Regulation of SRp20 exon 4 splicing

SR proteins are essential splicing factors involved in the use of both constitutive and alternative exons. We previously showed that the SR proteins SRp20 and ASF/SF2 have antagonistic activities on SRp20 pre-mRNA splicing. SRp20 activates exon 4 recognition in its pre-mRNA, whereas ASF/SF2 inhibits this recognition. In experiments aimed at testing the specificity of SRp20 and ASF/SF2 for exon 4 splicing regulation, we show here that this specificity lies in the RNA binding domains of SRp20 and ASF/SF2 and not in the RS domains. Surprisingly, a deletion of 14 amino acids at the end of ASF/SF2-RBD2 converts ASF/SF2 from an inhibitor to an activator of exon 4 splicing. We found that ASF3 also inhibits exon 4 recognition, thus acting similarly to ASF/SF2, while SC35 activates a cryptic 5' splice site downstream of exon 3 and, in doing so, represses exon 4 use. In contrast, Tra2 and the SR proteins 9G8 and SRp40 do not appear to affect exon 4 splicing.     

The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation

The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.     

Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.     

Exonic splicing enhancer-dependent selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site can be rescued in a cell lacking splicing factor ASF/SF2 through activation of the phosphatidylinositol 3-kinase/Akt pathway

Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5' splice site alternatively splices to a proximal 3' splice site (at nucleotide 3225) to express L2 or to a distal 3' splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3' splice sites and have been identified as regulating alternative 3' splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3' splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3' splice site while increasing use of the distal 3' splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3' splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3' splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.     

P68 RNA helicase (DDX5) alters activity of cis- and trans-acting factors of the alternative splicing of H-Ras

Background

H-Ras pre-mRNA undergoes an alternative splicing process to render two proteins, namely p21 H-Ras and p19 H-Ras, due to either the exclusion or inclusion of the alternative intron D exon (IDX), respectively. p68 RNA helicase (p68) is known to reduce IDX inclusion.

Principal Findings

Here we show that p68 unwinds the stem-loop IDX-rasISS1 structure and prevents binding of hnRNP H to IDX-rasISS1. We also found that p68 alters the dynamic localization of SC35, a splicing factor that promotes IDX inclusion. The knockdown of hnRNP A1, FUS/TLS and hnRNP H resulted in upregulation of the expression of the gene encoding the SC35-binding protein, SFRS2IP. Finally, FUS/TLS was observed to upregulate p19 expression and to stimulate IDX inclusion, and in vivo RNAi-mediated depletion of hnRNP H decreased p19 H-Ras abundance.

Significance

Taken together, p68 is shown to be an essential player in the regulation of H-Ras expression as well as in a vital transduction signal pathway tied to cell proliferation and many cancer processes.     

SRp30c is a repressor of 3' splice site utilization

Several intron elements influence exon 7B skipping in the mammalian hnRNP A1 pre-mRNA. We have shown previously that the 38-nucleotide CE9 element located in the intron separating alternative exon 7B from exon 8 can repress the use of a downstream 3' splice site. The ability of CE9 to act on heterologous substrates, combined with the results of competition and gel shift assays, indicates that the activity of CE9 is mediated by a trans-acting factor. UV cross-linking analysis revealed the specific association of a 25-kDa nuclear protein with CE9. Using RNA affinity chromatography, we isolated a 25-kDa protein that binds to CE9 RNA. This protein corresponds to SRp30c. Consistent with a role for SRp30c in the activity of CE9, recombinant SRp30c interacts specifically with CE9 and can promote splicing repression in vitro in a CE9-dependent manner. The closest homologue of SRp30c, ASF/SF2, does not bind to CE9 and does not repress splicing even when the intronic SRp30c binding sites are replaced with high-affinity ASF/SF2 binding sites. Only the first 7 nucleotides of CE9 are sufficient for binding to SRp30c, and mutations that abolish binding also prevent repression. Our results indicate that SRp30c can function as a repressor of 3' splice site utilization and suggest that the SRp30c-CE9 interaction may contribute to the control of hnRNP A1 alternative splicing.     

Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins.

