CN1631899A - Application of DLP involved in Pre-mRNA splicing and cell cycle regulation - Google Patents

Abstract

The invention relates to DLP protein, gene codzing DLP, and the application of DLP in preparation of splicing and medicine relating to agent of Pre-mRNA and adjustment of cell period, also, it relates to the application designed for DLP in RNAi preparing medicine against tumber.

Description

Application of DLP involved in Pre-mRNA splicing and cell cycle regulation

field of invention

The present invention relates to the DLP protein, the gene encoding DLP, the application of DLP in the preparation of reagents and medicines related to Pre-mRNA splicing and cell cycle regulation, and the present invention also relates to the use of RNAi (RNA interference) designed for DLP in the preparation of drugs for treating tumors Uses in medicine.

Background technique

Gene expression in eukaryotic cells is a multi-step complex process, including initiation, elongation and termination of transcription. During the transcription process, the primary Pre-mRNA undergoes capping at the 5' end, intron excision, exon joining, cleavage at the 3' end, and addition of polyadenylic acid to form mature mRNA, which is transported to the cytoplasm for translation into protein. The size of the proteome in an organism not only comes from the size of the number of genes, but also depends on the diversity of proteins. The research on the mechanism of protein diversity is of great significance to the understanding of biological evolution at the molecular level. It is predicted that the human genome may have about 35,000 genes, Drosophila about 14,000, and the simple model organism nematode about 19,000 genes. There appears to be a marked difference in the complexity of an organism and the number of genes in its genome. The reason lies in the proteome. Mechanisms such as gene rearrangement, use of different start sites for gene transcription, pre-mRNA editing, polyadenylation, alternative splicing, and post-transcriptional protein modification can generate multiple proteins from a single gene, thereby making proteins in the proteome The number of genes exceeds the number of genes in the genome. Among them, in terms of the number of genes affected and the range of biological species, alternative splicing is the most important mechanism for expanding protein diversity. The recent completion of the draft human genome suggests that alternative splicing of pre-mRNAs plays an important role in biological diversity, as more than 59% of human genes appear to have arisen through alternative splicing (Hastings ML and Krainer AR 2001) . In addition, it is estimated that 15% of all mutations that cause human genetic diseases are caused by the incorrect splicing of the Pre-mRNA of the corresponding gene, so that the gene cannot be expressed correctly (Philips AV and Cooper TA 2000). The removal of pre-mRNA introns thus plays an important role in gene regulation. Alternative pre-mRNA splicing and joining of different exons can form proteins with different functions, which is an important source of protein diversity (Maniatis and Tasic 2000).

The splicing reaction of pre-mRNA is through the spliceosome - consisting of five small nuclear RNAs (U1, U2, U4, U5 and U6) (Adams et al.1996; Kramer 1996; Reed 1996; Reed2000) and various protein subunits Ribosomal protein complex to complete (Ajuh et al.2001). These proteins include a set of general and specific proteins associated with snRNAs (Kramer et al., 1996, 1999; Will and Luhrmann 1997), as well as many non-snRNA-associated proteins, which contribute to the assembly and splicing of the complex The catalysis of is critical (Will andluhrmann 1997; Staley and Guthrie 1998). They may be involved in snRNP biosynthesis, spliceosome assembly and dissociation, and catalytic reactions of intron excision and exon junction (Raker VA and kastner B et al., 1999; Reed R 2000; Arenas JE and Abelson JN 1997). The splicing reaction consists of two consecutive transesterification reactions. In the first transesterification reaction, the 5' splice site is cleaved to generate splicing intermediates (exon 1 and lariat exon 2), and in the second In the first transesterification reaction, the 3' splice site is cleaved to generate the spliced product (lasso-shaped intron and spliced mRNA). The complete spliceosome then dissociates and its components re-engage in the formation of other spliceosomes, which is called the "splicing cycle" (Jurica M.S. and Moore M.J 2003). Assembly of the spliceosome involves the sequential assembly of snRNP particles and other proteins onto pre-mRNA substrates prior to catalysis (Reed and Ralandjian 1997). During spliceosome complex assembly, complexes E, A, B, and C are formed sequentially. Complex A is an early assembly intermediate that contains U1 and U2 snRNPs bound to unspliced pre-mRNA and assembled to the pre-mRNA substrate. Complex B prepares for the first step of the catalytic reaction and contains U2, U5 and U6 snRNAs in addition to the unspliced Pre-mRNA. Like complex B, complex C also contains U2, U5, and U6 snRNAs, but represents a stage after the first catalytic reaction (containing splicing intermediates), which is aggregated to the Pre- mRNA and connected to introns (Jurica et al., 2002). U1 snRNP is the first particle to bind to the pre-mRNA substrate, followed by U2 snRNP and U1 snRNP to form a pre-splice complex, followed by U4/U6 and U5 snRNPs to form a triple snRNP and pre-splice complex to form a splice body (Guthrie C 1991; Ruby S.W. and Abelson J 1991; Moore M and Query C 1993; Lamond A 1993; Newman A 1994). In this dynamic process, a large number of proteins are involved. As of 1999, approximately 100 splicing factors have been identified (Burge et al., 1999). With the development of mass spectrometry and improvements in spliceosome purification techniques, the number of splicing factors has multiplied. Satisfactorily, most of the previously identified splicing factors have been identified in recent mass spectrometry studies. But the emergence of many new proteins not previously associated with splicing was surprising. In 2002, the Molecular and Cellular Biology Laboratory of Harvard University isolated 145 different splice body proteins, 88 of which were known splicing factors, snRNP proteins and splice body proteins, and 43 existed in U1, U2, U4, U5, Among U6 sn RNPs, 58 are unknown novel proteins. 90 proteins have homologues in yeast. At least 30 proteins are known to function in steps of gene expression other than splicing. These proteins not only function in multiple steps of pre-mRNA splicing in multicellular organisms, but also mediate the extensive linkage between splicing and other steps of gene expression (Zhou et al. 2002). Splicing is associated with other biological processes in the cell, such as cell cycle, transcription and mRNA output. Pre-mRNA splicing and cell cycle regulation have become a hot field in cell biology and molecular biology research in recent years, and are also researches on genes. Expression and regulation, cell proliferation and differentiation, cell carcinogenesis and apoptosis, and the basis of biotechnology such as genetic engineering and cell engineering.

Contents of the invention

During the research on a cytokine-like factor-CKLF, a protein involved in Pre-mRNA splicing and cell cycle regulation was discovered, and the protein was named Dim1-likeprotein (DLP). Based on this finding, the present invention has been conceived as follows:

1. A DLP protein involved in Pre-mRNA splicing and cell cycle regulation, characterized in that it has the amino acid sequence shown in Sequence 2.

2. The nucleotide sequence encoding DLP protein, characterized in that it contains the nucleotide sequence shown in Sequence 1.

