Functional Pre- mRNA trans-Splicing of Coactivator CoAA and Corepressor RBM4 during Stem/Progenitor Cell Differentiation
2009; Elsevier BV; Volume: 284; Issue: 27 Linguagem: Inglês
10.1074/jbc.m109.006999
ISSN1083-351X
AutoresYang Brooks, Guanghu Wang, Zheqiong Yang, Kimberly K. Smith, Erhard Bieberich, Lan Ko,
Tópico(s)Cancer-related gene regulation
ResumoAlternative splicing yields functionally distinctive gene products, and their balance plays critical roles in cell differentiation and development. We have previously shown that tumor-associated enhancer loss in coactivator gene CoAA leads to its altered alternative splicing. Here we identified two intergenic splicing variants, a zinc finger-containing coactivator CoAZ and a non-coding transcript ncCoAZ, between CoAA and its downstream corepressor gene RBM4. During stem/progenitor cell neural differentiation, we found that the switched alternative splicing and trans-splicing between CoAA and RBM4 transcripts result in lineage-specific expression of wild type CoAA, RBM4, and their variants. Stable expression of CoAA, RBM4, or their variants prevents the switch and disrupts the embryoid body formation. In addition, CoAA and RBM4 counter-regulate the target gene Tau at exon 10, and their splicing activities are subjected to the control by each splice variant. Further phylogenetic analysis showed that mammalian CoAA and RBM4 genes share common ancestry with the Drosophila melanogaster gene Lark, which is known to regulate early development and circadian rhythms. Thus, the trans-splicing between CoAA and RBM4 transcripts may represent a required regulation preserved during evolution. Our results demonstrate that a linked splicing control of transcriptional coactivator and corepressor is involved in stem/progenitor cell differentiation. The alternative splicing imbalance of CoAA and RBM4, because of loss of their common enhancer in cancer, may deregulate stem/progenitor cell differentiation. Alternative splicing yields functionally distinctive gene products, and their balance plays critical roles in cell differentiation and development. We have previously shown that tumor-associated enhancer loss in coactivator gene CoAA leads to its altered alternative splicing. Here we identified two intergenic splicing variants, a zinc finger-containing coactivator CoAZ and a non-coding transcript ncCoAZ, between CoAA and its downstream corepressor gene RBM4. During stem/progenitor cell neural differentiation, we found that the switched alternative splicing and trans-splicing between CoAA and RBM4 transcripts result in lineage-specific expression of wild type CoAA, RBM4, and their variants. Stable expression of CoAA, RBM4, or their variants prevents the switch and disrupts the embryoid body formation. In addition, CoAA and RBM4 counter-regulate the target gene Tau at exon 10, and their splicing activities are subjected to the control by each splice variant. Further phylogenetic analysis showed that mammalian CoAA and RBM4 genes share common ancestry with the Drosophila melanogaster gene Lark, which is known to regulate early development and circadian rhythms. Thus, the trans-splicing between CoAA and RBM4 transcripts may represent a required regulation preserved during evolution. Our results demonstrate that a linked splicing control of transcriptional coactivator and corepressor is involved in stem/progenitor cell differentiation. The alternative splicing imbalance of CoAA and RBM4, because of loss of their common enhancer in cancer, may deregulate stem/progenitor cell differentiation. From the initial discovery of RNA splicing (1.Berget S.M. Moore C. Sharp P.A. Proc. Natl. Acad. Sci. U.S.A. 1977; 74: 3171-3175Crossref PubMed Scopus (865) Google Scholar, 2.Chow L.T. Gelinas R.E. Broker T.R. Roberts R.J. Cell. 1977; 12: 1-8Abstract Full Text PDF PubMed Scopus (709) Google Scholar) to recent advanced genomic studies (3.Modrek B. Lee C. Nat. Genet. 