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Arginine/Serine-rich Protein Interaction Domain-dependent Modulation of a Tau Exon 10 Splicing Enhancer

2005; Elsevier BV; Volume: 281; Issue: 5 Linguagem: Inglês

10.1074/jbc.m505809200

ISSN

1083-351X

Autores

Ian D’Souza, Gerard D. Schellenberg,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

Tau exon 10 splicing is altered by autosomal dominant mutations that cause frontotemporal dementia with parkinsonism chromosome 17-type and by unknown mechanisms in other related neurodegenerative disorders. Identifying cis- and trans-regulators of tau exon 10 splicing is therefore crucial for understanding disease mechanisms. We previously identified several splicing enhancers and silencers within exon 10 and intron 10. Here, we show that splicing factors SF2/ASF, Tra2β, and a 50-kDa nuclear protein bind in vitro to the polypurine enhancer at the 5′ end of exon 10. Disease splicing mutations N279K and Δ280K disrupt the enhancer and alter associations with these factors. N279K targets robustly bind Tra2β compared with the normal enhancer, which may explain why N279K enhances exon 10 splicing in vivo. In contrast, factor associations with Δ280K targets are nearly undetectable, explaining why Δ280K almost abolishes exon 10 splicing in vivo. Small interfering RNA-mediated suppression of endogenous SF2/ASF and Tra2β significantly reduces exon 10 splicing. Exogenous SF2/ASF dramatically enhances normal exon 10 splicing and efficiently rescues the Δ280K splicing defect. Domain deletion analyses show that the C-terminal RS domains of SF2/ASF and Tra2β are required for normal exon 10 splicing in vivo. In contrast to Tra2β, the SF2/ASF RS domain remains essential in the presence of a strengthened enhancer or when either weak splice site is strengthened. The data suggest that SF2/ASF has both essential and regulatory roles, whereas Tra2β has a supporting role in exon 10 splicing. Tau exon 10 splicing is altered by autosomal dominant mutations that cause frontotemporal dementia with parkinsonism chromosome 17-type and by unknown mechanisms in other related neurodegenerative disorders. Identifying cis- and trans-regulators of tau exon 10 splicing is therefore crucial for understanding disease mechanisms. We previously identified several splicing enhancers and silencers within exon 10 and intron 10. Here, we show that splicing factors SF2/ASF, Tra2β, and a 50-kDa nuclear protein bind in vitro to the polypurine enhancer at the 5′ end of exon 10. Disease splicing mutations N279K and Δ280K disrupt the enhancer and alter associations with these factors. N279K targets robustly bind Tra2β compared with the normal enhancer, which may explain why N279K enhances exon 10 splicing in vivo. In contrast, factor associations with Δ280K targets are nearly undetectable, explaining why Δ280K almost abolishes exon 10 splicing in vivo. Small interfering RNA-mediated suppression of endogenous SF2/ASF and Tra2β significantly reduces exon 10 splicing. Exogenous SF2/ASF dramatically enhances normal exon 10 splicing and efficiently rescues the Δ280K splicing defect. Domain deletion analyses show that the C-terminal RS domains of SF2/ASF and Tra2β are required for normal exon 10 splicing in vivo. In contrast to Tra2β, the SF2/ASF RS domain remains essential in the presence of a strengthened enhancer or when either weak splice site is strengthened. The data suggest that SF2/ASF has both essential and regulatory roles, whereas Tra2β has a supporting role in exon 10 splicing. In the central nervous system, the microtubule-associated protein tau is highly expressed in neuronal axons and at lower levels in glial cells. Tau functions in microtubule assembly, influences microtubule stability and is important for neurogenesis, axonal maintenance, and axonal transport (1Lee V.M.Y. