成将会通过干涉端粒酶的转位而破坏延伸过程。因为 hnRNP A2*能够高效解开 G-四链体，本发明使用专门为检测端粒酶进行性而设计的 TRAP方法分析了 hnRNP A2*如何影响端粒酶的进行性。 在稀溶液中， 端粒 酶显示出较强的进行性（图 3F, 7C , 第一泳道）。 本发明之前曾经发现一种 广泛用于模拟细胞内分子拥挤环境的试剂 PEG200 可以显著增强端粒 G-四 链体的热稳定性并抑制人端粒酶的进行性。 在 PEG200存在的条件下进行延 伸， 本发明像以前报道的那样， 观测到了进行性的显著下降（第二泳道）。 hnRNP A2*可以恢复这种进行性延伸 (第三泳道）， 而 POT1则不能（第四泳 道）。 因为两个蛋白均能解开 G-四链体， 因此这一差别可以解释为当结合到 端粒 DNA上时， hnRNP A2*可以暴露出自由的 3 端而 POT1则会覆盖 Ύ末 端。 细胞内 hnRNP A2*位于细胞核基质， 并且在端粒和卡佳尔体中与端粒酶 作用。 hnRNP A2*缺失的外显子 7-9序列均为亲水性（图 8A ),这使得 hnRNP A2*比起 hnRNP A2疏水性更强。 相较于主要出现在核质中的 hnRNP A2 , western印迹证明 hnRNP A2*仅仅出现在核基质中 （图 8B )。 这一区别说明 hnRNP A2*可能具有与 hnRNP A2不同的功能。
在体外实验中，一些 hnRNP家族的蛋白不仅可以与端粒 DNA结合，还 可以直接与端粒酶相互作用。本发明发现 hnRNP A2*可以像 hnRNP A2一样 拖拽下来细胞裂解物中的端粒酶活性（图 9A ) , 说明 hnRNP A2*同样物理 上可以结合端粒酶（图 4A ) 。 hnRNP A2*同样可以结合端粒酶（rTR ) 中的 RNA成分（图 4B ) 。 这种结合依赖于 RNA片段的大小， 这说明这种相互 作用受 rTR结构的影响。 hnRNP A2也可以与大鼠端粒酶相互作用， 但是其 作用方式与 hnRNP A2*不同，因为 hnRNP A2结合 0-269核苷酸片段（图 9B ), 而 hnRNP A2*则不结合（图 4B ) 。 为了证明 hnRNP A2*在细胞中是否能与端粒和端粒酶发生相互作用， 本发明在大鼠细胞中表达了使用 HA标签的 hnRNP A2*,并使用免疫荧光检 测了它的位置。 与体外实验结果一致， 本发明观测到了 hnRNP A2*与 RAP 1 和 TERT共定位（图 10A, 顶部和中间行图片）。 SiRNA对 TERT的荧光消 除（图 10B ) , 表达 HA标签的 hnRNP A2*的细胞选择性染色（图 10C )分 别证实了抗体对 HA标签的 hnRNP A2*和 TERT的特异性。 有趣的是， 本发 明还观测到了 hnRNP A2*与卡佳尔体的共定位（图 10A, 底部行图片）。 因 为卡佳尔体参与了将端粒酶全酶递送到端粒上， 这一结果说明 hnRNP A2* 可能参与了卡佳尔体调控端粒酶的活动中。
本发明随后进行了三种颜色的免疫荧光染色以进一步地探索这些相互 作用。 当同时进行 hnRNP A2*， TERT和端粒的检测时， 多数 hnRNP A2*与 端粒的共定位往往同时伴随着端粒酶的存在（图 4C,顶部行图片和图 11A )。 在观测到的 39个 hnRNP A2*/RAP1共定位中，有 37个伴随着端粒酶的存在
( hnRNPA2*/RAPl/TERT共定位）。 hnRNP A2*, TERT, 卡佳尔体的共定位 也揭示了 hnRNP A2*与端粒酶之间紧密的关系。在观测到的 21个 hnRNP A2* 与卡佳尔体的共定位中， 20个以 hnRNP A2* , 卡佳尔体， TERT复合物（图 4C, 底部行图片和图 11B )的形式出现。 另一方面， 端粒和卡佳尔体处一半 的 TERT都与 hnRNP A2*相结合。这一 hnRNP A2*和端粒酶在端粒与卡佳尔 体处出现的高度共定位强烈意味着 hnRNP A2*与端粒酶之间有着紧密的关 系。很有可能 hnRNP A2*在卡佳尔体中被组装进入端粒酶全酶并被递送至端 粒处。 巧合的是， hnRNP A2*与大鼠端粒酶的结合（图 4B )明显与大鼠端粒 酶的 3 端区域（269-419核苷酸）有关， 而正是这一区域含有的卡佳尔体盒
( Cajal body Box, CAB ) 负责它在卡佳尔体中的积累。 体内 hnRNP A2*的表达水平与端粒酶活性相关， hnRNP A2*的 t^J^长 了端粒长度。 为了证实 hnRNP A2*与端粒酶之间的关系， 本发明研究了体内 hnRNP A2*表达水平与端粒酶活性的相关性。 本发明分别使用 RT-PCR检测 了大鼠组织中的 hnRNP A2, hnRNP A2*和 TERT的含量， 使用 TRAP检测 了端粒酶活性（图 5A, 12A )。 本发明发现端粒酶活性并不是总是与 TERT 的 mRNA水平正相关。例如肝细胞具有极高的 TERT的 mRNA水平，而 TRAP 检测到的端粒酶活性很低。 而端粒酶活性与 hnRNP A2*的表达水平高度相 关。 这种相关性说明了 hnRNP A2*在体内端粒酶活性中扮演着重要的角色。 相反地， 这种相关性没有出现在 hnRNP A2与端粒酶之间。
