Mutations in Tau Gene Exon 10 Associated with FTDP-17 Alter the Activity of an Exonic Splicing Enhancer to Interact with Tra2β
2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês
10.1074/jbc.m301800200
ISSN1083-351X
AutoresZhi‐Hong Jiang, Hao Tang, Necat Havlioglu, Xiaochun Zhang, Stefan Stamm, Riqiang Yan, Jane Y. Wu,
Tópico(s)Signaling Pathways in Disease
ResumoMutations in the human tau gene leading to aberrant splicing have been identified in FTDP-17, an autosomal dominant hereditary neurodegenerative disorder. Molecular mechanisms by which such mutations cause tau aberrant splicing were not understood. We characterized two mutations in exon 10 of the tau gene, N279K and Del280K. Our results revealed an exonic splicing enhancer element located in exon 10. The activity of this AG-rich splicing enhancer was altered by N279K and Del280K mutations. This exonic enhancer element interacts with human Tra2β protein. The interaction between Tra2β and the exonic splicing enhancer correlates with the activity of this enhancer element in stimulating splicing. Biochemical studies including in vitro splicing and RNA interference experiments in transfected cells support a role for Tra2β protein in regulating alternative splicing of human tau gene. Our results implicate the human tau gene as a target gene for the alternative splicing regulator Tra2β, suggesting that Tra2β may play a role in aberrant tau exon 10 alternative splicing and in the pathogenesis of tauopathies. Mutations in the human tau gene leading to aberrant splicing have been identified in FTDP-17, an autosomal dominant hereditary neurodegenerative disorder. Molecular mechanisms by which such mutations cause tau aberrant splicing were not understood. We characterized two mutations in exon 10 of the tau gene, N279K and Del280K. Our results revealed an exonic splicing enhancer element located in exon 10. The activity of this AG-rich splicing enhancer was altered by N279K and Del280K mutations. This exonic enhancer element interacts with human Tra2β protein. The interaction between Tra2β and the exonic splicing enhancer correlates with the activity of this enhancer element in stimulating splicing. Biochemical studies including in vitro splicing and RNA interference experiments in transfected cells support a role for Tra2β protein in regulating alternative splicing of human tau gene. Our results implicate the human tau gene as a target gene for the alternative splicing regulator Tra2β, suggesting that Tra2β may play a role in aberrant tau exon 10 alternative splicing and in the pathogenesis of tauopathies. Microtubule-associated protein tau plays an important role in microtubule assembly and stabilization (1Cleveland D.W. Hwo S.Y. Kirschner M.W. J. Mol. Biol. 1977; 116: 227-247Crossref PubMed Scopus (635) Google Scholar, 2Drubin D.G. Kirschner M.W. J. Cell Biol. 1986; 103: 2739-2746Crossref PubMed Scopus (584) Google Scholar, 3Kanai Y. Takemura R. Oshima T. Mori H. Ihara Y. Yanagisawa M. Masaki T. Hirokawa N. J. Cell Biol. 1989; 109: 1173-1184Crossref PubMed Scopus (297) Google Scholar, 4Kanai Y. Chen J. Hirokawa N. 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These observations suggest that a proper ratio of Tau4R/3R is essential for normal function of Tau in the human brain. It remains unclear how alterations in Tau4R/3R ratio lead to neuronal degeneration and, in some cases, also glial dysfunction and death. However, the discovery of these splicing mutations in the tau gene indicates the critical importance of controlling the balance of different Tau isoforms by alternative splicing for normal brain function. We are interested in understanding molecular mechanisms underlying tau alternative splicing regulation. Our previous work established a tau minigene system (17Jiang Z. Cote J. Kwon J.M. Goate A.M. Wu J.Y. Mol. Cell. Biol. 2000; 20: 4036-4048Crossref PubMed Scopus (98) Google Scholar). Many of the tau intronic mutations described so far are near the splice donor site (5′ splice site) of the intron following exon 10. Our biochemical studies support the model that some intronic mutations near the 5′ splice site following exon 10 destabilize a stem-loop structure and lead to enhanced U1 snRNP 1The abbreviations used are: snRNP, small nuclear ribonucleoprotein; Ad, adenovirus; RT, reverse transcriptase; WT, wild type; RNAi, RNA interference; ESE, exonic splicing enhancer. interaction with the 5′ splice site of exon 10 and thus an increase in splicing between exon 10 and exon 11 and an increase in the ratio of Tau4R to Tau3R (15Hutton 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. Heutink P. et al.Nature. 