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2162) Google Scholar, 2Ebneth A. Godemann R. Stamer K. Illenberger S. Trinczek B. Mandelkow E.M. Mandelkow E. J. Cell Biol. 1998; 143: 777-794Crossref PubMed Scopus (668) Google Scholar). In the human tau gene (MAPT), exons 9–12 each encode a 31–32-amino acid imperfect repeat that comprises the microtubule-binding domain of tau. In adult human brain, exons 2, 3, and 10 are alternatively spliced to produce six different tau isoforms (3Andreadis A. Brown W.M. Kosik K.S. Biochemistry. 1992; 31: 10626-10633Crossref PubMed Scopus (497) Google Scholar, 4Goedert M. Wischik C.M. Crowther R.A. Walker J.E. Klug A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4051-4055Crossref PubMed Scopus (863) Google Scholar, 5Goedert M. Spillantini M.G. Rotier M.C. Ulrich J. Crowther R.A. EMBO J. 1989; 8: 393-399Crossref PubMed Scopus (846) Google Scholar). Exon 10 (E10) 2The abbreviations used are: E10, exon 10; ESE, exon splicing enhancer; FTDP-17, frontotemporal dementia with parkinsonism chromosome 17-type; I10, intron 10; PPE, polypurine enhancer; RRM, RNA recognition motif; RS, arginine/serine-rich protein interaction domain; siRNA, small interfering RNA; SR, Arg/Ser-rich splicing factors; 3R, three-repeat tau isoforms; 4R, four-repeat tau isoforms; RT, reverse transcription. inclusion generates isoforms with four microtubule repeats called four-repeat (4R) tau. When E10 is skipped, the result is 3R tau isoforms. The 4R/3R ratio in normal adult brain is ∼1. In fetal brain, only the shortest 3R tau isoform is expressed because of constitutive exclusion of exons 2, 3, and 10 (6Kosik K.S. Orecchio L.D. Bakalis S. Neve R.L. Neuron. 1989; 2: 1389-1397Abstract Full Text PDF PubMed Scopus (521) Google Scholar, 7Janke C. Beck M. Stahl T. Holzer M. Brauer K. Bigl V. Arendt T. Mol. Brain Res. 1999; 68: 119-128Crossref PubMed Scopus (78) Google Scholar). Pre-mRNA splicing regulation is complex requiring multiple interactions between small ribonucleoprotein particles and non-small ribonucleoprotein particle splicing factors with conserved and nonconserved splicing elements in the unprocessed RNA transcript (8Reed R. Curr. Opin. Cell Biol. 2000; 12: 340-345Crossref PubMed Scopus (188) Google Scholar). Conserved elements are the 3′ and 5′ splice site signals that demarcate the exonintron boundary. Nonconserved sequences that promote splicing are called exon splicing enhancers (ESE) and intron splicing enhancers (ISE) (9Blencowe B.J. Trends Biochem. Sci. 2000; 25: 106-110Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). Serine/arginine-rich (SR) proteins usually bind to enhancer elements and are essential at multiple steps in the splicing pathway (10Graveley B.R. RNA. 2000; 6: 1197-1211Crossref PubMed Scopus (884) Google Scholar). Most alternatively spliced exons contain a weak splice site, whose recognition requires SR factor associations with ESEs and ISEs (11Berget S.M. J. Biol. Chem. 1995; 270: 2411-2414Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar). Alternative splicing is also influenced by splicing inhibitory sequences called exon splicing silencers and intron splicing silencers. An additional function of some enhancers is to counteract the inhibitory effects of neighboring silencer elements (12Kan J.L.C. Green M/R. Genes Dev. 1999; 13: 462-471Crossref PubMed Scopus (122) Google Scholar, 13Guil S. Gattoni R. Carrascal M. Abian J. Stevenin J. BachElias M. Mol. Cell Biol. 2003; 23: 2927-2941Crossref PubMed Scopus (105) Google Scholar, 14D'Souza I. Schellenberg G.D. J. Biol. Chem. 