3. An expression vector containing the nucleotide sequence of item 2.

4. The use of DLP in the preparation of reagents or medicines related to Pre-mRNA splicing or cell cycle regulation.

5. Antibody to DLP.

6. RNAi designed for DLP.

7. The RNAi according to item 6, characterized in that it has the gene sequence shown in sequence 3 or 4.

8. A prokaryotic or eukaryotic expression vector containing the RNAi of item 6 or 7.

9. The use of the RNAi according to Item 6, 7 or 8 in the preparation of a drug for treating tumors.

Brief description of the drawings

Figure 1: Nucleotide sequence and deduced amino acid sequence of DLP cDNA. The orientation of the nucleotide sequence is from 5'-3'. The predicted amino acid sequence is shown below the nucleotide sequence. The stop codon is indicated by *, the protein kinase C phosphorylation site is indicated by '△', the casein kinase II phosphorylation site is indicated by '○', and the N-myristoylation site is indicated by '▲'.

Figure 2: GST Pull-down detection of the interaction between DLP and splicing-related factors APC4, hnRNPF, PQBP and Prp6 in vitro.

Figure 3: The upper panel is a schematic representation of the deletion body of DLP. Del1 (1-128aa), Del2 (21-126aa), Del3 (33-149aa).

The figure below shows the results of different deletions of DLP and the GST Pull-down of Prp6. DLPDel3 interacts with Prp6 in vitro.

Figure 4: Diagram of the involvement of DLP in the splicing of Adml-M3 Pre-mRNA.

Figure 5 RT-PCR detection results of DLP RNAi in MCF-7, HepG2 and ISH.

GAPDH was used as an internal reference.

Figure 6 Effect of DLP on cell cycle. The right side is the result of the corresponding protein level detected by Western Blotting.

DLP has a DIM1 domain. The amino acid sequence homology analysis found that DLP has a weak homology with a 15KD splice body protein U5 of yeast, and has 39% and 24% homology with a mitotic protein DIM1 and human thioredoxin, respectively. At the same time, it is found that it is quite conserved among various genera such as human, mouse, Arabidopsis thaliana, Oryza, melanogaster, fruit fly, and yeast. Evolutionary analysis also showed that DLP is a conserved protein and is evolutionarily related to DIM1 protein. Further research found that its function is related to splicing and cell cycle.

The full length of DLP nucleotides is 2540bp, encoding 149 amino acids, the molecular weight is about 17KD, the isoelectric point is 5.63, and it is located at 16q22.3. Northern Blot hybridization showed that DLP was highly expressed in skeletal muscle, liver, heart and pancreas, moderately expressed in kidney, brain and placenta, and lowly expressed in lung. RT-PCR confirmed that DLP was also highly expressed in human breast cancer cell MCF-7, liver cancer cell HepG2 and endometrial cancer cell Ishikawa. It shows that DLP is widely distributed in the body and is a broad-spectrum gene.

The inventors analyzed the localization of DLP, and used green fluorescent protein to do localization, indicating that DLP is localized in the nucleus, which is consistent with the process that DLP may participate in splicing in the nucleus.

Flow testing was also done. DLP overexpression and RNAi plasmids were transiently transfected into MCF-7 cells respectively, and the cell cycle changes were detected by flow cytometry. It was found that the overexpression of DLP in MCF-7 cells could increase the percentage of cells in the G2/M phase, When the effect of DLP was inhibited by RNAi, the percentage of cells in S phase increased, and the percentage of cells in G2/M phase decreased. The cells were arrested in the G0/G1 phase with the estrogen inhibitor ICI182780, and then the DLP overexpression and RNAi plasmids were transiently transfected into MCF-7 cells, and it was found that there was no significant effect on the cell cycle, indicating that DLP has an important effect on the cell cycle. Entry has no effect, but may play a role in the S-G2/M phase transition of the cell cycle. The results showed that DLP is necessary and sufficient for the transition from S phase to G2/M. This result is consistent with Dim1 regulating G2 phase progression in fission yeast and S. cerevisiae (Stevens SW and Abelson J1999). Therefore, RNAi designed against DLP is of great significance in the treatment of tumors. In addition, MTT analysis found that DLP can promote the proliferation of certain cells such as Hela, C2C12 cells and the differentiation of C2C12 cells. This may be an indirect effect of DLP affecting the cell cycle. DLP was transiently transfected into 293T cells, Hela cells, MCF-7 cells and C2C12 cells, and the cell proliferation was analyzed by MTT. It was found that DLP could significantly promote the proliferation of C2C12 and Hela cells (p<0.05). DLP can also promote the differentiation of C2C12 cells and induce the formation of myotubes.

GST Pull-down assay proved the interaction between DLP and splicing factors. First, DLP was expressed in prokaryotic cells, and New Zealand white rabbits were immunized to prepare polyclonal antibodies. Using the prepared polyclonal antibody for Western Blot detection, the expression of endogenous DLP protein in MCF-7 cells can be detected. GST Pull-down experiments found that there was a direct interaction between DLP and the splicing-related factor U5-102KD (Prp6) in vitro. In order to verify the region where DLP interacts with U5-102KD (Prp6), the inventors designed different deletions of DLP, and the GST Pull-down study of the deletion confirmed that the 33 amino acids at the end of DLPN interacted with U5-102KD (Prp6) area. It shows that the N-terminus of DLP is the part where it plays a role. U5-102KD (Prp6) is a U4/U6-specific protein (Evgen 1999), Prp6 has been shown to interact with hDim1p homologue Dib1 in yeast (Uetz 2000), Prp6 contains at least 10 TPR (tetratrico peptiderepeats) domain (Abovich 1990; Galisson 1993), U5-102KD contains 19 TPRs (Evgen 2000). The TPR domain is the binding site for multiple protein-protein interactions (Sikorslci 1990; Vockhart 1994; Chung 1999). This result further confirms that DLP may be involved in Pre-mRNA splicing. Next, co-immunoprecipitation, yeast two-hybrid and confocal methods were used to further verify the direct interaction between DLP and U5-102KD (Prp6) protein. These three methods all confirmed the conclusion of GST Pull-down. Moreover, other proteins interacting with DLP were screened out by yeast two-hybrid method, among which Homo sapiens nuclear receptor coactivator 7 (ERAP140) was cloned and identified by Harvard University Myles. A coactivator for receptors that enhances the transcriptional activity of nuclear receptors it interacts with. Like other coactivators, estrogen can periodically recruit ERAP140 to the promoter, but it does not have any structural and sequence similarities to previously identified nuclear receptor coactivators (Wenlin Shao 2002). This result is not inconsistent with the role of DLP in pre-mRNA splicing, and suggests that DLP may be linked to the process of transcription. The role of DLP with transcriptional activators is consistent with the localization of DLP in the nucleus.

