The RNA-Binding Protein TIA-1 Is a Novel Mammalian Splicing Regulator Acting through Intron Sequences Adjacent to a 5′ Splice Site

Abstract

Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependent on the U-rich sequence IAS1 lying immediately downstream from its 5 splice site. We show that IAS1 can activate the use of several heterologous 5 splice sites in vitro. Addition of the RNA-binding protein TIA-1 to splicing extracts preferentially enhances the use of 5 splice sites linked to IAS1. TIA-1 can provoke a switch to use of such sites on pre-mRNAs with competing 5 splice sites, only one of which is adjacent to IAS1. Using a combination of UV cross-linking and specific immunoprecipitation steps, we show that TIA-1 binds to IAS1 in cell extracts. This binding is stronger if IAS1 is adjacent to a 5 splice site and is U1 snRNP dependent. Overexpression of TIA-1 in cultured cells activates K-SAM exon splicing in an IAS1-dependent manner. If IAS1 is replaced with a bacteriophage MS2 operator, splicing of the K-SAM exon can no longer be activated by TIA-1. Splicing can, however, be activated by a TIA-1-MS2 coat protein fusion, provided that the operator is close to the 5 splice site. Our results identify TIA-1 as a novel splicing regulator, which acts by binding to intron sequences immediately downstream from a 5 splice site in a U1 snRNP-dependent fashion. TIA-1 is distantly related to the yeast U1 snRNP protein Nam8p, and the functional similarities between the two proteins are discussed.Many eucaryotic genes are made up of exons and introns (43). They are transcribed into pre-mRNAs, from which the intron sequences are removed by splicing. Exons to be included in mRNA must be identified as such. This involves interaction of short sequences at or close to the exon's 5Ј and 3Ј splice sites (5Јss and 3Јss, respectively) with spliceosome components such as snRNPs and associated proteins (for reviews, see references 4, 29, and 43). Exon splicing can be controlled, and several sequences which participate in the control of tissue-specific or developmentally controlled alternative splicing events have been described (for a review, see reference 32). These sequences are particularly interesting to study, as they may yield information on both splicing activation mechanisms and tissuespecific control mechanisms of gene expression. We have been studying fibroblast growth factor receptor 2 (FGFR-2) premRNA splicing for this reason.FGFR-2 alternative exons K-SAM and BEK are spliced in a tissue-specific, mutually exclusive manner, and the two types of FGFR-2 obtained bind different subsets of FGF family members (38). The K-SAM exon is under complex control. It has weak splice sites, and it contains an exon splicing silencer (ESS) which functions by recruiting hnRNP A1 (13). To overcome the activity of this silencer, at least three activating sequences in the downstream intron are required (6,10,12). One of these, IAS1, lies immediately downstream of the 5Јss and is a U-rich sequence (10). In the absence of IAS1 (10), or if IAS1 is moved further downstream from the 5Јss (F. Del GattoKonczak, unpublished data), the K-S...

Predicting the sequence specificities of DNA- and RNA-binding proteins by deep learning

“…For example, exons known to be downregulated by Nova had higher Nova scores in their upstream introns, and exons known to be upregulated by Nova had higher Nova scores in their downstream intron 48 . Similarly, TIA has been shown to upregulate exons when bound to the downstream intron 49 , and PTBP1 has been shown to suppress exon inclusion when bound to upstream introns of weaker splice sites 50 .…”

Modulation of msl-2 5′ splice site recognition by Sex-lethal

“…Inhibition of TIA-1 crosslinking to msl-2 59 splice site region+ A: Radioactively labeled RNAs corresponding to msl-2 or 5SBM 59 splice site region were incubated with HeLa nuclear extracts (NE) in the presence of the indicated concentrations of GST-SXL, the mixtures irradiated with 254 nm UV light and TIA-1 immunoprecipitated using specific antibodies+ The immunoprecipitates were then fractionated by electrophoresis on SDS-denaturing polyacrylamide gels, and TIA-1 crosslinking quantified by Phosphorimager analysis+ The positions of TIA-1 and of residual amounts of crosslinked GST-SXL present in the TIA-1 immunoprecipitates are indicated+ B: Quantification of four independent experiments carried out as described in A+ Filled circles indicate crosslinking of TIA-1 to msl-2 RNA; empty circles indicate crosslinking of TIA-1 to 5SBM RNA+ Standard deviations are indicated+ C: msl-2 or msl-2 5SBM were incubated with GST-TIA1 (6+6 ng/mL) in the absence or in the presence of GST-SXL at the indicated concentrations, the mixtures irradiated with 254 nm UV light, fractionated by electrophoresis on SDS-denaturing polyacrylamide gels, and analyzed by autoradiography+ The positions of the recombinant proteins are indicated+ The difference in TIA-1 displacement between msl-2 and msl-2 5SBM appears to be quantitatively more pronounced in the reconstituted system than in nuclear extracts, most likely due to stabilizing effects of TIA-1 binding by other components of the nuclear extract+ of the splice site+ For instance, in both yeast and higher eukaryotic pre-mRNAs, the presence of uridine-rich sequences downstream from the 59 splice site makes U1 snRNP binding responsive to a family of related proteins that includes Nam8 and TIA-1 (Puig et al+, 1999;del Gatto-Konczak et al+, 2000;Förch et al+, 2000)+ Recruitment of Nam8 by the regulatory factor Mer1p facilitates U1 snRNP binding and splicing activation during meiosis in Saccharomyces cerevisiae (Spingola & Ares, 2000)+ Conversely, the strong dependence of the rather weak 59 splice site of msl-2 on TIA-1 for U1 snRNP binding and splicing (Förch et al+, 2000) can make blockage of TIA-1 binding by SXL an efficient mechanism to prevent U1 snRNP recruitment and significantly contribute to splicing inhibition+ We have observed, however, that SXL can also reduce U1 snRNA crosslinking to a msl-2 mutant RNA in which substitution of uridine at intronic position 5 by the consensus guanosine decreases the dependence of the 59 splice site on TIA-1 (Förch et al+, 2000; data not shown)+ These results argue that SXL can also block other interactions that U1 snRNP makes with the 59 splice site region+ Although SXL can repress a 39 splice site in tra premRNA by binding exclusively to its associated Py-tract, SXL appears to target both splice sites to regulate msl-2 splicing+ One difference between the regulatory outcome of these two RNAs is that although SXL induces a switch in 39 splice site choice in tra, it causes intron retention in msl-2 transcripts+ It seems likely that retention of an intron requires more stringent control of splice site utilization than a switch between two alternative 39 splice sites, where repression of one of the competing sites may suffice to achieve regulation+…”