2002; 277: 26587-26599Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 15Zahler A.M. Damgaard C.K. Kjems J. Caputi M. J. Biol. Chem. 2004; 279: 10077-10084Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 16Zhu J. Mayeda A. Krainer A.R. Mol. Cell. 2001; 8: 1351-1361Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 17Eperon I.C. Makarova O.V. Mayeda A. Munroe S.H. Caceres J.F. Hayward D.G. Krainer A.R. Mol. Cell Biol. 2000; 20: 8303-8318Crossref PubMed Scopus (148) Google Scholar). For MAPT, cis-regulation of E10 alternative splicing has been extensively studied, where multiple enhancers and silencers regulate use of the weak E10 3′ and 5′ splice sites (14D'Souza I. Schellenberg G.D. J. Biol. Chem. 2002; 277: 26587-26599Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 18D'Souza I. Schellenberg G.D. J. Biol. Chem. 2000; 275: 17700-17709Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). E10 sequences include three nonredundant, weak ESEs in the first half of the exon that include an SC35-like ESE, a polypurine enhancer (PPE), and an A/C-rich enhancer separated by a central exon splicing silencer from additional ESE sequences at the 3′ end of E10. Intronic sequences immediately downstream of the E10 5′ splice site include an intron splicing silencer and an adjacent intron splicing modulator, which counteracts the intron splicing silencer. Mutations in MAPT cause frontotemporal dementia with parkinsonism chromosome 17-type (FTDP-17), an autosomal dominant neurodegenerative disease (19Foster N.L. Wilhelmsen K. Sima A.A.F. Jones M.Z. D'Amato C.J. Gilman S. Ann. Neurol. 1997; 41: 706-715Crossref PubMed Scopus (603) Google Scholar, 20Poorkaj P. Bird T.D. Wijsman E. Nemens E. Garruto R.M. Anderson L. Andreadis A. Wiederholt W.C. Raskind M. Schellenberg G.D. Ann. Neurol. 1998; 43: 815-825Crossref PubMed Scopus (1233) Google Scholar, 21Hutton M. Lendon C.L. Rizzu P. Baker M. Froelich S. Houlden H. Pickering-Brown S. Chakraverty S. Isaacs A. Grover A. Hackett J. Adamson J. Lincoln S. Dickson D. Davies P. Petersen R.C. Stevens M. de Graaff E. Wauters E. van Baren J. Hillebrand M. Joosse M. Kwon J.M. Nowotny P. Che L.K. Norton J. Morris J.C. Reed L.A. Trojanowski J. Basun H. Lannfelt L. Neystat M. Fahn S. Dark F. Tannenberg T. Dodd P.R. Hayward N. Kwok J.B.J. Schofield P.R. Andreadis A. Snowden J. Craufurd D. Neary D. Owen F. Oostra B.A. Hardy J. Goate A. van Swieten J. Mann D. Lynch T. Heutink P. Nature. 1998; 393: 702-705Crossref PubMed Scopus (2927) Google Scholar, 22Spillantini M.G. Murrell J.R. Goedert M. Farlow M.R. Klug A. Ghetti B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7737-7741Crossref PubMed Scopus (1315) Google Scholar). FTDP-17 belongs a group of disorders called tauopathies, in which hyperphosphorylated tau forms pathological neuronal aggregates called neurofibrillary tangles. Other tauopathies include Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, and Pick disease. Over 30 coding and intronic MAPT mutations are known that cause FTDP-17 (23D'Souza I. Schellenberg G.D. Biochim. Biophys Acta. 2005; 1739: 104-115Crossref PubMed Scopus (114) Google Scholar, 24Ingram E.M. Spillantini M.G. Trends Mol. Med. 2002; 8: 555-562Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). One class includes missense mutations that alter tau protein function. The second mutation class includes missense, silent, deletion, and intronic mutations that alter E10 splicing by disrupting either the 5′ splice site (14D'Souza I. Schellenberg G.D. J. Biol. Chem. 2002; 277: 26587-26599Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 21Hutton M. Lendon C.L. Rizzu P. Baker M. Froelich S. Houlden H. Pickering-Brown S. Chakraverty S. Isaacs A. Grover A. Hackett J. Adamson J. Lincoln S. Dickson D. Davies P. Petersen R.C. Stevens M. de Graaff E. Wauters E. van Baren J. Hillebrand M. Joosse M. Kwon J.M. Nowotny P. Che L.K. Norton J. Morris J.C. Reed L.A. Trojanowski J. Basun H. Lannfelt L. Neystat M. Fahn S. Dark F. Tannenberg T. Dodd P.R. Hayward N. Kwok J.B.J. Schofield P.R. Andreadis A. Snowden J. Craufurd D. Neary D. Owen F. Oostra B.A. Hardy J. Goate A. van Swieten J. Mann D. Lynch T. Heutink P. Nature. 