2002; 30: 13-19Crossref PubMed Scopus (1056) Google Scholar), a large body of evidence suggests that alternative splicing as an integral part of gene regulation profoundly impacts biological and pathological functions (4.Sanford J.R. Caceres J.F. J. Cell Sci. 2004; 117: 6261-6263Crossref PubMed Scopus (27) Google Scholar). In the human genome, although ∼20,000 protein-coding genes exist, more than 90% of multi-exon genes are alternatively spliced (3.Modrek B. Lee C. Nat. Genet. 2002; 30: 13-19Crossref PubMed Scopus (1056) Google Scholar, 5.Pan Q. Shai O. Lee L.J. Frey B.J. Blencowe B.J. Nat. Genet. 2008; 40: 1413-1415Crossref PubMed Scopus (2620) Google Scholar, 6.Wang E.T. Sandberg R. Luo S. Khrebtukova I. Zhang L. Mayr C. Kingsmore S.F. Schroth G.P. Burge C.B. Nature. 2008; 456: 470-476Crossref PubMed Scopus (3636) Google Scholar). Thus, alternative pre-mRNA splicing contributes greatly to proteome diversity without increasing the number of genes (7.Stetefeld J. Ruegg M.A. Trends Biochem. Sci. 2005; 30: 515-521Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 8.Stamm S. Ben-Ari S. Rafalska I. Tang Y. Zhang Z. Toiber D. Thanaraj T.A. Soreq H. Gene. 2005; 344: 1-20Crossref PubMed Scopus (693) Google Scholar). Alternative splicing can be cell- and tissue-specific, which is particularly important at the early developmental stages (9.Smith C.W. Patton J.G. Nadal-Ginard B. Annu. Rev. Genet. 1989; 23: 527-577Crossref PubMed Scopus (567) Google Scholar). The wild type and variant proteins often exert overlapping but distinct functions whose balance is controlled by the choice of alternative splicing. Aberrant splicing patterns may disrupt the normal functional balance of isoforms, which in turn impairs cell differentiation and induces disease or cancer (10.Kalnina Z. Zayakin P. Silina K. Linē A. Genes Chromosomes Cancer. 2005; 42: 342-357Crossref PubMed Scopus (164) Google Scholar, 11.Venables J.P. BioEssays. 2006; 28: 378-386Crossref PubMed Scopus (256) Google Scholar). Pre-mRNA splicing is a process of joining exons and splicing out introns. In addition to constitutive splicing events, exons can be alternatively spliced. This includes exon skipping, alternative 5′ and 3′ splicing, mutually exclusive exons, intron retention, and alternative transcription start or stop sites (12.Xing Y. Lee C. Nat. Rev. Genet. 2006; 7: 499-509Crossref PubMed Scopus (209) Google Scholar). The cis-splicing mechanism involves splicing within one pre-mRNA molecule. In contrast, significant evidence supports that trans-splicing can occur at a much lower frequency by joining exons from two independent pre-mRNA molecules through the usage of canonical splicing sites in the absence of DNA recombination (13.Mayer M.G. Floeter-Winter L.M. Mem. Inst. Oswaldo. Cruz. 2005; 100: 501-513Crossref PubMed Scopus (34) Google Scholar). Many examples of trans-splicing have been described (14.Akiva P. Toporik A. Edelheit S. Peretz Y. Diber A. Shemesh R. Novik A. Sorek R. Genome Res. 2006; 16: 30-36Crossref PubMed Scopus (219) Google Scholar). However, the biological functions of a large amount of trans-splicing events remain to be characterized. Among the products of some alternative splicing and trans-splicing are non-coding RNAs (15.Eddy S.R. Nat. Rev. Genet. 2001; 2: 919-929Crossref PubMed Scopus (1047) Google Scholar, 16.Mattick J.S. Makunin I.V. Hum. Mol. Genet. 