1998; 393: 702-705Crossref PubMed Scopus (2927) Google Scholar, 17Jiang Z. Cote J. Kwon J.M. Goate A.M. Wu J.Y. Mol. Cell. 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In this study, we used in vitro biochemical assays to dissect the cis-elements and trans-splicing factors involved in the regulation of tau Exon 10 inclusion. Our results have revealed an exonic splicing enhancer element located in exon 10. The activity of this AG-rich splicing enhancer is affected by N279K and Del280K mutations. Proteins interacting with this AG-rich sequence were analyzed using UV cross-linking and immunoprecipitation assays. A 40-kDa protein containing a RNA-binding domain and a domain rich in serine and arginine (the SR domain), Tra2β, has been identified among the proteins interacting with this exonic enhancer element. The interaction of Tra2β with the exonic splicing enhancer correlates with the activity of this enhancer element in stimulating tau exon 10 splicing. Finally, down-regulation of Tra2β expression by RNA interference in transfected cells led to a reduction in tau exon 10 inclusion. These observations suggest that human Tra2β may act as an important regulator for facilitating exon 10 inclusion in tau splicing. Plasmid, Oligonucleotides, and Antibodies—The tau genomic DNA fragments containing exons 9–11 as well as intronic sequences flanking exon 10 were amplified by PCR from the normal adult human or FTDP-17 patient brain genomic DNA to make wild-type or mutant tau constructs. Tau minigene constructs were made by inserting the genomic fragments into a mammalian expression vector pcDNA3 (Invitrogen) between HindIII and XhoI sites under the control of the cytomegalovirus promoter (17Jiang Z. Cote J. Kwon J.M. Goate A.M. Wu J.Y. Mol. Cell. Biol. 2000; 20: 4036-4048Crossref PubMed Scopus (98) Google Scholar). Tau minigene plasmids with N279K and Del280K mutation were made by site-directed mutagenesis with specific oligonucleotides. TauEx10–11d5(dEn) was made by deletion of sequence GTG CAG ATA ATT AAT AAG AAG C as indicated by the thick line in Fig. 1A from TauEx10–11d5, and TauEn-IchEx9–10(WT, N279K, Del280K) were made by inserting this sequence upstream of the caspase-2 exon 9–10 splicing unit (44Cote J. Dupuis S. Jiang Z. Wu J.Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 938-943Crossref PubMed Scopus (52) Google Scholar). Ad-TauEx10d5(WT, N279K) were made by replacing the upstream exon 9 and associated intronic sequences with the first exon region (L1) of the adenovirus (Ad) major late transcription unit. DNA sequence analysis of tau genomic fragments and different expression plasmids was carried out on an ABI 373A automatic sequencer using a PRISM Ready reaction DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems). Oligonucleotides were purchased from Dharmacon and IDT Integrated DNA Technologies, Inc. Anti-SR monoclonal antibody 1H4 was purchased from ATCC. A polyclonal antibody specific for human Tra2β was as described (67Beil B. Screaton G. Stamm S. DNA Cell Biol. 1997; 16: 679-690Crossref PubMed Scopus (69) Google Scholar). Transfections and RT-PCR—HeLaRB, HEK293, and N2a cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and were seeded at 2 × 105 cells/well in a six-well dish 24 h prior to transfection. Transfection was done using a standard calcium phosphate precipitation procedure with 1–3 μg of DNA as described (70Jiang Z.H. Zhang W.J. Rao Y. Wu J.