SR proteins recognize exonic splicing enhancer (ESE) elements and promote exon use, whereas certain hnRNP proteins bind to exonic splicing silencer (ESS) elements and block exon recognition. We investigated how ESS3 in HIV-1 tat exon 3 blocks splicing promoted by one SR protein (SC35) but not another (SF2/ASF). hnRNP A1 mediates silencing by binding initially to a required high-affinity site in ESS3, which then promotes further hnRNP A1 association with the upstream region of the exon. Both SC35 and SF2/ASF recognize upstream ESE motifs, but only SF2/ASF prevents secondary hnRNP A1 binding, presumably by blocking its cooperative propagation along the exon. The differential antagonism between a negative and two positive regulators exemplifies how inclusion of an alternative exon can be modulated.     

Hydrogen peroxide alters splicing of soluble guanylyl cyclase and selectively modulates expression of splicing regulators in human cancer cells

Background

Soluble guanylyl cyclase (sGC) plays a central role in nitric oxide (NO)-mediated signal transduction in the cardiovascular, nervous and gastrointestinal systems. Alternative RNA splicing has emerged as a potential mechanism to modulate sGC expression and activity. C-α1 sGC is an alternative splice form that is resistant to oxidation-induced protein degradation and demonstrates preferential subcellular distribution to the oxidized environment of endoplasmic reticulum (ER).

Methodology/Principal Findings

Here we report that splicing of C-α1 sGC can be modulated by H₂O₂ treatment in BE2 neuroblastoma and MDA-MD-468 adenocarcinoma human cells. In addition, we show that the H₂O₂ treatment of MDA-MD-468 cells selectively decreases protein levels of PTBP1 and hnRNP A2/B1 splice factors identified as potential α1 gene splicing regulators by in silico analysis. We further demonstrate that down-regulation of PTBP1 by H₂O₂ occurs at the protein level with variable regulation observed in different breast cancer cells.

Conclusions/Significance

Our data demonstrate that H₂O₂ regulates RNA splicing to induce expression of the oxidation-resistant C-α1 sGC subunit. We also report that H₂O₂ treatment selectively alters the expression of key splicing regulators. This process might play an important role in regulation of cellular adaptation to conditions of oxidative stress.     

A unique glutamic acid-lysine (EK) domain acts as a splicing inhibitor

SRrp86 is a unique member of the SR protein superfamily of splicing factors containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK) rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins as long as the entire region encompassing the RS-EK-RS domains was intact. To further investigate the function and domains of SRrp86, we generated a series of chimeric proteins by swapping the RNA recognition motif and RS domains between SRrp86 and two canonical members of the SR superfamily, ASF/SF2 and SRp75. Although domain swaps between SRrp86 and ASF/SF2 showed that the RRMs primarily determined splicing activity, swaps between SRrp86 and SRp75 demonstrated that the RS domains could also determine activity. Because SRp75 also has two RS domains but lacks the EK domain, we further investigated the role of the EK domain and found that it acts to repress splicing and splice-site selection, both in vitro and in vivo. Incubation of extracts with peptides encompassing the EK-rich region inactivated splicing and insertion of the EK region into SRp75 abolished its ability to activate splicing. Thus, the unique EK domain of SRrp86 plays a modulatory role controlling RS domain function.     

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.     

Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.     

The gene encoding the splicing factor SF2/ASF is a proto-oncogene

Alternative splicing modulates the expression of many oncogene and tumor-suppressor isoforms. We have tested whether some alternative splicing factors are involved in cancer. We found that the splicing factor SF2/ASF is upregulated in various human tumors, in part due to amplification of its gene, SFRS1. Moreover, slight overexpression of SF2/ASF is sufficient to transform immortal rodent fibroblasts, which form sarcomas in nude mice. We further show that SF2/ASF controls alternative splicing of the tumor suppressor BIN1 and the kinases MNK2 and S6K1. The resulting BIN1 isoforms lack tumor-suppressor activity; an isoform of MNK2 promotes MAP kinase-independent eIF4E phosphorylation; and an unusual oncogenic isoform of S6K1 recapitulates the transforming activity of SF2/ASF. Knockdown of either SF2/ASF or isoform-2 of S6K1 is sufficient to reverse transformation caused by the overexpression of SF2/ASF in vitro and in vivo. Thus, SF2/ASF can act as an oncoprotein and is a potential target for cancer therapy.