2006; 15: R17-29Crossref PubMed Scopus (1852) Google Scholar). Although non-coding RNAs do not encode a protein product, many of them play important regulatory roles at multiple levels of RNA metabolism. Evidence has also suggested that the majority of the mammalian genome is transcribed into non-coding RNAs, many of which are alternatively spliced products. Altered splicing patterns are prevalent in diseases and cancers. It has been shown that germ-line sequence variations at splice sites are present in cancer genes such as BRCA1 and APC (17.Brose M.S. Volpe P. Paul K. Stopfer J.E. Colligon T.A. Calzone K.A. Weber B.L. Genet. Test. 2004; 8: 133-138Crossref PubMed Google Scholar, 18.Neklason D.W. Solomon C.H. Dalton A.L. Kuwada S.K. Burt R.W. Fam. Cancer. 2004; 3: 35-40Crossref PubMed Scopus (29) Google Scholar, 19.Ozcelik H. Nedelcu R. Chan V.W. Shi X.H. Murphy J. Rosen B. Andrulis I.L. Hum. Mutat. 1999; 14: 540-541Crossref PubMed Scopus (12) Google Scholar). Differentially expressed alternative splice variants are associated with severe clinical outcome in cancer patients (20.Adamia S. Reiman T. Crainie M. Mant M.J. Belch A.R. Pilarski L.M. Blood. 2005; 105: 4836-4844Crossref PubMed Scopus (58) Google Scholar). Faulty alternative splicing signals impair apoptosis pathways. The high relevance between alternative splicing and cancer is a reflection of the fundamental role of alternative splicing in controlling stem cell or progenitor cell differentiation, as cancer is considered a stem cell disease. Recent studies on human embryonic stem cells and neural progenitor cells indicate that a large amount of alternative splicing events normally occur (21.Yeo G.W. Xu X. Liang T.Y. Muotri A.R. Carson C.T. Coufal N.G. Gage F.H. PLoS Comput. Biol. 2007; 3: 1951-1967Crossref PubMed Scopus (104) Google Scholar). In addition, splicing of a number of exons has been shown to be reprogrammed due to the switch of alternative splicing regulators during neural development (22.Boutz P.L. Stoilov P. Li Q. Lin C.H. Chawla G. Ostrow K. Shiue L. Ares Jr., M. Black D.L. Genes Dev. 2007; 21: 1636-1652Crossref PubMed Scopus (401) Google Scholar). Thus, alternative splicing control may play a key role in stem cell and progenitor cell differentiation. Alternative splicing is coupled with transcriptional regulation which is in part regulated by transcriptional coactivators and corepressors (23.Auboeuf D. Dowhan D.H. Li X. Larkin K. Ko L. Berget S.M. O'Malley B.W. Mol. Cell. Biol. 2004; 24: 442-453Crossref PubMed Scopus (128) Google Scholar, 24.Kornblihtt A.R. Curr. Opin. Cell Biol. 2005; 17: 262-268Crossref PubMed Scopus (186) Google Scholar). We have previously characterized coactivator CoAA 2The abbreviations used are: CoAAcoactivator activatorCoAMcoactivator modulatorCoAZcoactivator with zinc fingerncCoAZnon-coding CoAZRBM4RNA binding motif protein 4ESembryonic stemECembryonic carcinomaEBembryoid bodyRRMRNA recognition motifGFAPglial fibrillary acidic proteinRTreverse transcriptionRACErapid amplification of cDNA endsCMVcytomegalovirusGAPDHglyceraldehyde-3-phosphate dehydrogenaseMMTVmurine mammary tumor virusMAP-2microtubule-associated protein-2. (gene symbol RBM14) in regulating transcription-coupled pre-mRNA alternative splicing (23.Auboeuf D. Dowhan D.H. Li X. Larkin K. Ko L. Berget S.M. O'Malley B.W. Mol. Cell. Biol. 2004; 24: 442-453Crossref PubMed Scopus (128) Google Scholar, 25.Iwasaki T. Chin W.W. Ko L. J. Biol. Chem. 2001; 276: 33375-33383Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 26.Kang Y.K. Schiff R. Ko L. Wang T. Tsai S.Y. Tsai M.J. O'Malley B.W. Cancer Res. 2008; 68: 7887-7896Crossref PubMed Scopus (16) Google Scholar). The CoAA pre-mRNA is alternatively spliced to yield several alternative splicing variants, one of which is a functional dominant negative variant termed CoAM. In stem cells, CoAA and CoAM regulate early stem cell differentiation through a switch of their alternative splicing at the stage of embryoid body cavitation (27.Yang Z. Sui Y. Xiong S. Liour S.S. Phillips A.C. Ko L. Nucleic Acids Res. 2007; 35: 1919-1932Crossref PubMed Scopus (26) Google Scholar). In human cancers the CoAA gene is amplified with the recurrent deletion of its 5′ upstream regulatory sequence (28.Sui Y. Yang Z. Xiong S. Zhang L. Blanchard K.L. Peiper S.C. Dynan W.S. Tuan D. Ko L. Oncogene. 