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9155-9160Crossref PubMed Scopus (129) Google Scholar). Transfection efficiency was routinely around 60%, as evaluated by cotransfection of a green fluorescent protein-expressing plasmid. Cells were harvested 48 h after transfection, and the RNA was extracted using Trizol reagent (Invitrogen). Splicing products derived from the expressed minigenes were detected using RT-PCR as previously described (70Jiang Z.H. Zhang W.J. Rao Y. Wu J.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9155-9160Crossref PubMed Scopus (129) Google Scholar). cDNAs were also prepared from the frontal cortex region in human brain tissues obtained at autopsy. RT-PCR was also performed using primers specific for human Tra2β to detect the expression of this gene. In Vitro Splicing Assays—Splicing substrates were synthesized using T7 RNA polymerase (Promega). Final concentrations of different reagents were as follows in a 10-μl reaction: 500 ng of linearized DNA template, 0.4 mm ATP and CTP, 0.1 mm GTP and UTP, 0.84 mm GpppG cap analogue (Amersham Biosciences), 10 mm dithiothreitol, 0.5 units/μl RNasin (Promega), 1× transcription buffer (Promega), 20 μCi of [α-32P]UTP, and 1 unit/μl RNA polymerase. Samples were then treated with 0.1 unit/μl DNase I (Promega) for 15 min and ethanol-precipitated. The full-length transcripts were gel-purified. Synthesis of cold competitor RNAs was in a scaled-up 100-μl reaction with the following modifications: 0.5 mm ATP, 0.5 mm CTP, 0.5 mm GTP, 0.5 mm UTP, 0.42 mm cap analogue, and a trace amount of [α-32P]UTP for quantification. HeLa cell nuclear extracts were prepared according to previously established protocols and contained 20 mg/ml total proteins (71Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Splicing reactions were set up and processed as previously (72Krainer A.R. Maniatis T. Cell. 1985; 42: 725-736Abstract Full Text PDF PubMed Scopus (247) Google Scholar) except that some batches of nuclear extracts were supplemented with 1 unit of creatine kinase (Sigma). 2–3 fmol of RNA substrates were added, and the samples were incubated at 30 °C for 2 h unless otherwise mentioned. Splicing products were resolved on 8% polyacrylamide, 8 m urea gels. The identity of lariat molecules was determined by performing a debranching reaction in a S100 extract (73Ruskin B. Krainer A.R. Maniatis T. Green M.R. Cell. 1984; 38: 317-331Abstract Full Text PDF PubMed Scopus (470) Google Scholar), followed by gel migration alongside molecular weight markers. The intensity of the products was quantified by the PhosphorImager. In some splicing assays, purified SR protein or U2AF65 or cell lysates containing overexpressed Tra2β or SRp40 was added. UV Cross-linking and Immunoprecipitation Assays—Splicing reactions were set up as described above except using 20 fmol of RNA substrates. 10-μl aliquots were transferred onto a 96-well microtiter plate and irradiated with 1 J in UV Stratalinker 1800 (Stratagene). For site-specific cross-linking, a specific label was put inside the second AAG sequence following a protocol previously described (74Lapham J. Yu Y.T. Shu M.D. Steitz J.A. Crothers D.M. RNA. 1997; 3: 950-951PubMed Google Scholar, 75Yu Y.T. Methods. 1999; 18: 13-21Crossref PubMed Scopus (22) Google Scholar). Various amounts of competitive transcripts were added to demonstrate the specifically bound proteins. Samples were then treated for 30 min at 37 °C with equal volume of RNase A (5 mg/ml). Radiolabeled cross-linked proteins were boiled for 5 min in 1× SDS-PAGE loading buffer and separated on 12.5% SDS-PAGE. For immunoprecipitation, following the RNase treatment, samples were incubated with 1H4 anti-SR monoclonal antibody (ATCC). Protein A/G-agarose beads were then added with further incubation and gentle rocking. The RNA-labeled proteins retained on the beads after several washings were eluted and resolved on SDS-PAGE. Site-specific Label Incorporation and Cross-linking—Human tau exon 10 DNA with N279K mutation was in vitro transcribed into RNA with trace-labeled [α-32P]UTP. 40 pmol of synthesized RNA was annealed with 60 pmol of chimeric oligonucleotide (3′-(UAUUAAU)2′-O-methyl-TCTT(CUUC)2′-O-methyl-5′) and cleaved with RNase H. The 3′-half RNA was treated with calf intestinal phosphatase and labeled with [γ-32P]ATP. The 5′-half RNA was ligated with 3′-half RNA using a DNA ligation kit (Roche Applied Science) by bridge oligonucleotide (5′-CTGGACGTTGCTAAGATCCAGCTTCTTCTTAATTATCTGCACCTTTGG-3′). 