1998; 393: 702-705Crossref PubMed Scopus (2927) Google Scholar, 25Grover A. Houlden H. Baker M. Adamson J. Lewis J. Prihar G. Pickering-Brown S. Duff K. Hutton M. J. Biol. Chem. 1999; 274: 15134-15143Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar), PPE, A/C-rich enhancer, and exon splicing silencer sequences in E10 (18D'Souza I. Schellenberg G.D. J. Biol. Chem. 2000; 275: 17700-17709Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 26D'Souza I. Poorkaj P. Hong M. Nochlin D. Lee V.M.Y. Bird T.D. Schellenberg G.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5598-5603Crossref PubMed Scopus (423) Google Scholar, 27Spillantini M.G. Yoshida H. Rizzini C. Lantos P.L. Khan N. Rossor M.N. Goedert M. Brown J. Ann. Neurol. 2000; 48: 939-943Crossref PubMed Scopus (127) Google Scholar, 28Grover A. Deture M. Yen S.H. Hutton M. Neurosci. Lett. 2002; 323: 33-36Crossref PubMed Scopus (45) Google Scholar, 29Yoshida H. Crowther R.A. Goedert M. J. Neurochem. 2002; 80: 548-551Crossref PubMed Scopus (48) Google Scholar) or the intron splicing silencer and intron splicing modulator sequences in intron 10 (I10) (14D'Souza I. Schellenberg G.D. J. Biol. Chem. 2002; 277: 26587-26599Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 30Stanford P.M. Shepherd C.E. Halliday G.M. Brooks W.S. Schofield P.W. Brodaty H. Martins R.N. Kwok J.B.J. Schofield P.R. Brain. 2003; 126: 814-826Crossref PubMed Scopus (109) Google Scholar). Most splicing mutations increase 4R tau isoforms and alter the 4R/3R ratio from 1 to 2–3. However, mutations Δ280K and E10+19, which decrease E10 inclusion in splicing assays, are expected to reduce the 4R/3R ratio to as low as 0.33. Silent and intronic mutations do not change the protein sequence of tau but affect E10 splicing and ultimately tau function. Thus, subtle changes in the ratio of normal tau isoforms cause severe neurodegeneration. Identifying proteins that associate with cis-elements is necessary to elucidate how E10 splicing is regulated. Here we focus on identifying protein interactions with the E10 PPE, a 9-nucleotide purine-rich ESE sequence between E10 positions +16 and +24 (see Fig. 1A). The PPE sequence contains two copies of an AAG motif and one copy of a GAR (where R is a purine) motif that occur frequently in natural, purine-rich ESEs. These motifs are often high affinity binding sites for SR and SR-related proteins. FTDP-17 mutations N279K and Δ280K alter the normal PPE sequence by adding or removing an AAG copy, respectively (see Fig. 1A). Consequently these mutations have opposite effects on E10 splicing, where N279K increases and Δ280K decreases E10 inclusion. Our earlier work predicted that the normal PPE function requires SR factor interactions, which are altered by disease mutations Δ280K and N279K (26D'Souza I. Poorkaj P. Hong M. Nochlin D. Lee V.M.Y. Bird T.D. Schellenberg G.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5598-5603Crossref PubMed Scopus (423) Google Scholar). Indeed, the SR-like splicing factor Tra2β is essential for E10 inclusion in splicing assays and associates in vitro with both normal and mutant PPE templates (31Jiang Z.H. Tang H. Havlioglu N. Zhang X.C. Stamm S. Yan R.Q. Wu J.Y. J. Biol. Chem. 2003; 278: 18997-19007Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Here we show specific in vitro associations of a trio of factors with the normal and strengthened (mutation N279K) PPE but not with a disrupted PPE (mutation Δ280K) and identify two of these proteins as the SR domain-containing factors SF2/ASF and Tra2β. We corroborate their specific effects on E10 splicing by gene knock-down assays and present in vivo mechanisms for their varied effects on E10 splicing. Overexpression of SF2/ASF rescues the splicing defect in mutation Δ280K. Domain deletion assays in non-neuronal and neuronal cells show that the C-terminal protein-interacting domains of both SF2/ASF and Tra2β are required for E10 splicing. Our data reveal PPE-dependent and PPE-independent roles for SF2/ASF for use of the weak 3′ and 5′ splice sites, respectively. Thus, SF2/ASF has complex essential and regulatory roles on E10 splicing, whereas the role of Tra2β appears secondary to SF2/ASF. Plasmid Construction and DNA Mutagenesis—Splicing vector hN has been described (18D'Souza I. Schellenberg G.D. J. Biol. Chem. 2000; 275: 17700-17709Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and contains the entire 93-bp MAPT E10 sequence with 33 and 51 bp of flanking introns (see Fig. 3A). Minigene construct E10AH (see Fig. 2) contains entire MAPT exons 9, 10, and 11 as well as 324 and 559 bp of intron 9 and I10 sequences, respectively, inserted into expression vector pRcRSV (Invitrogen). Intron 9 in E10AH contains 224 bp of sequence immediately 5′ of E9 and 100 bp of sequence immediately 3′ of E10. I10 in E10AH contains 100 bp of sequence immediately 5′ of E10 and 459 bp of sequence immediately 5′ of E11. FTDP-17 mutations Δ280K and N279K were introduced into hN (18D'Souza I. Schellenberg G.D. J. Biol. Chem. 2000; 275: 17700-17709Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and E10AH by PCR mutagenesis.FIGURE 2Knock-down effects of SF2/ASF and Tra2β on E10 splicing. A, immunoblots of HeLa cells treated with increasing concentrations of siR-NAs show reduction of endogenous SF2/ASF (si-SF2/ASF, left panel) and Tra2β (si-Tra2β, middle panel) compared with β-tubulin expression. The siRNA concentrations used per lane are 25, 50, 75, and 100 nmol for si-SF2/ASF and 50, 75, and 100 nmol for si-Tra2β. si-SF2/ASF and si-Tra2β show gene specificity (right panel, lanes 3 and 4). The control siRNA (siCONTROL nontargeting siRNA number 1; Dharmacon, Inc.) lacks identity to known gene targets and controls for possible nonspecific effects of siRNA treatment. si-SF2/ASF and si-Tra2β cotreatment (50 nmol each) decreases the levels of both factors (right panel, lane 5) less efficiently than treatments with 100 nmol of individual siRNAs. Monoclonal antibodies αSF2/ASF and E7 (β-tubulin) and the polyclonal antibody Rb3505 (Tra2β) were used for detection. B, E10AH minigene splicing construct containing E10 separated from flanking tau exons E9 and E11 by 0.35 and 0.55 kilobase pairs of total intronic sequences, respectively. C, bar graph showing E10+ levels from triplicate transfections. The error bars represent the standard deviations. A corrected significance criteria of p < 0.0125 was used, and p values comparing E10 inclusion in siRNA-treated samples to control samples within the same cell line are represented by symbols: ‡, p < 1 × 10–3; δ, p < 1 × 10–4; and †, p < 1 × 10–5. D, autoradiograph of E10+ and E10– products from representative RT-PCRs obtained by transient transfection of minigene splicing vector E10AH in mock or siCON-TROL-treated HeLa and PC12 cells (left panel) as well as in individual or combined (si-SF2/ASF + si-Tra2β) gene-specific siRNA-treated HeLa (middle panel) and PC12 cells (right panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT) SF2/ASF, Tra2β, and hnRNP G Expression Constructs—SF2/ASF cDNA was amplified by RT-PCR from HeLa RNA using primers ASF5′F (5′-AGCTGGATCCATGTCGGGAGGTGGTG-3′) and ASF3′R (5′-CGATCTCGAGTTATGTACGAGAGCGAGATCTGC-3′). The PCR product was digested at BamHI and XhoI sites in the primer sequences and inserted into vector pcDNA3.1/Zeo+ (Invitrogen) to generate expression construct SF2/ASF. The Tra2β expression vector contains a cDNA sequence obtained by PCR amplification from vector Htra2-β1-V5 (32Hofmann Y. Wirth B. Hum. Mol. Genet. 2002; 11: 2037-2049Crossref PubMed Google Scholar) using primers Tra2b5′F (5′-AGCTGGATCCATGAGCGACAGCGGCGGCAG-3′) and Tra2b3′R (5′-CGATCTCGAGTTAATAGCGACGAGGTGAGTATGATCG-3′). The BamHI/XhoI-digested product was inserted into pcDNA3.1/Zeo+. We generated domain deletion constructs by PCR mutagenesis of full-length SF2/ASF and Tra2β expression vectors (see Fig. 4A). Primers used to delete the N-terminal RRM domains from SF2/ASF (construct SF2/ASF-ΔRRM) were SF2dRRMF (5′-AGCTGGATCCATGCGTGAAGCAGGTGATGTA-3′) and SF23′R. For RS domain deletion construct SF2/ASF-ΔRS, the primer pair SF25′F and SF2dRS (5′-GCGCTCGAGTTATGGGCCCATCAACTTTAACC-3′) was used. The N-terminal RS domain in construct Tra2β-ΔRS1 (see Fig. 4A) was deleted using primers Tra2dRS1 (5′-CGCGGATCCATGCATGTTGGGAATCGGGCAAATCC-3′) and Tra2b3′R. The central RRM domain in Tra2β-ΔRRM (see Fig. 4A) was deleted using primers Tra2b5′F and Tra2b3′R with expression construct pCMVhtl1 + 5′ as template (kind gift from William Mattox). In Tra2β-ΔRS2 (see Fig. 4A) the C-terminal RS domain was deleted using primers Tra2b5′F and Tra2bdRS2 (5′-GCGCTCGAGTTATCCTGGTGTTGGCGTATGTGG-3′). Construct hnRNP-G-V5 is described elsewhere (32Hofmann Y. Wirth B. Hum. Mol. Genet. 2002; 11: 2037-2049Crossref PubMed Google Scholar). Cell Culture, Transfections, and RNA Isolation—Maintenance and transfection of PC12 cells is described elsewhere (14D'Souza I. Schellenberg G.D. J. Biol. Chem. 2002; 277: 26587-26599Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen). All of the transfections were performed in triplicate with Lipfectamine (Invitrogen) in six-well plates. HeLa cells were transfected with 2 μg of plasmid DNA and 6 μl of Lipofectamine in 1 ml of OptiMEM (Invitrogen) for 5 h at 37 °C (5% CO2), after which 1 ml of Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum was added. For coexpression experiments in PC12 cells, 2.5 μg of total plasmid containing 0.5 μgof an E10 splicing vector and 2 μg (4-fold excess) of either pSKII control vector (Stratagene) or individual splicing factor expression constructs were used with 6 μl of Lipofectamine. Total RNA was isolated with TRIzol reagent (Invitrogen) as previously described (26D'Souza I. Poorkaj P. Hong M. Nochlin D. Lee V.M.Y. Bird T.D. Schellenberg G.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5598-5603Crossref PubMed Scopus (423) Google Scholar). siRNA Synthesis and Transfection—Commercially synthesized (Dharmacon Inc.) siCONTROL nontargeting siRNA number 1 and gene-specific siRNA pools were used for endogenous suppression of SF2/ASF and Tra2β. siRNA transfections were optimized in 24-well plates containing cells at 70% confluency using Oligofectamine (Invitrogen) with 25, 50, 75, and 100 nmol of each siRNA in the pool (see Fig. 2A). Maximal inhibition was observed with 100 nmol of each siRNA. Protein was harvested 48–72 h post-transfection, and expression of targeted factors was tested by immunoblot analyses (described below). To test the effects of siRNA treatment on E10 splicing vectors, the cells were seeded into 12-well plates, and siRNA transfections were scaled up. After 24 h, the cells were