2007; 26: 822-835Crossref PubMed Scopus (23) Google Scholar). We have shown that deletion of this regulatory sequence leads to defective switching between CoAA and CoAM (27.Yang Z. Sui Y. Xiong S. Liour S.S. Phillips A.C. Ko L. Nucleic Acids Res. 2007; 35: 1919-1932Crossref PubMed Scopus (26) Google Scholar). coactivator activator coactivator modulator coactivator with zinc finger non-coding CoAZ RNA binding motif protein 4 embryonic stem embryonic carcinoma embryoid body RNA recognition motif glial fibrillary acidic protein reverse transcription rapid amplification of cDNA ends cytomegalovirus glyceraldehyde-3-phosphate dehydrogenase murine mammary tumor virus microtubule-associated protein-2. In this study we describe the trans-splicing events between CoAA and its downstream RBM4 genes, both of which encode transcription and alternative splicing coregulators that participate in neural stem cell or progenitor cell differentiation. The human RBM4 gene is ∼10 kilobases distal to CoAA at chromosome 11q13. RBM4 protein has been previously shown to regulate alternative splicing of the microtubule-associated protein Tau at its exon 10 (29.Kar A. Havlioglu N. Tarn W.Y. Wu J.Y. J. Biol. Chem. 2006; 281: 24479-24488Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and to antagonize polypyrimidine tract-binding protein alternative splicing activities (30.Lai M.C. Kuo H.W. Chang W.C. Tarn W.Y. EMBO J. 2003; 22: 1359-1369Crossref PubMed Scopus (92) Google Scholar, 31.Lin J.C. Tarn W.Y. Mol. Cell. Biol. 2005; 25: 10111-10121Crossref PubMed Scopus (60) Google Scholar). RBM4 is a mammalian ortholog of the Drosophila melanogaster Lark, which is critical for viability, fertility, development, and circadian rhythms output (32.Jackson F.R. Banfi S. Guffanti A. Rossi E. Genomics. 1997; 41: 444-452Crossref PubMed Scopus (27) Google Scholar, 33.McNeil G.P. Schroeder A.J. Roberts M.A. Jackson F.R. Genetics. 2001; 159: 229-240Crossref PubMed Google Scholar, 34.McNeil G.P. Zhang X. Genova G. Jackson F.R. Neuron. 1998; 20: 297-303Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). We found that RBM4 represses nuclear receptor-mediated transcription. The CoAA and RBM4 gene transcripts are trans-spliced to produce a novel zinc finger-containing coactivator CoAZ and a non-coding splice variant ncCoAZ. CoAZ and ncCoAZ are derived by 5′ competitive alternative splicing and display switched expression patterns during neural stem cell differentiation. Both CoAZ and ncCoAZ activate transcription and balance coactivator CoAA and corepressor RBM4 splicing activities. In particular, CoAA and CoAZ promote the skipping of Tau exon 10, and RBM4 and ncCoAZ promote the inclusion of Tau exon 10. The imbalance of Tau alternative splicing at its exon 10 is known to be involved in neurodegenerative diseases (35.Pittman A.M. Fung H.C. de Silva R. Hum. Mol. Genet. 