30,000 cpm of RNA was incubated with 20–40 μg of HeLa cell nuclear extracts for cross-linking. Various amounts (0, 4, and 40 pmol) of competitor oligonucleotide (5′-GTGCAGATAATTAAGAAGAAGCTGGATC-3′) corresponding to the enhancer element were added. RNA-Protein Interaction Assays—40 fmol of biotin-conjugated RNAs corresponding to TauEx10–11dEn, TauEx10–11Del280K, TauEx10–11wt, and TauEx10–11N279K were incubated for 30 min on ice with various amounts of cell lysates prepared from HEK293 cells transiently transfected with a Tra2β-Myc expression construct. After affinity selection with streptavidin-agarose beads (Sigma), the beads were washed four times in cold phosphate-buffered saline with 75 mm NaCl and 0.1% Nonidet P-40. Proteins associated with the biotinylated RNAs were dissolved in sample buffer and analyzed by Western blotting using 9E10 anti-Myc monoclonal antibody (Babco). U snRNP Blocking Assays—Partial inhibition of U snRNAs was achieved by incubation with 2′-O-methyl oligoribonucleotides as described (17Jiang Z. Cote J. Kwon J.M. Goate A.M. Wu J.Y. Mol. Cell. Biol. 2000; 20: 4036-4048Crossref PubMed Scopus (98) Google Scholar). 2′-O-methyl oligonucleotides (U1, 8 μm; U2, 0.3 μm; U5, 12 μm; U6, 13 μm) were added to splicing reactions and incubated at 30 °C for 10 min prior to the addition of RNA substrates. Incubations were then carried on for 1.5 h. Splicing products were resolved on 8% denaturing gels. U1 snRNP RNase H Protection Assay—The TauEx10–11 RNA substrate (3 fmol) was added to a 12.5 μl of splicing reaction containing mock- or U1 snRNP-partially depleted HeLa cell cytoplasmic extracts S100 (53Black D.L. RNA. 1995; 1: 763-771PubMed Google Scholar) and incubated for 15 min at 30 °C. Oligonucleotides (20 pmol) directed against exon 10 5′ splice sites (17Jiang Z. Cote J. Kwon J.M. Goate A.M. Wu J.Y. Mol. Cell. Biol. 2000; 20: 4036-4048Crossref PubMed Scopus (98) Google Scholar) were then added along with 0.2 units of RNase H (Invitrogen), and the incubation continued for 10 min at 37 °C. Resulting RNA fragments were resolved on 6% polyacrylamide, 8 m urea gels (76Eperon I.C. Ireland D.C. Smith R.A. Mayeda A. Krainer A.R. EMBO J. 1993; 12: 3607-3617Crossref PubMed Scopus (169) Google Scholar, 77Chabot B. Blanchette M. Lapierre I. La Branche H. Mol. Cell. Biol. 1997; 17: 1776-1786Crossref PubMed Scopus (111) Google Scholar). SR proteins were prepared as described (78Zahler A.M. Lane W.S. Stolk J.A. Roth M.B. Genes Dev. 1992; 6: 837-847Crossref PubMed Scopus (626) Google Scholar). Tra2β protein-containing cell lysates were prepared from HEK293 cells transfected with Tra2β-Myc expression plasmid. RNA Interference Experiments—An RNA interference assay was carried out in HEK293 cells as described (79Elbashir S.M. Lendeckel W. Tuschl T. Genes Dev. 2001; 15: 188-200Crossref PubMed Scopus (2712) Google Scholar). The oligonucleotides were custom-synthesized by Dharmacon (Boulder, CO). An Exonic Splicing Enhancer Located in Exon 10 of the Human Tau Pre-mRNA—The N279K mutation in exon 10 of the tau gene was reported to affect exon 10 splicing (Fig. 1A) (20Clark L.N. Poorkaj P. Wszolek Z. Geschwind D.H. Nasreddine Z.S. Miller B. Li D. Payami H. Awert F. Markopoulou K. Andreadis A. D'Souza I. Lee V.M. Reed L. Trojanowski J.Q. Zhukareva V. Bird T. Schellenberg G. Wilhelmsen K.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13103-13107Crossref PubMed Scopus (450) Google Scholar, 27D'Souza I. Poorkaj P. Hong M. Nochlin D. Lee V.M. Bird T.D. Schellenberg G.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5598-5603Crossref PubMed Scopus (423) Google Scholar). It was proposed that a T to G change in the N279K mutation might improve an exonic splicing enhancer because of the sequence change from AATAAGAAG to AAGAAGAAG. Consistent with the hypothesis that this AG-rich region acts as a splicing enhancer, is the discovery of the Del280K mutation that changes the sequence from AATAAGAAG to AATAAG in FTDP-17 patients. In these pat
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