考虑到 hnRNP A2*在大鼠细胞中表达水平较低， 而在组织中较多的 hnRNP A2*与更高的端粒酶活性相关（图 5A, 12A ),本发明推测 hnRNP A2* 的过表达会导致端粒的延长。 为了验证这一假定， 本发明利用逆转录病毒转 染培养的海拉（HeLa )和大鼠细胞， 使 hnRNP A2*过表达。 比起空载体转染 的对照组，端粒在 hnRNP A2*过表达的实验组细胞中得到延长（图 5B, 12B )。 有趣的是， 随着细胞培养时间的延长， 大鼠细胞中出现了 hnRNP A2*表达量 的逐渐下降，同时伴随着端粒的逐渐缩短（图 12B底部）。端粒长度对 hnRNP A2*表达水平的依赖性进一步证实了 hnRNP A2*是端粒酶调控的端粒延伸中 一个正因子的结论。 以上所述仅为本发明的较佳实施例， 并不用以限制本发明， 凡在本发明 的精神和原则之内， 所作的任何修改、 等同替换、 改进等， 均应包含在本发 明的保护范围之内。
SEQ.l hnRNP A2*氨基酸序列
1 MEREKEQFRK LFIGGLSFET TEESLRNYYE QWGKLTDCW MRDPASKRSR 51 GFGFVTFSSM AEVDAAMAAR PHS IDGRWE PKRAVAREES GKPGAHVTVK 101 KLFVGGIKED TEEHHLRDYF EEYGKIDTIE I ITDRQSGKK RGFGFVTFDD 151 HDPVDKIVLQ KYHTI GHNA EVRKALSRQE MQEVQSSRSG RGGNYGSGNY 201 NDFGNYNQQP SNYGPMKSGN FGGSR丽 GGP YGGGNYGPGG SGGSGGYGGR 251 SRY
SEQ.2 hnRNP A2*核苷酸（mRNA)序列
1 atggagagag aaaaggaaca gttccgtaag ctctttattg gtggcttaag ctttgaaacc
61 acagaagaaa gtttgaggaa ctactacgaa caatggggaa agcttacaga ctgtgtggta
121 atgagggatc ctgcaagcaa aagatcaaga ggatttggtt ttgtaacttt ttcatccatg
181 gctgaggttg atgctgccat ggctgcaaga cctcattcaa ttgatgggag agtagttgag
241 ccaaaacgtg ctgtagcaag agaggaatct ggaaaaccag gggctcatgt aactgtgaag
301 aagctgtttg ttggcggaat taaagaagat actgaggaac atcaccttag agattacttt
361 gaggaatatg gaaaaattga taccattgag ataattactg ataggcagtc tggaaagaaa
421 agaggctttg gctttgttac ttttgatgac catgatcctg tggataaaat cgtattgcag
481 aaataccata ccatcaatgg tcataatgca gaagtaagaa aggctttgtc tagacaagaa
541 atgcaggaag ttcagagttc taggagtgga agaggaggca attatggaag tggaaattac
601 aatgattttg gaaattataa ccagcaacct tctaactacg gtccaatgaa gagtggaaac
661 tttggtggta gcaggaacat ggggggacca tatggtggag gaaactatgg tccaggaggc
721 agtggaggaa gtgggggtta tggtgggagg agccgatact ga

Claims

权 利 要 求 书
1、 一种 hnRNP A2*蛋白质， 其特征在于， 所述蛋白质包含有 SEQ ID NO. 1 所示的氨基酸序列的蛋白质、 蛋白质片段、 蛋白质的类似物或衍生物。
2、 根据权利要求 1 所述的蛋白质， 其特征在于， 所述的蛋白质片段、 类似物或衍生物的氨基酸序列具有与 SEQ ID NO. 1 所示的氨基酸序列至少 95%的相同性。
3、 根据权利要求 2所述的蛋白质， 其特征在于， 所述蛋白质包含具有 SEQ ID NO. 1所示的氨基酸序列的蛋白质。
4、 一种核酸， 其特征在于， 编码具有 SEQ ID NO. 1 所示的氨基酸序列 的蛋白质、 蛋白质片段、 蛋白质的类似物或衍生物的核酸。
5、 根据权利要求 4所述的核酸， 其特征在于， 所述核酸包含编码具有 SEQ ID NO. 1所示的氨基酸序列的核酸。
6、 根据权利要求 4所述的核酸， 其特征在于， 所述核酸包含有 SEQ ID NO. 2中的 1-762位的序列。
7、 一种如权利要求 1-3任一所述的 hnRNP A2*蛋白质的用途， 其特征在 于， 所述 hnRNP A2*蛋白质用于解开端粒 G-四链体，促进端粒酶对端粒的延 长。
8、 根据权利要求 7所述的用途， 其特征在于， 所述 hnRNP A2*蛋白质能 够识别端粒 5' -TAGGGTTAGG-3'的核酸序列， hnRNP A2*蛋白质与端粒 DNA 结合后， 解开端粒 G-四链体的结构， 并暴露出 5' -GTTAG-3'的末端， 所述 5' -GTTAG-3'能和脊推动物端粒酶 RNA模板 5' -CUAAC-3'配对，使端粒酶对端 粒进行延长， 维持端粒长度。
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