2006; 15: R188-195Crossref PubMed Scopus (98) Google Scholar). Thus, the regulation of coactivator CoAA and corepressor RBM4 through alternative trans-splicing variants may have functional importance in normal cell differentiation as well as disease. Total RNA from HeLa cells was isolated using Trizol reagent, treated with DNase I, and converted to cDNA using first-strand cDNA synthesis kit (Invitrogen). The presence of CoAZ and ncCoAZ was initially detected by RT-PCR followed by sequence analysis. Full-length cDNAs were subsequently obtained by 3′ and 5′-RACE (Invitrogen). As controls, the absence of CoAA transcript in 5′-RACE and the absence of RBM4 transcript in 3′-RACE were confirmed using CoAA or RBM4-specific primers. Primers crossing splicing junctions were used in RACE to ensure specificity. The 5′ and 3′ ends of CoAZ and ncCoAZ transcripts were subjected to sequence analysis. The primers for 5′-RACE were: CoAZ primers, GSP1, tctatcggacactctttg; GSP2, acgtgcattcgtttgcctttca; GSP3, tgtgcacgaaggcgaactgtt; ncCoAZ-specific, ttgcaactctgtgttatc; The primers for 3′-RACE primers were: CoAZ primers, GSP1, gattcagaattcaagatattcgtgggcaa; GSP2, tgacgtggtga-aaggcaa; GSP3, ctggtccaaagagtgtccgataga; ncCoAZ-specific, gataacacagagttgcaa. Identified cDNAs sequences have been deposited in GenBank with accession numbers as follows: human CoAZ, EU287938; human ncCoAZ, EU287939; mouse CoAZ, EU287940; mouse ncCoAZ, EU287941. The CoAA minigenes with alternative splicing capacity have been previously described (27.Yang Z. Sui Y. Xiong S. Liour S.S. Phillips A.C. Ko L. Nucleic Acids Res. 2007; 35: 1919-1932Crossref PubMed Scopus (26) Google Scholar). To facilitate trans-splicing analysis, RBM4 minigenes were constructed under the control of CMV or its native RBM4 promoter (from −1113 to +1) as diagrammed in Fig. 2A. The RBM4 minigenes contain four intact exons, nucleotides 1–71, 1019–1442, 4769–5459, and 7346–7787. Deletions were introduced to the first intron (519–813), the second intron (1623–4558), and the third intron (5728–7065). A minimum 160-bp intronic sequence around each splicing site was left intact to preserve necessary binding sites for splicing factors. A SpeI restriction enzyme recognition site was introduced into the third exon of RBM4 (5125–5131) to distinguish minigene transcripts from the endogenous. The splicing capacity of RBM4 minigene cassettes was evaluated by RT-PCR. The spliced transcripts were gel-purified (Qiagen) and SpeI-digested (New England Biolabs). In trans-splicing assays, 293 cells were cotransfected with both CoAA and RBM4 minigenes using vector as controls. RT-PCR analyses using primers spanning the CoAA first exon and the RBM4 third exon were performed. Primers used gattcagaattcaagatattcgtgggcaa and ctcaagctttaaaaggctgagtacc. PCR products corresponding to the correct size of CoAZ and ncCoAZ were gel-purified and SpeI-digested before gel analysis. Endogenous CoAZ, ncCoAZ, and RBM4 mRNA expression patterns were analyzed using first-strand cDNAs from multiple normal human tissues and cancer cell lines (MTCTM panels, Clontech). For P19 cells, total RNA was isolated at each differentiation stage using Trizol reagent (Invitrogen), treated with DNase I, and normalized for their concentrations before use. RNA was reverse-transcribed to cDNA using SuperScript III first-strand synthesis system (Invitrogen). Real-time PCR (iCycler, Bio-Rad) was performed using SYBR Green dye in duplicate in a 25-μl reaction. The results were normalized to GAPDH. Primer pairs used were as follows: primers common to endogenous CoAA and CoAM, atgaagatttttgtgggcaa, ctaaacgccggtcg-gaacc; CoAM-specific, tctcaaccaagggtatggtt, ctac-atgcggcgctggta; ncCoAZ, atcgagtgtgacgtggtaaaag, aagctttgctcttattcttgctg; CoAZ, tgacgtggtaaaaggcaa, atctattggacactctttggac; RBM4, gccattttagcgttttgtc-ag, atctattggacactctttggac; total Tau, ccaccaaaatccggagaacgaa, gcttgtgatggatgttccctaa; Tau exon 10, gtgcagataattaataagaagctg, gcttgtgatggatgttccctaa; Nanog, agggtctgctactgagatgctctg, caaccactggtttttctgccaccg; MAP2, ggacatcagcctcactcacaga, gcagcatgttcaaagtcttcacc; Sox6, cagcggatggagaggaagcaatg, ctttttctgttcatcatgggctgc; glial fibrillary acidic protein (GFAP), gaatgactcctccactccctgc, cgctgtgaggtctggcttggc; GAPDH, accacagtccatgccatcac, tccaccaccctgttgctgta. Polyclonal anti-CoAZ (anti-ZnF) antibody was generated by immunizing rabbits with GST-CoAZ fusion protein (113–339 amino acids) (Covance). The antibody titer of each bleed was monitored by enzyme-linked immunosorbent assay. His-tagged CoAZ as antigen was cross-linked to the Affi-gel 10 resin and was used for affinity purification according to the manufacturer's protocol (Bio-Rad). Endogenous or overexpressed CoAA, CoAZ, and RBM4 were detected by immunoblotting using nuclear extracts from overexpressed 293 cells or from P19 stem cells or using whole cell extracts from rat cortical cell culture. Immunoblots were probed with anti-CoAA, anti-RRM, anti-ZnF, and anti-FLAG (Sigma) at a dilution of 1:200, 1:400, 1:400, and 1:10000, respectively. The blots were detected with the ECL system (Amersham Biosciences). Polyclonal anti-CoAA (CoAA-specific, against 307–545 aa of CoAA) and anti-RRM antibody (against 1–156 amino acids of CoAM and recognizes CoAA, CoAM, and CoAZ) were previously prepared and affinity-purified (27.Yang Z. Sui Y. Xiong S. Liour S.S. Phillips A.C. Ko L. Nucleic Acids Res. 2007; 35: 1919-1932Crossref PubMed Scopus (26) Google Scholar). Anti-ZnF is against 113–339 amino acids of CoAZ and recognizes CoAZ and RBM4. The ES-derived embryoid bodies were paraffin-embedded, and the sections were stained with purified anti-CoAA, anti-RRM, anti-ZnF, and anti-active caspase-3 antibodies (United States Biological, C2087-16A). Sagittal sections of mouse embryonic tissue at E12.5 and E15.5 were stained with affinity-purified anti-ZnF at a dilution of 1:200. Antibody binding was detected using biotinylated anti-rabbit or anti-mouse IgG F(ab)2 secondary antibody followed by detecting reagents (DAKO). Sections were counterstained with hematoxylin. Immunofluorescence double staining was carried out using rabbit polyclonal anti-CoAA, anti-RRM, and anti-ZnF and counter-stained with mouse monoclonal anti-Nestin, anti-GFAP, and anti-MAP-2 antibodies. Cy3- and fluorescein isothiocyanate-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were applied at a dilution of 1:200. Mouse embryonal carcinoma P19 cells were maintained in α-modified minimum essential medium supplemented with 7.5% bovine calf serum and 2.5% fetal bovine serum, 100 units/ml penicillin, and 0.1 μg/μl streptomycin. Cells were incubated in 5% CO2 at 37 °C. Undifferentiated P19 cells (EC) were induced to differentiate by 500 nm all-trans retinoic acid (Sigma) up to 4 days in suspension culture to form embryoid bodies (EB2–4). The EBs were trypsinized and further differentiated (D3–15) for an additional 15 days in the absence of retinoic acid. Stably transfected P19 cells were selected using 400 μg/ml G418 for 3 weeks. Positive clones were identified by the presence of plasmid DNA and Western blot analysis. More than two stable clones for each transfection were identified and analyzed. For ES-derived embryoid bodies, pre-implantation blastocyst-derived ES cells J-1 were grown on γ-irradiated feeder fibroblasts, and neural differentiation was induced by serum deprivation of embryoid bodies as previously described (36.Bieberich E. MacKinnon S. Silva J. Noggle S. Condie B.G. J. Cell Biol. 2003; 162: 469-479Crossref PubMed Scopus (103) Google Scholar, 37.Wang G. Silva J. Krishnamurthy K. Tran E. Condie B.G. Bieberich E. J. Biol. Chem. 2005; 280: 26415-26424Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). To generate neural progenitors, embryoid bodies were trypsinized and plated on polyornithine/laminin-coated dishes to further culture 3 (NP3) or 5 (NP5) days. Cells were grown in DMEM/F-12 with N2 supplement and fibroblast growth facto-2. Cortical neural cells from embryonic rat brain at E18.5 were isolated and cultured in vitro for 7 days as previously described (38.Redmond L. Kashani A.H. Ghosh A. Neuron. 