“…Factors that regulate pre-mRNA splicing affect primarily the efficiency of E complex formation (reviewed by Blencowe, 2000;Graveley, 2000;Smith & Valcárcel, 2000)+ One example is SXL, which blocks U2AF binding to the Py-tracts associated with a 39 splice site in transformer (tra;Valcárcel et al+, 1993;Granadino et al+, 1997) and in msl-2 pre-mRNAs (Merendino et al+, 1999)+ Another example is the protein TIA-1 (Tian et al+, 1991), which promotes U1 snRNP binding to weak 59 splice sites followed by U-rich sequences (del Gatto-Konczak et al+, 2000;Förch et al+, 2000)+ Here we have investigated the molecular mechanisms causing retention of msl-2 59 UTR intron in vitro using HeLa nuclear extracts+ The results indicate that, in addition to the uridine-rich Py-tract at the 39 splice site region, a second stretch rich in uridines, immediately downstream from the 59 splice site, is required for efficient inhibition by SXL+ SXL binding to this sequence blocks binding of the factor TIA-1 and of U1 snRNP to the 59 splice site+ RESULTS AND DISCUSSION SXL binding at both ends of msl-2 intron is required for efficient splicing inhibition…”

The sequence complementarity between HIV-1 5′ splice site SD4 and U1 snRNA determines the steady-state level of an unstable env pre-mRNA

“…Most eukaryotic primary transcripts contain introns that have to be removed prior to their nucleocytoplasmic export to ensure translation of a continuous open reading frame (ORF)+ Splicing takes place in two catalytic steps within a large multicomponent complex, the spliceosome (for reviews, see Moore et al+, 1993;Burge et al+, 1998;Reed, 2000)+ One of the earliest steps in spliceosome assembly is the duplex formation between the free 59 end of the U1 snRNA and the 59 splice site+ The RNA duplex is not only stabilized through its hydrogen bonding but almost certainly through additional interactions of protein components with the pre-mRNA also in the vicinity of the 59 splice site (Puig et al+, 1999;Zhang & Rosbash, 1999;Del Gatto-Konczak et al+, 2000)+ Beside its role in the splicing reaction, the integrity of a 59 splice site is important for nuclear premRNA stability as has been shown by point mutations within the 59 splice site of, for example, the HIV-1 tat/ rev intron (Lu et al+, 1990) or polyoma virus late transcripts (Barrett et al+, 1995) leading to a decrease in the accumulation of unspliced transcripts+ Moreover, binding of snRNPs leads to nuclear retention of the RNA and it is generally recognized that completion of the splicing reaction removes this obstacle to RNA nucleocytoplasmic export (Chang & Sharp, 1989;Legrain & Rosbash, 1989;Hamm & Mattaj, 1990;Huang & Carmichael, 1996)+ There remains a conceptual problem of how the nucleocytoplasmic export of unspliced RNA equipped with suitable snRNP binding sites occurs+ This is the case for the nucleocytoplasmic export of a variety of retroand pararetrovirus mRNAs (Cullen, 1992;Kiss-Laszlo & Hohn, 1996;Bodem et al+, 1997)+ The overlapping of their genes entails that one and the same sequence may be part of an intron or of an exon, depending on which gene is being expressed+ To enable the use of their full genomic potential, the excision of introns has to be restrained by regulatory processes, for instance, through inefficient 39 splice sites (Katz & Skalka, 1990;Fu et al+, 1991;McNally & Beemon, 1992;Staffa & Cochrane, 1994;Dyhr-Mikkelsen & Kjems, 1995;O'Reilly et al+, 1995;Zhang & Stoltzfus, 1995;Si et al+, 1997) or cis-acting splicing enhance...…”