The inventors designed in vitro splicing experiments. It was found that the addition of the GST-DLP fusion protein can stimulate the splicing of the Adml-M3 splicing substrate, and when the anti-GST-DLP antibody was added, the splicing was inhibited, and the addition of the GST-DLP fusion protein restored the splicing activity of DLP. It proved that DLP can indeed participate in the process of splicing.

The transcription of mRNA includes splicing of pre-mRNA, which produces mature mRNA, which is conducive to the normal transmission of genetic information, and abnormal splicing can lead to diseases such as genetics and tumors. The DLP of the present invention is involved in the splicing process, so it can be used to prepare medicines and diagnostic reagents for such diseases related to splicing abnormalities.

Expression vectors and host cells containing DLP nucleotides can be used to produce large quantities of DLP. The host cells can be prokaryotic cells such as bacterial cells such as Escherichia coli; they can also be eukaryotic cells such as yeast cells, higher eukaryotic cells such as mammalian cells. Transformation of host cells can be carried out by conventional methods in the art. Antibodies against DLP can be used to antagonize the effects of DLP. Includes monoclonal and polyclonal antibodies. Polyclonal antibodies against DLP can be obtained by direct immunization of animals, and monoclonal antibodies can be obtained by hybridoma technology.

Example

Reagents and instruments used in the examples are listed below.

1.1. Plasmid vector and recombinant cloning

The prokaryotic expression vector pGEX-4T-3 was purchased from Amersham Biosciences. The eukaryotic expression vectors pcDNA3.1(-)/myc/hisB, pEGFP, and pDsRed-C1 were provided by the Disease Gene Center of Peking University Health Science Center. The RNA interference vector pSURER was purchased from Brummelkamp.

DNA binding domain vectors pGBKT7, pGBKT7-53, pGBKT7-Lam, DNA activation domain vectors pGADT7, pGADT7-T were purchased from Clontech.

The recombinant plasmid pADML-M3 was donated by Professor Reed R. of Harvard University.

1.2. Strains

1.2.1. Bacterial strains

DH5α, genotype FΦdlacZΔM15 recA endA1 gyrA96 thi-1hsdR17(rK-mK+)supE44 relAl deoRΔ(lacZYA-argF)U169, Invitrogen.

BL21, genotype E.coil B F ⁻ , ompT, hsdS (r _B ⁻ , m _B ⁻ ), gal, dcm were purchased from Amersham Biosciences.

1.2.2. Yeast strains strain genotype reporter gene AH109 Mata, trp1-901, leu2-3, 112, ura3-52His3-200, gal4△, gal80△, LYS2::GAL1 _USA -GAL1 _TATA -HIS3, GAL2 _USA -GAL2 _TATA -ADE2, URA3::MEL1 _USA -MEL1 _TATA - lacZ ADE2HIS3MEL1 (or LacZ) Y187 MATα, ura3-52, ade2-101, trp1-901, His3-200, gal4△, gal80△, Leu2-3, 112, ^Met- , URA3::GAL1 _USA -GAL1 _TATA -lacZ1 LacZ CG-1945 MATa, ura3-52, his3-200, ade2-101, lys2-801Trp1-901, leu2-3, 112, gal4-542, gal80-538, cyh ^r 2LYS2::GAL1 _USA ^- GAL1 _TATA -HIS3, URA3::GAL4 _{17 -mers(x3)} -CYC1 _TATA -LACZ HIS3, LaCZ

1.3. Cell lines

C2C12 cells are mouse skeletal muscle cells, Hela cells are human cervical cancer cells, and 293T are human embryonic kidney cells. MCF-7 cells are human breast cancer cells, HepG2 cells are human liver cancer cells, and Ishikawa cells are human endometrial cancer cells are preserved in our laboratory.

1.4. Tool enzymes and reagents

(1) Restriction enzymes, DNA modification enzymes, T4DNA ligase, and T7RNA polymerase were purchased from New England Biolabs in the United States; calf intestinal alkaline phosphatase (CIP) was purchased from Promega; Taq polymerase and pfu enzyme were purchased from Takara Company; RNaseA was purchased from Sigma; RNaseA inhibitor was purchased from invitrogen.

(2) Nucleotides, dNTP and ATP were purchased from promega and invitrogen companies.

(3) Oligonucleotides, the following are oligonucleotides for RNAi

oligo1,

5'-GATCCCCCCTGCAGTTTATACACAGTATTCAAGAGATACTGTGTATAAACTGCAGTTTTTGGAAA-3'

oligo2,

5'-

AGCTTTTCCAAAAACTGCAGTTTATACACAGTATCTCTTGAATACTGTGTATAAATGCAGGGG-3’

1.5. Molecular weight standard

DNA molecular weight standard DL2000 was purchased from Takara Company, and protein molecular weight standard was purchased from Novangen Company.

1.6. Kit

QIAGEN Plasmid Extraction Kit, Gel Recovery Kit, and PCR Purification Kit were purchased from GENE Company; Adventure cDNA PCR Kit was purchased from Clontech Company. Trizon RNA extraction kit was purchased from Invitrogen. ProtoScriptTM First Strand cDNA Synthesis Kit was purchased from NEB Company. HelaScribe Nuclear Extract in vitro Transcription System, TNT Coupled Reticulocyte Lysate Systems were purchased from Promega. Human adult multiple tissue Northern (MTN) blots, Human cDNA normal tissue first strand, and The spotlight ^TM Random Primer Labeling Kit were purchased from Clontech.

1.7. Radioisotopes

L-[ ³⁵ S]methionine, α-[ ³² P]dCTP (10 mci/ml), γ-[ ³² P]dGTP (10 mci/ml) were purchased from Amersham Biosciences.

1.8. Media and library sources

Trypton, Yeast Extract, and Agar were purchased from Oxoid. Yeast YPD medium (liquid) and SD medium (liquid) were prepared according to the requirements of Clontech Company. Human mammary gland library was purchased from Clontech Company.

1.9. Chemical reagents

Tris (Tris) was purchased from GIBCO-BRL, USA. PEG3350, salmon sperm DNA was purchased from sigma company. LIPOFECTAMINE2000 Reagent was purchased from Invitrogen Company. Other reagents are imported aliquots or domestic analytical reagents. Lithium acetate, various amino acids, and salmon sperm DNA were purchased from Clontech, and X-gal was purchased from Sigma.

1.10. Main instruments

(1) DNA amplification instrument (Perkin-Elmer, USA), PCR amplification.

(2) Ultraviolet gel imaging system (UVP GSD5000, UK).

(3) Electroporator (Gene Pulser, Bio-Rad USA).

(4) ABI3700 automatic sequencer from PE Company.

(5) Image Master Desk Top Scanner (Pharmacia) scanner.