rinsed in phosphate-buffered saline and fed 450 μl of antibiotic-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. One μg of normal or mutant splicing vector E10AH was mixed with 3 μl of Lipofectamine in 50 μlof OptiMEM and added to each well. Total RNA was isolated 48 h later for RT-PCR analyses with TRIzol reagent (Invitrogen). Quantitation of Tau E10 Splicing by RT-PCR—Tau E10 splicing from transiently transfected splicing vectors was analyzed by RT-PCR assays as described previously (26D'Souza I. Poorkaj P. Hong M. Nochlin D. Lee V.M.Y. Bird T.D. Schellenberg G.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5598-5603Crossref PubMed Scopus (423) Google Scholar). E10+ and E10– transcripts were amplified by RT-PCR using vector-specific primer pairs SD6/SA2 for hN and pREP/BGHPA2 for E10AH as previously reported (18D'Souza I. Schellenberg G.D. J. Biol. Chem. 2000; 275: 17700-17709Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 33Poorkaj P. Kas A. D'Souza I. Zhou Y. Pham O. Olson M.V. Schellenberg G.D. Mamm. Genome. 2001; 12: 700-712Crossref PubMed Scopus (57) Google Scholar). Amplified products from hN are 261 bp (E10–) and 354 bp (E10+) and from E10AH are 447 bp (E10–) and 540 bp (E10+). The products were resolved on 5–7.5% acrylamide gels and quantitated using a Molecular Imager® system (Bio-Rad). For each mutant construct, the values presented are the averages of at least three different transfection experiments with the normal E10 construct transfected in parallel. Statistical comparisons were made using a two-tailed Student's t test. Criteria for significance were calculated using a Bonferroni correction for multiple comparisons. In Vitro RNA Synthesis—A primer-based strategy was used to in vitro synthesize short, radiolabeled RNAs containing normal or mutant PPE sequences. Forward and reverse primers were designed such that on annealing they form a double-stranded T7 promoter region upstream of the RNA target to be transcribed. A universal forward primer, T7F3 (5′-CGCTCTAGATAATACGACTCACTATAGGGAGTAT-3′) containing the core T7 promoter sequence (italics) was used. Nine additional nucleotides upstream of the T7 promoter sequence maximize transcription efficiency, and five additional nucleotides downstream of the promoter serve as a linker that overlaps with the RNA target. The reverse primer contains the reverse complement of the core T7 promoter (underlined) with tau sequences (italics) that include the normal or mutant PPE element (bold type). The reverse primers used were: T7PPE (5′-GCCTCCTGGATCCAGCTTCTTATTATACTCCCTATAGTGAGTCGTATTAGCG-3′), T7279K (5′-GCCTCCTGGATCCAGCTTCTTCTTATACTCCCTATAGTGAGTCGTATTAGCG-3′), and T7Δ280K (5′-GCCTCCTGGATCCAGCTTATTATACTCCCTATAGTGAGTCGTATTAGCG-3′). To anneal universal and reverse primers, 100 ng of each in 0.1 m NaCl was heated to 94 °C for 3 min and cooled at room temperature for 10 min. For in vitro transcription, 1 μlof the annealed primer reaction was added to 19 μl of a transcription mixture containing 1× T7 polymerase buffer (Invitrogen), 20 mm dithiothreitol, 20 units of RNasin (Promega), 5 μm [32P]αATP or [32P]αGTP (800 Ci/mmol), 0.5 mm each of the remaining three NTPs and 50 units of T7 RNA polymerase. The reactions were incubated at 31 °C for 2 h, heat-denatured at 75 °C for 2 min, quenched on ice for 2 min, and treated with 10 units of DNase I at 37 °C for 20 min to remove primers. 