2002; 34: 999-1010Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). For astrocytes, neural cells from P3 neonates were dissected, filtered with a 40 λm filter and cultured for 7 days. The culture was shaken overnight in the incubator at 180–220 rpm to remove contaminating cell types and retain astrocyte populations (39.Schwartz J.P. Wilson D.J. Glia. 1992; 5: 75-80Crossref PubMed Scopus (129) Google Scholar). CV-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 0.1 μg/μl streptomycin and incubated in 5% CO2 at 37 °C. Cells were cultured in 24-well plates and transfected with MMTV luciferase reporter and various amounts of glucocorticoid receptor, pcDNA3 vector, CoAA, CoAM, CoAZ, ncCoAZ, and RBM4 using Lipofectamine 2000 (Invitrogen). Cells were incubated with the ligand dexamethasone (100 nm) to induce the MMTV-luciferase reporter, when applicable, for 16 h before harvest. Total amounts of DNA for each well were balanced by adding vector DNA. Relative luciferase activities were measured by a Dynex luminometer. Data are shown as the means of triplicate transfections ± S.D. Protein sequences of the RNA recognition motif (RRM) domains were aligned with ClustalW 2.0.10 at EMBL European Bioinformatics Institute. The amino acid sequences within conserved RRMs containing four anti-parallel β-strands, and two α-helices were used to build the phylogenetic tree. The Cluster algorithm using matrix Blosum62 was performed at GeneBee Molecular Biology Server. The bootstrap analysis was carried out with 100 replicates. We have previously reported that gene amplification and its associated genetic alterations in oncogene CoAA deregulate CoAA pre-mRNA alternative splicing and potentially impact stem cell differentiation (27.Yang Z. Sui Y. Xiong S. Liour S.S. Phillips A.C. Ko L. Nucleic Acids Res. 2007; 35: 1919-1932Crossref PubMed Scopus (26) Google Scholar, 28.Sui Y. Yang Z. Xiong S. Zhang L. Blanchard K.L. Peiper S.C. Dynan W.S. Tuan D. Ko L. Oncogene. 2007; 26: 822-835Crossref PubMed Scopus (23) Google Scholar). At the 5′ boundary of all identified amplicons, CoAA (RBM14), RBM4, and RBM4B coregulator genes are consecutively aligned and are conserved in mammals. Supported by the NCBI EST data base (supplemental Fig. 1) as well as a genome-wide study (14.Akiva P. Toporik A. Edelheit S. Peretz Y. Diber A. Shemesh R. Novik A. Sorek R. Genome Res. 2006; 16: 30-36Crossref PubMed Scopus (219) Google Scholar), there are trans-splicing events between CoAA and its downstream RBM4 gene, which is ∼10 kilobases distal to CoAA. To identify these trans-splicing variants, total RNA from HeLa cells was extracted. RT-PCR analysis spanning both CoAA and RBM4 followed by sequencing analysis confirmed the presence of two trans-splicing events. As shown in Fig. 1A, CoAA contains three exons, and RBM4 contains four exons. In addition to the alternatively spliced transcripts corresponding to the CoAA and CoAM transcripts we previously reported (25.Iwasaki T. Chin W.W. Ko L. J. Biol. Chem. 2001; 276: 33375-33383Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 27.Yang Z. Sui Y. Xiong S. Liour S.S. Phillips A.C. Ko L. Nucleic Acids Res. 2007; 35: 1919-1932Crossref PubMed Scopus (26) Google Scholar), we identified two additional transcripts, joining the CoAA first exon and the RBM4 second or third exons. Further 5′-RACE and 3′-RACE analyses were performed, and the two full-length trans-spliced cDNAs were cloned and designated as CoAZ (coactivator with zinc finger) and ncCoAZ (non-coding CoAZ). Both CoAZ and ncCoAZ share identical transcripts with CoAA at the 5′-untranslated region. The 3′-untranslated region of CoAZ and ncCoAZ is identical to that of the RBM4 transcript, indicating that they