(6) Gyrotory Water BathShaker G76 (New Brunswick Scientific Co., Inc) shaker, used for the amplification of bacteria and yeast.

(7) Hybridization furnace A10284 (Gene company)

(8) EL 311 SX ELISA.

(9) Vibra Cell Ultrasonic Crusher (Sonic&Material Inc.).

(10) BECTON DICKINSON FACScan flow cytometer: Germany.

1.11. Primer names and sequences

pcDNA3.1-DLP:

Forward: 5'ATGAGCTTCCTACTGCCCAAG 3'

Reverse: 5'GCTCTAGATGTGCCTTCTTTCTTCATC 3'

pcDNA3.1-Prp6:

Forward: 5’

ATAAGAATGCGGCCGCTAAACTATACTTTGCTACGGAGTGCAT 3’

Reverse: 5'CCGGAATTCCGGCTGACACGAGACATAAAAACT 3'

pcDNA3.1-PQBP:

Forward: 5’

ATAAGAATGCGGCCGCTAAACTATGGGAGAGGAACAGGGCCCT 3’

Reverse: 5'CCGGAATTCCGGGGTGGGCAGGATCACCAGAA 3'

pcDNA3.1-hnRNPF:

Forward: 5’

ATAAGAATGCGGCCGCTAAACTATTCTGGCCATTTCTCTTGAAAC 3’

Reverse 5’CCGGAATTCCGGTCAAGTTGAAAAACAAACAAATCT 3’

pcDNA3.1-APC4:

Forward: 5’

ATAAGAATGCGGCCGCTAAACTATATGTTGCGTTTTCCGACCTG 3’

Reverse: 5'CCGGAATTCCGGTTAGGAGTCTAGCTCAGGGTC 3'

pcDNA3.1-Flag-Prp6:

Forward: 5’

ATAAGAATGCGGCCGCGCCACCATGGACTACAAGGACGACGATGAC

AAGGAATTCACTTTGCTACGGAGTGCAT 3’

Reverse: 5'CCGGAATTCCGGCTGACACGAGACATAAAAACT 3'

pEGFPN1-DLP:

Forward: 5'GGAATTCATGAGCTTCCTACTGCCCAAG 3'

Reverse: 5'CGGGATCCAATGTCTTGATAGCGAAGGTCATATTTG 3'

pGEX-4T-3-DLP:

Forward: 5'GGAATTCCATGAGCTTCCTACTGCCCAAG 3'

Reverse: 5'AAGGAAAAAAGCGGCCGCAAAAGGAAAACTAAATGTCTTGATAGAGAAGGTCA 3'

pGEX-4T-3-DLPDEL1:

Forward: 5'GGAATTCCATGAGCTTCCTACTGCCCAAG 3'

Reverse: 5'AAGGAAAAAAGCGGCCGCAAAAGGAA

AATTAAAGCTTCCCCCCTCATTGC 3’

pGEX-4T-3-DLPDEL2:

Forward: 5'GGAATTCCATGAGCTTCCTACTGCCCAAG 3'

Reverse: 5'AAGGAAAAAAGCGGCCGCAAAGGAAAAAAAGCTTCCCCCTCATTGC 3'

pGEX-4T-3-DLPDEL3:

Forward: 5'GGAATTCCATGGAAGATCCTGTCTGTCTGC 3'

Reverse: 5'AAGGAAAAAAGCGGCCGCAAAAGGAAAACTAAATGTCTTGATAGAGAAGGTCA 3'

pDsRedC1-Prp6:

Forward: 5'GGAATTCCACTTTGCTACGGAGTGCAT 3'

Reverse: 5'GGGGTACCCTGACACGAGACATAAAAACT 3'

pGBKT7-DLP:

Forward: 5'GGAATTCATGAGCTTCCTACTGCCCAAG 3'

Reverse: 5'ACGCGTCGACGCTAAATGTCTTGATAGAGAAGG 3'

pGADT7-Prp6:

Forward: 5'GGAATTCACTTTGCTACGGAGTGCAT 3'

Reverse: 5'CATCGATCTGACACGAGACATAAAAACT 3'

1.12. Nucleic acid sequence analysis

Sequences were analyzed using the sequence analysis software DNASTAR. Homology comparisons were performed using BLAST.

Example 1

Extraction of total mRNA

Total cellular mRNA was extracted using Trizol ^TM reagent from Invitrogen Company. Take 1×10 ⁷ MCF-7 cells, HepG2 and Ishikawa cells, collect by centrifugation, add 1ml Trizol ^TM reagent, blow repeatedly to break the cells, add 0.2ml chloroform after standing at room temperature for 5 minutes, shake vigorously for 15 seconds, 4 ℃12000rpm for 15 minutes, collect the supernatant, precipitate with isopropanol, wash once with 70% ethanol, dry at room temperature, resuspend the total RNA in DEPC-treated _H2O , quantify it with a spectrophotometer and use it for the first strand of cDNA Synthesis.

cDNA duplex synthesis

NEB's ProtoScript ^TM First Strand cDNA Synthesis Kit was used to synthesize the first and second strands of cDNA using the mRNA mentioned in 2.3 as a template. The brief description is as follows: 1ng-2ugmRNA, 2ul Primer dT23VN, 4ul dNTP, add H ₂ O to 16ul reaction system, 70°C for 5 minutes, quickly cool in ice bath, after short centrifugation, add 2ul 10xRT Buffer, 1ul RNase inhibitor, 1ul M-MulV Reverse Transcriptase, mix gently, react at 42°C for one hour, inactivate at 95°C for 5 minutes, add 1ul (2u) RNase H at 37°C for 20 minutes, digest RNA. Inactivate DNase at 95°C for 5 minutes. Dilute the reaction to 50ul, take 2-5ul for PCR reaction.

RT-PCR reaction

The reaction system is as follows: 2.5ul 10x buffer, 4ul dNTP mix, 5ul (10uM) DLP upstream and downstream primers or GAPDH positive control primer, 5ul cDNA double strand or human adult tissue cDNA first strand (Clontech), 0.25ul pfu, 3.25ul water . The PCR reaction conditions were: 94°C for 1 minute; 94°C for 30 seconds, 50°C for 30 seconds, 72°C for 1 minute, 28 rounds, and 72°C for 10 minutes. For the positive control reaction conditions: 94°C for 2 minutes, 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, 25 rounds, 72°C for 10 minutes. After the PCR product was separated by agarose electrophoresis prepared by TAE, the PCR band was cut off, and three times the volume of binding buffer was added, sol at 55°C, shortly centrifuged, washed three times with PE washing buffer, and eluted with elution buffer .

