hnRNP A1 Recruited to an Exon In Vivo Can Function as an Exon Splicing Silencer

Abstract

Some exons contain exon splicing silencers. Their activity is frequently balanced by that of splicing enhancers, and this is important to ensure correct relative levels of alternatively spliced mRNAs. Using an immunoprecipitation and UV-cross-linking assay, we show that RNA molecules containing splicing silencers from the human immunodeficiency virus type 1 tat exon 2 or the human fibroblast growth factor receptor 2 K-SAM exon bind to hnRNP A1 in HeLa cell nuclear extracts better than the corresponding RNA molecule without a silencer. Two different point mutations which abolish the K-SAM exon splicing silencer's activity reduce hnRNP A1 binding twofold. Recruitment of hnRNP A1 in the form of a fusion with bacteriophage MS2 coat protein to a K-SAM exon whose exon splicing silencer has been replaced by a coat binding site efficiently represses splicing of the exon in vivo. Recruitment of only the glycine-rich C-terminal domain of hnRNP A1, which is capable of interactions with other proteins, is sufficient to repress exon splicing. Our results show that hnRNP A1 can function to repress splicing, and they suggest that at least some exon splicing silencers could work by recruiting hnRNP A1.Many eucaryotes make extensive use of alternative splicing to create more than one version of a protein from a single transcription unit. Alternative splicing can be controlled in a cell-type-specific fashion, allowing different cell types to make those versions of a protein best adapted to their particular needs. Such control acts on competing splice sites and can involve activation or repression.Two interesting cases of splicing activation involve construction of multiprotein complexes on the pre-mRNAs. In Drosophila, activation of splicing of a female-specific dsx exon requires assembly on the exon of a complex including the femalespecific protein tra, tra-2, and SR proteins (32, 33). Neuronspecific activation of splicing of the mouse c-src exon N1 is achieved by assembly on downstream intron sequences of a multiprotein complex including the protein KSRP (39). In vitro, KSRP induces the assembly of five other proteins, including hnRNP F, on the intronic splicing enhancer (38). Other exonic splicing enhancers have also been shown to interact with SR proteins (30,34,50,55). SR proteins are known to engage in protein-protein contacts important for splicing (34). Splicing activation thus often involves installation of multiprotein complexes on pre-mRNA sites in such a manner as to allow them to interact productively with spliceosome components.Intron sequences involved in splicing repression have been described for several systems. In Drosophila, the female-specific sxl protein represses use of a male-specific 3Ј splice site on the tra pre-mRNA by binding to the associated polypyrimidine sequence and blocking binding of U2AF (51). sxl blocks splicing of a male-specific sxl exon by binding to multiple pyrimidine-rich sites in the flanking introns (28). Splicing of some exons is repressed by binding of polypyrimidine tract binding pr...

Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA

“…In transcript C3, the second exon (positions 5359 -5408) contained the identified cis inhibitory element ESS2p that binds hnRNP H (11). However, the downstream ESS2 inhibitory element (7-9), which binds hnRNP A1 (14,16), and the recently described ESE2 activator element (41) were not present in this transcript (Fig. 1A).…”

“…(iii) Their accessibility is limited by their sequestering in stable secondary structures (24). (iv) Several cis-inhibitory elements have been identified that down-regulate the A2, A3, and A7 sites by binding of hnRNP A1 or hnRNP H proteins (11,12,14,16,18,19,21). Despite these numerous handicaps, the utilization of several of the HIV-1 acceptor sites is essential for virus multiplication.…”

“…The combination of these various sites gives rise to at least 35 different mRNAs (1). Although the relative efficiencies of the HIV-1 donor sites seem to depend mainly upon their complementarity to the U1 snRNA 5Ј-terminal sequence (3,4), efficiencies of HIV-1 acceptor sites depend upon the presence of cis-regulatory elements (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Several studies have shown that HIV-1 acceptor sites are suboptimal as follows.…”

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulatesSMN2gene expression in patient fibroblasts

“…To determine whether the increase in exon 7 inclusion was a specific consequence of attaching the GGA repeat sequence to SMN2 exon 7, we tested other tailed oligonucleotides that lacked ESE sequences but would anneal to the same target site. In these oligonucleotides (called 5ЈPTB and 5ЈA1), the ESE sequences were replaced with sequences that bind to inhibitors of splicing such as PTB or hnRNP A1, respectively (24)(25)(26)(27). In contrast to the 5ЈGGA oligonucleotide, both these oligonucleotides and the NT oligonucleotide demonstrated that annealing at the 5Ј end of SMN2 exon 7 was insufficient to stimulate incorporation (Fig.…”

Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged

“…Two hnRNP proteins frequently overexpressed in cancer-hnRNP A1 and PTB-have been implicated in silencing of the epithelial-specific IIIb exon, while hnRNP H/F proteins can contribute to IIIc silencing (Del Gatto-Konczak et al 1999;Carstens et al 2000;Mauger et al 2008). There is currently no strong evidence to suggest that changes in the levels of these hnRNP proteins provide an important means of regulating switching from exon IIIb to IIIc.…”