utilize the same polyadenylation signals. RACE analyses specific to ncCoAZ utilized primers in the RBM4 second exon (not shown). RACE analyses specific to CoAZ utilized splicing junction primers to enhance specificity (Fig. 1B). The results suggested that CoAZ is an in-frame fusion transcript that encodes a protein of 339 amino acids containing the first RRM domain of CoAA and a CCHC retroviral type zinc finger-containing C terminus of RBM4 (Fig. 1C). In contrast, the ncCoAZ transcript encodes a premature stop codon by joining the CoAA first exon and the RBM4 s exon. Thus, ncCoAZ does not yield a protein product that was confirmed by further analysis. In addition to their identification in human cells including HeLa and 293 cells, CoAZ and ncCoAZ were also identified in the mouse P19 stem cells we used in this study. Human and mouse CoAZ have significant identity in their primary sequences including a highly homologous RRM domain and an identical CCHC zinc finger (Fig. 1C). These results suggested that CoAZ and ncCoAZ were trans-splicing products of the CoAA and RBM4 genes with potentially conserved protein function in CoAZ. The donor and acceptor sites for the trans-splicing events in CoAZ and ncCoAZ are separated by ∼20 kilobases of sequence in both human and mouse. The possibility that CoAZ and ncCoAZ are produced by trans-splicing events from two independent transcripts rather than by cis-splicing events of a single long transcript is supported by the RT-PCR analyses using primers on different exons of CoAA and RBM4 (supplemental Fig. 2). To further confirm the trans-splicing events, we constructed the minigenes of CoAA and RBM4 and tested them in cells. As shown in Fig. 2A, each minigene contained shortened intron regions to facilitate the subsequent transfection. Both CoAA and RBM4 minigenes were driven by either a CMV promoter or their native promoter sequences. Our rationale was that trans-splicing events would be detected in cells between two independent minigenes if the wild type genes were naturally trans-spliced. Without trans-splicing events, PCR products will not be detected as the DNA templates are on two separated plasmids. The CoAA minigenes were previously described to retain alternative splicing capacities (27.Yang Z. Sui Y. Xiong S. Liour S.S. Phillips A.C. Ko L. Nucleic Acids Res. 2007; 35: 1919-1932Crossref PubMed Scopus (26) Google Scholar). Accordingly, RBM4 minigenes with either the native promoter or CMV promoter were similarly constructed. A SpeI restriction site was introduced into the RBM4 third exon to distinguish the minigene transcripts from the endogenous transcripts by restriction digestion. The RBM4 minigenes were first evaluated for their splicing capacities using RT-PCR after transfection of 293 cells. The size of PCR products indicated that all three RBM4 introns were correctly spliced out, although intron 1 splicing (494 bp) was more efficient (Fig. 2B). The splicing events of RBM4 intron 2 (1117 bp) and intron 3 (884 bp) were present but less efficient, leaving most of the products unspliced. The PCR products in the vector control represented the endogenous spliced transcripts. The gel-purified spliced transcripts were further digested by SpeI restriction enzyme. Our data clearly indicated the presence of digested fragments with the expected sizes that could only be derived from the minigenes (Fig. 2B and supplemental Fig. 3). The endogenous transcripts were not digestible. Because the minigenes retained splicing capacity, we then tested the trans-splicing events by co-transfection of both CoAA and RBM4 minigenes. RT-PCR analyses using primers at the CoAA first exon and RBM4 third exon were performed. The results dem
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