Plasmid construction

According to the mRNA sequence of DLP analyzed by computer, primers at both ends of the open reading frame were designed, and the ORF of DLP was amplified with the primers, and the amplified product was connected to the pGEM-T-Easy vector for sequencing. The open reading frame of DLP was excised from the pGEM-T-easy vector with EcoRI, then digested with EcoRV and BamHI, after purification, it was connected to the eukaryotic expression vector pcDNA3.1 digested with EcoRv and BamHI (-)/myc/HisB, resulting in pCDNA3.1-DLP. Design primers for the DLP coding region, design EcoRI and BamHI restriction sites at both ends, use PCR to amplify the coding region of DLP from pCDNA3.1-DLP, and connect to pEGFP-N1 after digestion with EcoRI-BamHI In the expression vector, the pEGFP-DLP recombinant plasmid was obtained. This vector can express the fusion protein of enhanced green fluorescent protein (EGFP) and DLP in eukaryotic cells. The reading frame of DLP is consistent with that of EGFP. Transfect this vector into eukaryotic cells After the cells are removed, the site where the DLP is located can be observed under a fluorescent microscope. Design primers for the DLP coding region, design EcoRI and NotI restriction sites at both ends, use PCR to amplify the coding region of DLP from pCDNA3.1-DLP, and connect to pGEX-4T- after digestion with EcoRI-NotI 3, the recombinant plasmid pGEX-DLP was obtained. This vector can express GST and DLP fusion protein in E.coil BL21. The cDNA sequences of hnRNPF, PQBP, APC4 and Prp6 were amplified from the human cDNA panel using Clontech's Advantage ^TM -cDNA PCR kit, and NotI and EcoRI restriction sites were designed at both ends of the primers, and then ligated to pcDNA3 after digestion .1(-)/myc/HisB vector, get pcDNA3.1-hnRNPF, PQBP, APC, Prp6 recombinant plasmids, which can be transcribed and translated in vitro, and use GST Pull-down to study the interaction with DLP. For the construction of DLP deletions, EcoRI and NotI restriction sites were designed at both ends of the primers for deletion 1 (1-128), deletion 2 (21-126), and deletion 3 (33-149), from pcDNA3.1 -Amplified on DLP, digested and connected to the processed pcDNA3.1(-)/myc/HisB to obtain recombinant plasmids of different deletions. In order to study whether DLP and Prp6 co-localize in eukaryotic cells, the recombinant plasmid of Prp6 and red fluorescent protein was constructed, and the primers of Prp6 were designed, and the primers at both ends were introduced into the restriction sites of EcoRI and KpnI. -Amplify on Prp6, connect to the pDsred-C1 (Clontech) vector digested with EcoRI and KpnI after enzyme digestion, and obtain the recombinant plasmid of pDsred-Prp6. The vector can express the fusion protein of red fluorescent protein (Red) and Prp6 in eukaryotic cells, and after the vector is transfected into eukaryotic cells, the location of Prp6 can be observed under a fluorescent microscope. In order to study the interaction between DLP and Prp6 in vivo, a recombinant plasmid with Flag tag was constructed. Design primers, introduce NotI site and Flag tag sequence upstream, introduce EcoRI restriction site downstream, amplify from pcDNA3.1-prp6, and connect to pcDNA3.1 vector after digestion.

For yeast two-hybrid studies, the bait protein pGBKT7-DLP was constructed as follows: the reading frame of DLP was amplified from pSUPER-DLP by PCR, the PCR product was digested with EcoRI and SalI, and ligated into the pGBKT7 (Clontech) vector. The coding region of Prp6 was amplified from pcDNA3.1-Prp6 by PCR, and the PCR product was digested with EcoRI and CalI, and ligated into pGADT7 (Clontech) vector.

Plasmid extraction and purification

For the purification of plasmids for sequencing and gene cloning operations, the alkaline lysis method of "Molecular Cloning (Second Edition)" and the small plasmid kit of Tianwei Times were used. Use Qiagentip-20 Minipreparation Kit (plasmid purification) for transfection of eukaryotic cells: pick a single colony in 3ml LB medium, culture for 12-16 hours, centrifuge, resuspend in 0.3ml Buffer P2, reverse 4-6 Once, place at room temperature for 5 minutes. Add to 4°C pre-cooled Buffer P3, invert 4-6 times, and ice bath for 5 minutes. Centrifuge at high speed, add the supernatant to a Qiagen-tip 20 column equilibrated with 1ml Buffer QBT, wash the column with 1ml Buffer QC, repeat three times, add 0.8ml Buffer QF to elute the plasmid, add 0.7 times the volume of isopropanol, and run at room temperature at high speed Centrifuge for 30 minutes. Discard the supernatant and wash twice with 70% ethanol. Air dry for 5 min and dissolve in _H2O .

sequencing

Sequencing was performed using ABI's 3700 DNA sequencer. The sequencing results are shown in Figure 1.

Northern Blot

Probe labeling and hybridization operations refer to the instructions of the Spotlight ^TM Random Primer Labeling Kit kit from Clontech Company. The process is briefly described as follows:

(1) Probe labeling: Dissolve 25ng-1ug DNA in 20ul volume, incubate at 90-100°C for 2-3 minutes, quickly ice-bath for 5 minutes, add 5ul [α- ³² P]dCTP-labeled Random Primers and ReactionBuffer Mix, replenish water To 50ul, incubate at 37°C for 5-10 minutes.

(2) Hybridization: Preheat the hybridization solution at 68°C, put the nylon membrane adsorbed with nucleic acid into the hybridization bottle containing the hybridization solution, add 0.1ml/cm ² of the hybridization solution containing 100ug/ml salmon sperm DNA, and heat at 68°C Prehybridize for 30 minutes. 20ng/ml probe was mixed into the hybridization solution, kept at 95°C for 2-5 minutes, and ice-bathed for 5 minutes. Add to the hybridization flask and hybridize at 68°C for 1 hour.

(3) Membrane washing: wash the membrane with Wash Buffer 1 at room temperature for 30-40 minutes, and wash the membrane with Wash Buffer 2 at 50°C for 40 minutes.

(4) Color development: Take out the film with tweezers, wash and dry Wash Buffer 2, cover with plastic wrap, expose at -70°C for 1-3 days, develop and fix.

Expression and purification of DLP in prokaryotic cells

GST-DLP can induce the expression of GST-DLP fusion protein in Escherichia coli BL21 strain at 30°C. First, prepare competent cells of BL21: pick a single clone into an appropriate amount of medium, and cultivate it at 37°C and 250rpm to A600 0.4-0.5. Centrifuge at 2500g for 15 minutes and set aside. Transform 1-50ngGST-DLP into competent cells and purify with Glutathione Sepharose 4B. The purification steps are as follows:

(1) Pick a single clone and culture it in 2-3ml LBA medium for 4-5 hours. Transfer to a 100ml Erlenmeyer flask and grow overnight.

(2) Transfer to a 500ml Erlenmeyer flask and cultivate for 3-5 hours.