7 μl of formamide loading dye was added to samples, heated at 95 °C for 5 min, and resolved on 12% urea-PAGE gels. Full-length transcripts were visualized by autoradiography, excised, and eluted in 400 μl of elution mix (0.5 m ammonium acetate, 2.5 mm EDTA, 0.5% SDS) for 1 h at room temperature. The eluate was ethanol-precipitated and resuspended in 100 μl of RNase-free H2O, and 1 μl was used for quantitation in a scintillation counter. HeLa Nuclear Extract Preparation, UV Cross-linking, and Affinity Purification—HeLa nuclear extracts were prepared as described (34Dignam JD Lebovitz RM Roeder RG Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). The cell pellets were obtained from HeLa-S3 spinner cultures grown at the National Cell Culture Center, a research resource facility established by the National Center of Resources for Research, National Institutes of Health. For UV cross-linking, radiolabeled (30–100 × 105 cpm) gel-purified, full-length RNA targets were incubated with HeLa nuclear extract in 10-μl reactions under splicing-permissive conditions (0.6 mm ATP, 0.1 m creatinine phosphate, 1.5 mm MgCl2, 50–75 μg of HeLa extract, 4 μg each of tRNA and heparin, and 2 μlof10× binding buffer containing 20 mm HEPES (pH 7.9), 1 m potassium glutamate, 0.2 mm EDTA, 0.5 mm dithiothreitol, and 20% glycerol). The reactions were incubated at 30 °C for 30 min, exposed to UV light (0.2 J for 12 min) at a distance of 4 cm on ice to form covalent RNA-protein complexes before treating with RNase A to remove unbound RNA sequences. 11 μlof2× SDS protein loading buffer was added to the samples before boiling for 5 min. The bound proteins were resolved on 10–15% SDS-PAGE gels and visualized by autoradiography. For RNA affinity purification, 5′-Ome-modified normal PPE and mutant (Δ280K,279K) RNA target sequences were commercially synthesized (Dharmacon Inc.) and are identical in sequence to in vitro transcribed templates used in UV cross-linking assays. RNA-agarose affinity columns were prepared according to methods described before (35Caputi M. Mayeda A. Krainer A.R. Zahler A.M. EMBO J. 1999; 18: 4060-4067Crossref PubMed Scopus (226) Google Scholar) and incubated with 200 μl of HeLa nuclear extract under splicing conditions at 30 °C for 20 min to allow binding of splicing factors. Protein-RNA-agarose complexes were collected without exposure to UV light and washed extensively in Dignam Buffer D before eluting bound factors in 2× SDS gel loading buffer. The samples were boiled for 5 min, resolved on 10–12% SDS-PAGE gels, and electroblotted for immunodetection assays. Immunoblotting—Monoclonal antibody mAb104 (1:100) was used to detect the SR family of splicing factors that range in size from 20 to 85 kDa (36Zahler A.M. Lane W.S. Stolk J.A. Roth M.B. Genes Dev. 1992; 6: 837-847Crossref PubMed Scopus (626) Google Scholar). SF2/ASF was detected by a monoclonal antibody (1:5000) (37Hanamura A. Caceres J.F. Mayeda A. Franza Jr., B.R. Krainer A.R. RNA. 1998; 4: 430-444PubMed Google Scholar). Tra2β was detected using a polyclonal antibody Rb3505 (1:1500) raised against a synthetic peptide containing the N-terminal 15 amino acids (MSDSGEQNYGERESR) (Invitrogen Antibody Services). Anti-β-tubulin monoclonal antibody E7 was obtained from the NICHD Developmental Studies Hybridoma Bank maintained by The University of Iowa (Department of Biological Sciences, Iowa City, IA) and was used at dilution 1:3000 as an internal protein control. Rabbit secondary antibodies (Jackson ImmunoResearch) used at 1:2500 dilution were anti-mouse IgM for mAb104 and anti-mouse IgG for both SF2/ASF and β-tubulin. Chemiluminescent immunodetection was achieved with horseradish peroxidase conjugated to protein A (Sigma) at a dilution of 1:3000 and the ECL plus detection system (Amersham Biosciences). The following experiments were designed to identify the trans-acting proteins that recognize the normal PPE, to determine whether this protein-RNA interaction is di

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