(3) Add 0.5ml 1M IPTG and incubate at 30°C for 3 hours.

(4) Collect the cells by centrifugation at 3000rpm for 10 minutes.

(5) Resuspend the cells with 25ml PBS. Sonicate for 10 minutes.

(6) Add 1.25ml 20% Triton X-100 and mix for 1 hour.

(7) Centrifuge at 1000rpm for 10 minutes, add 0.5ml 50% slurry of Glutathione Sepharose 4B, and keep at room temperature for 30 minutes.

(8) Centrifuge at 1000rpm for 5 minutes, wash with PBS three times.

(9) Elute with 0.5ml elution buffer, and collect the supernatant after brief centrifugation.

As a result, DLP was successfully expressed in Escherichia coli with a purity of over 90%.

Example 2

Preparation of anti-GST-DLP polyclonal antibody and Western blot hybridization

The purified GST-DLP fusion protein was used to immunize New Zealand white rabbits to prepare polyclonal antibodies against DLP. For Western blot, the extracted protein was quantified by Bio-Rad's DC protein quantification reagent, and then subjected to 15% SDS-PAGE electrophoresis, and the protein was transferred to a nitrocellulose membrane after electrophoresis. After the membrane was blocked with 5% skimmed milk powder for 2 hours, a 1:30 dilution of rabbit anti-DLP polyclonal antibody or a 1:5000 dilution of anti-c-myc monoclonal antibody (Invitrogen) was added at 4°C overnight, and washed three times with TBST After) (10 minutes/time), add horseradish enzyme-labeled anti-rabbit IgG (Amersham Pharmacia Biotech) or goat anti-mouse IgG (Santa Cruz) diluted 1:4000, incubate for 1 hour, and wash the membrane three times with TBS Add substrate solution for color development.

Example 3

GST Pull-down

Using Promega's TNT Rabbit Reticulocyte lysate and TNT T7polymerase labels kit, in vitro transcription and translation of pcDNA-Prp6, hnRNPF, PQBP and APC4. Proceed as follows:

(1) Add the reagents to the 1.5ml tube in the following order

25ul TNT lysate

2ul TNT reaction buffer

1ul TNT RNA polymerase

1ul amino acid mixture, minus Met(1mM)

2ul [ ³⁵ S]methionine (>1000ci/mmol)

1ul Rnasin Ribonuclease inhibitor

2ul pcDNA-Prp6, hnRNPF, PQBP and APC4

16ul Nuclease-free water

50ul total volume

30°C for 90 minutes.

(2) Add 50ul glutathione agarose beads, 500ng-10ug GST or GST-DLP, and 4-5ul in vitro transcription and translation products into a 1.5ml tube.

(3) Incubate at 4°C for 2 hours, 13000rpm for 2 minutes, discard the supernatant. Wash glutathione agarose beads with 1ml of cold lysis buffer.

(4) Centrifuge at 13,000 rpm for 1 minute, discard the supernatant, and repeat washing twice.

(5) Elution with 50ul 20mM reduced glutathione, room temperature for 10 minutes.

(6) Centrifuge at 13000 rpm for 2 minutes.

(7) Add 2xSDS-PAGE loading buffer and boil at 100°C for 5 minutes. Load on 12% SDS-PAGE polyacrylamide gel.

(8) Remove the gel and fix it at room temperature for 30 minutes.

(9) Dry on a gel dryer for 30-90 minutes.

(10) Expose at -70°C, develop after 4-16 hours.

It was found that DLP interacted with U5-102KD (the homologue in yeast is Prp6), but did not interact with PQBP, hnRNPF and APC4 (Figure 2). To investigate the site where DLP interacts with Prp6, different deletions of DLP were constructed. GST Pull-down experiments proved that the N-terminal 1-33aa of DLP interacted with Prp6 (Figure 3).

Example 4

co-immunoprecipitation

(1) MCF-7 cells were cultured to 10 ⁶ , and the pcDNA-Flag-Prp6 recombinant plasmid was transfected with LIPOFECTAMINE 2000 reagent.

(2) After 48 hours, the cells were collected, washed twice with pre-cooled PBS, scraped off with a cell scraper, and resuspended in PBS.

(3) Centrifuge at 2000g for 5 minutes, discard the supernatant, add lysis buffer [50mM Tris-HCL PH7.5, 100mM Nacl, 10% (v/v) glycerol, 0.1% (v/v) NP-40, 1mM dithiothreitol , 1mM EDTA, protease inhibitor cocktail] for 10 minutes.

(4) Sonicate twice, 10 seconds each time to lyse the cells, and incubate the lysate and Protein-A-Sepharose at 4°C for 2 hours.

(5) Centrifuge at 14000 g for 10 minutes, add pre-immune serum or anti-Flag antibody (invitrogen) (lug) to the supernatant, and incubate at 4°C for 16 hours. Immune complexes were captured with Protein-A-Sepharose.

(6) Centrifuge at 6000g for 10 minutes, wash Protein-A-Sepharose with NETN Buffer [20mM Tris (PH8.0), 1mM EDTA, 900mM Nacl, 0.5% (v/v) NP-40, Protease inhibitor cocktail] for 4-5 all over.

(7) 95-100°C for 5 minutes, add 2xSDS loading buffer. Immune complexes were separated on a 12% SDS denaturing polyacrylamide gel.

(8) Remove the gel, electrotransfer to the nitrocellulose membrane, add 5% skim milk powder to block at room temperature for 1-2 hours.

(9) Add anti-GST-DLP polyclonal antibody (1:30), room temperature for 1-2 hours.

(10) Wash the membrane 3 times with TBST, 10 minutes each time.

(11) Add the secondary antibody of monkey anti-rabbit IgG (Amersham Pharmacia Biotech) coupled with horseradish enzyme, and incubate at room temperature for 1-2 hours.

(12) Wash the membrane three times with TBST, 10 minutes each time.

(13) Expose with ECL reagent from Pharmacia Company.

To detect the intracellular interaction between DLP and Prp6, MCF-7 cells were transfected with pcDNA3.1-Flag-Prp6, total protein was extracted, immunoprecipitated with anti-Flag monoclonal, and then Western blotting with anti-GST-DLP polyclonal antibody Detect DLP. It was found that DLP can indeed interact with Flag-tagged Prp6, which is consistent with the results of GST Pull-down.

Localization of DLP in cells and co-localization with Prp6

The pEGFPN1-DLP vector was constructed using the pEGFP-N1 N-terminal protein fusion vector from CLONTECH, which can express the GFP-DLP fusion protein in eukaryotic cells, and these vectors were transfected into MCF-7 cells by Lipofectin ^TM After 24 hours, the slides were washed three times with PBS, fixed with PBS/4% paraformaldehyde, room temperature for 15 minutes, and then washed three times with PBS. Permeabilize with 0.1% Triton/PBS for 15 minutes, then wash three times with PBS, add 200ul 2.5mg/ml DAPI, react at room temperature for 30 minutes, then wash three times with PBS, and use conventional fluorescent wavelengths under a fluorescence microscope Observe with DAPI wavelength and take pictures.

In order to study whether DLP and Prp6 interact in eukaryotic cells, the pDsredC1-Prp6 vector was constructed using the pDsred-C1 C-terminal protein fusion vector of CLONTECH, which can express the fusion protein of pDsred-Prp6 in eukaryotic cells. The pEGFP-DLP and pDsred-Prp6 vectors were transfected into MCF-7 cells by the method of Lipofectin ^TM , and the transfected cells were sliced. After 24 hours, the slices were washed three times with PBS, and washed with PBS/4% paraformaldehyde. For fixation, room temperature for 15 minutes, and then washed three times with PBS. Under a fluorescence microscope, the samples were observed with conventional fluorescence wavelengths and photographed.

In order to further confirm the interaction between DLP and Prp6, the subcellular colocalization of DLP and Prp6 was done. The fusion expression vector of pEGFP-DLP and pDsRed-Prp6 was co-transfected into MCF-7 cells. 24 hours after transfection, the colocalization of the two was observed by confocal microscopy. It was found that the labeled green and red fluorescence were mainly observed in the nucleus and overlapped, indicating that DLP and Prp6 interacted and were related to Pre-mRNA splicing.

Example 5

in vitro splicing experiment

First, the Pre-mRNA splicing substrate was transcribed in vitro using HelaScribe Nuclear Extract in vitro Transcription System from Promega. Proceed as follows:

(1) Splicing substrate pAdML-M3 mRNA was linearized with XbaI. The Gel Recovery Kit from Gene Company was used to recover and purify.

(2) Add the following ingredients in sequence to a 1.5ml sterile siliconized tube

6ul Hela Nuclear Extract 1X Transcription Buffer

2ul Mgcl ₂ 50mM

1ul 25X rNTP Mix

5ul lined pAdML-M3 mRNA subtracts

1ul [α- ^32P ]rGTP (3000Ci/mmol, 10mCi/ml)

5ul Nuclease-Free ddH ₂ O

5ul HelaScribe Nuclear Extract

25ul total volume

(3) Incubate at 30°C for 60 minutes.

(4) Preheat Hela Extract stop buffer at 25°C for 1 hour, add 175ul to the reaction system to terminate the reaction.

(5) Add 200ul TE-saturated phenol, mix for 60 seconds, and centrifuge at 14000g for 5 minutes.

(6) Transfer 150ul supernatant to a clean 1.5ml tube.

(7) Add 200ul stop buffer again, and re-extract once with phenol. Mix well and centrifuge as above. Add 300ul chloroform: isoamyl alcohol (24:1) to the supernatant twice for extraction, mix well and centrifuge briefly.

(8) Transfer the supernatant to a new sterile siliconized tube.

(9) Add 700ul of 100% ethanol and mix well. -70°C for at least 15 minutes.

(10) Transfer the ethanol mixture to a sterile 1.5ml microcentrifuge tube. Centrifuge at 14000g for 10 minutes at 4°C.

(11) Carefully remove the supernatant and dry at room temperature. Dissolve RNA with 10-20ul nuclease-free water.

(12) Add 2X SDS loading buffer, 100°C for 5 minutes.

(13) RNA was isolated on 6% denatured polyacrylamide gel containing 7M urea.

(14) Cut the full-length RNA from the gel, add elution buffer (0.75M ammonium acetate, 10mM magnesium acetate, 0.1% SDS, 0.1mM EDTA) overnight.

(15) The eluted RNA was separated with a 0.45um filter, ethanol precipitated, and the RNA was dissolved with nuclease-free water.

The splicing process is briefly described as follows:

(1) Add the following solutions in sequence to a 1.5ml sterile microcentrifuge tube:

6-8ul Hela Nuclear Extract

5ul[α- ^32P ]-radioabled transcription products

1.25ul 50mM Mgcl ₂

3.75ul 100mM ATP

1.28ul 20mM creatine phosphate

1.25ul 10mM DTT

1ul Rnasin inhibitor (40u/ul)

5-6ul Nuclease-Free ddH ₂ O (or pre-immune, GST, GST-DLP,

Anti GST-DLP antibody) --

25ul total volume

(2) Incubate at 30°C for 1 hour.

(3) Add proteinase K and SDS at a final concentration of 4ug/ml and 0.1% to terminate the reaction. Incubate at 37°C for 20 minutes. Dilute the volume to 100ul with 125mM Tris (PH8.0), 1mM EDTA, 0.3M sodium acetate.

(4) Extract once with 200ul TE-saturated phenol/chloroform: isoamyl alcohol. Centrifuge at 14000g for 5 minutes.

(5) Transfer the supernatant to a new microcentrifuge tube, and extract it once with 200ul chloroform. Centrifuge at 14000g for 5 minutes.

(6) Transfer the supernatant to a new microcentrifuge tube, use 300ul of absolute ethanol to precipitate the RNA, and overnight at -80°C.

(7) Centrifuge at 14000 g for 10 minutes, discard the supernatant, and dry at room temperature.

(8) 20ul DEPC-treated water dissolved RNA. Add an equal volume of 2x SDS loadingbuffer.

(9) 90-100°C for 5 minutes, isolate RNA on 12% denatured polyacrylamide gel containing 7M urea.

(10) Fix at room temperature for 30 minutes, and dry the glue for 90 minutes.

(11) After exposure at -80°C for 1-2 days, develop.

In vitro splicing experiments were used to confirm the role of DLP in splicing. ³² p-labeled Adml-M3Pre-mRNA was synthesized and incubated with nuclear extracts of Hela cells under standard splicing conditions. In splicing experiments, a polyclonal antibody against GST-DLP was used to neutralize the effect of endogenous DLP in HeLa cells. As shown in Figure 4, the addition of non-specific serum had no significant effect on splicing. Splicing was significantly inhibited after adding anti-GST-DLP polyclonal antibody, and excess prokaryotic purified GST-DLP fusion protein could restore the splicing of Adml-M3 Pre-mRNA. These results suggest that DLP is involved in the splicing of Adml-M3 Pre-mRNA.

Example 6

Construction of RNAi recombinant plasmid and Western blotting

Use the pSUPER vector to construct the recombinant plasmid of DLPRNAi, as follows:

(1) Select 197-215 bp of the DLP open reading frame and design the oligonucleotide sequence.

Oligo1:

5'GATCCCCCCTGCAGTTTATACACAGTATTCAAGAGATACTGTGTATAAACTGCAGTTTTTGGAAA 3'

Oligo2:

5’AGCTTTTCCAAAACTGCAGTTTATACACAGTATCTCTTGAATACTGTGTATAAATGCAGGGG 3’

(2) The oligonucleotides were resuspended in annealing buffer (100mM potassium acetate, 30mM HEPES-KOH, PH7.4, 2mM acetate), 95°C for 4 minutes, 70°C for 10 minutes, and then cooled to room temperature to generate double-stranded DNA.

(3) The double-stranded DNA was phosphorylated with phosphorylase and inserted into the pSUPER vector treated with BglII/HindIII.

(4) The recombinant plasmid of DLP RNAi was transfected into MCF-7 cells with LIPOFECTAMINE 2000 reagent. After 48 hours, the cells were collected, and the degree of DLP inhibition was detected with the polyclonal antibody of GST-DLP. The steps are as described above.

In order to further verify whether DLP RNAi can inhibit the endogenous DLP mRNA, MCF-7, HepG2 and Ishikawa cells were transfected with DLP RNAi, the mRNA was extracted, reverse-transcribed for RCR, and it was found that DLP RNAi could indeed inhibit the endogenous DLP in these three cells Original expression (Figure 5).

Example 7

Cell cycle detection by flow cytometry

(1) Preparation of cells: MCF-7 cells were transfected with recombinant plasmids of pcDNA-DLP and pSUPER-DLP. 12 hours after transfection, the estrogen inhibitor ICI182780 was added to a final concentration of 10 nM. After 48 hours, the adherent cells were treated with Digest with 0.25% trypsin for about 10 minutes, blow off the cells and collect them by centrifugation at 2000 rpm for 10 minutes. Wash twice with PBS, add 70% ethanol to fix overnight.

(2) Staining procedure: the cells were centrifuged, washed once with PBS, added PI staining solution to make the final concentration 50ug/ml, RNaseA to make the final concentration 100U/ml, and stained at room temperature for 30 minutes. Cell cycle changes were detected by flow cytometry.

Determination of cell proliferation by MTT assay

To detect the effect of DLP on cell growth, pcDNA3.1-DLP and pSUPER-DLP recombinant plasmids were transiently transfected into C2C12, 293T, Hela and MCF-7 cells. 4 hours before the termination of cell culture, add 10ul of 10mg/ml MTT (dissolved in PBS) in the culture well, and add the lysate (50%DMSO, 20%SDS, PH4.4) after the cells continue to culture for 4 hours overnight, The next day, the absorbance value at 570 mM wavelength was measured with a microplate reader.

MCF-7 cells were transfected with pSUPER-DLP or pcDNA3.1-DLP. After 24 hours, the estrogen inhibitor ICI182780 was added for 16 hours, and the cells were arrested in G0/G1 phase. Cells were collected and cell cycle changes were detected by flow cytometry. The corresponding protein levels were detected by Western Blotting. As shown in Figure 6, after endogenous DLP was silenced, the percentage of cells in G2/M phase decreased significantly, and the percentage in S phase increased. On the other hand, the overexpression of DLP resulted in a significant increase in the number of cells in G2/M phase. However, when the cell cycle was arrested in G0/G1 phase by the estrogen inhibitor ICI182780, the overexpression of DLP had no obvious effect on the cell cycle. These results suggest that DLP plays an important role in the S-G2/M phase of the cell cycle.

Cell Differentiation Experiment

C2C12 cells were transfected with pcDNA-DLP cells. After 24 hours, the culture medium containing 2% horse serum was replaced for 4-5 days, and the same medium was replaced every day. After 48 hours, the cells were washed twice with PBS, stained with Wright-Gimsa at room temperature for 1 hour, and observed under a fluorescence microscope.

DLP was overexpressed in C2C12, 293T, Hela and MCF-7 cells, and then the effect on cell proliferation was analyzed by MTT. Overexpression of DLP can stimulate the proliferation of C2C12 and Hela cells, but the effect is not obvious in 293T and MCF-7. It was also found that DLP can promote the differentiation of mouse skeletal muscle cells C2C12 and the formation of skeletal muscle myotubes. These results are consistent with a process by which DLP affects the cell cycle.

Sequence Listing

SEQUENCE LISTING

<110> Shang Yongfeng

<120> Application of DLP involved in Pre-mRNA splicing and cell cycle regulation

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<170>PatentIn version 3.1

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gcg ata aaa agt act gct gag aag gtg ttg gtt ctc agg ttt ggg aga 96

Ala Ile Lys Ser Thr Ala Glu Lys Val Leu Val Leu Arg Phe Gly Arg

20 25 30

gat gaa gat cct gtc tgt ctg cag cta gat gat att ctt tct aag acc 144

Asp Glu Asp Pro Val Cys Leu Gln Leu Asp Asp Ile Leu Ser Lys Thr

35 40 45

tct tct gac tta agt aaa atg gct gct ata tac ctg gta gat gtg gac 192

Ser Ser Asp Leu Set Lys Met Ala Ala Ile Tyr Leu Val Asp Val Asp

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caa act gca gtt tat aca cag tat ttt gac atc agt tat att cca tct 240

Gln Thr Ala Val Tyr Thr Gln Tyr Phe Asp Ile Ser Tyr Ile Pro Ser

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Thr Val Phe Phe Phe Asn Gly Gln His Met Lys Val Asp Tyr Gly Ser

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cca gat cac act aag ttt gtg gga agc ttc aaa acc aaa caa gac ttc 336

Pro Asp His Thr Lys Phe Val Gly Ser Phe Lys Thr Lys Gln Asp Phe

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Ile Asp Leu Ile Glu Val Ile Tyr Arg Gly Ala Met Arg Gly Lys Leu

115 120 125

att gtc caa agt cct att gat ccc aag aat att ccc aaa tat gac ctt 432

Sequence Listing

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Ser Ser Asp Leu Ser Lys Met Ala Ala Ile Tyr Leu Val Asp Val Asp

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Gln Thr Ala Val Tyr Thr Gln Tyr Phe Asp Ile Ser Tyr Ile Pro Ser

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Claims (9)

1. A DLP protein involved in Pre-mRNA splicing and cell cycle regulation, characterized in that it has the amino acid sequence shown in Sequence 2. 2. The nucleotide sequence encoding DLP protein, characterized in that it contains the nucleotide sequence shown in Sequence 1. 3. An expression vector comprising the nucleotide sequence of claim 2. 4. The use of the DLP of claim 1 in the preparation of reagents or medicines related to Pre-mRNA splicing or cell cycle regulation. 5. An antibody to the DLP of claim 1. 6. RNAi designed for DLP. 7. The RNAi according to claim 6, characterized in that it has the gene sequence shown in sequence 3 or 4. 8. A prokaryotic or eukaryotic expression vector containing the RNAi according to claim 6 or 7. 9. The use of RNAi according to claim 6, 7 or 8 in the preparation of drugs for treating tumors.

Images