U2 snRNP auxiliary factor 65 kDa (U2AF 65 ) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF 65 stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5′ splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF 65 . U2AF 65 overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF 65 inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF 65 effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF 65 even inhibits general splicing of adenovirus major late (Ad ML) or β-globin pre-mRNA. Thus, we conclude that U2AF 65 possesses a splicing Inhibitory function that leads to alternative exon skipping.U2AF 65 | pre-mRNA splicing | splicing inhibition | exon exclusion | SMN P re-mRNA splicing is a process in which noncoding intron sequences are removed and exon sequences are then ligated together (1, 2). Pre-mRNA splicing is carried out by spliceosome, a large RNA-protein complex that contains five small nuclear ribonucleoproteins (U snRNPs) and more than 100 additional proteins (3). Pre-mRNA splicing occurs in the consensus sequences at the 5′ splice-site, 3′ splice-site, and branch point that are necessary for splicing. The sequence between 3′ AG dinucleotide and branch point is the polypyrimidine (Py) tract that directs spliceosome assembly on the 3′ splice-site. Alternative splicing provides an important regulatory mechanism in higher eukaryotes for multiple proteins produced from a single gene (4, 5).The U2 snRNP auxiliary factor 65 kDa (U2AF 65 ) exists as a heterodimer with U2AF 35 (6). U2AF 65 contains three C-terminal RNA recognition motifs (RRMs) and an N-terminal arginine/ serine-rich (RS) domain (7,8). Using U2AF 65 depletion/adding back technology with in vitro HeLa nuclear extract, it was demonstrated that U2AF 65 is an essential splicing factor (9). Whereas U2AF 65 binds to Py tract to promote prespliceosome assembly and branchpoint/U2 snRNA base pairing, U2AF 35 plays a role in the 3′ splice-site (10, 11). As U2AF 65 prefers high C/U-rich sequences in the Py tract, a stronger interaction between U2AF 65 and Py tract promotes prespliceosome assembly (12). U2AF 65 is also essential in vertebrate development (13,14). Its expression level is related to myotonic dystrophy, cystic fibrosis, and cancers (15, 16).Proximal spinal muscular atrophy (SMA) is an autosomal recessive genetic disease (17) and a leading cause of infant mortality. The motor neurons in the anterior horn of spinal cord are severely damaged in patients with type 1 SMA, usually leading to death before age 2 y as a result of a lack of respiratory support (18,19). In patients with SMA, the SMN1 gene is deleted or mutated, whereas the SMN2 gene, a duplicate of the SMN1 gene, is included (20). SMN2 genomic DNA...

Abstract. CD44 is a transmembrane receptor for hyaluronic acid. CD44 pre-mRNA contains 19 exons, 9 of which are alternatively spliced. Among the CD44 spliced variants, the v4-7 variant, one of the v6 exon-containing isoforms that contains variable exon 4, 5, 6 and 7, confers metastatic potential to nonmetastatic cells. Splicing of CD44 and the function of CD44 isoforms are different in breast cancer cells. hnRNP A1 is a ubiquitously expressed protein with an inhibitory function in pre-mRNA splicing. We showed that CD44v6 isoform, which includes all of the v6-containing mRNA isoforms, had the highest expression level in non-metatatic breast cancer cells (MCF7) when compared to the level in metastatic breast cancer cells (MDA-MB-231) and normal breast cells (MCF10A). Furthermore we showed that hnRNP A1 knockdown regulated splicing of CD44 differently in breast cancer cells. We showed here that CD44 isoform expression is completely different in MDA-MB-231 cells than that in MCF7 and MCF10A cells, whereas MCF7 and MCF10A cells had a similar expression pattern of CD44 isoforms. RT-PCR analysis of CD44v6 showed that MCF7 and MCF10A cells predominantly expressed the c5v6v7v8v9v10c6 isoform. However, in addition to this isoform, MDA-MB-231 cells also expressed the c5v6v8v9v10c6 and c5v6c6 isoforms. We also found that knockdown of hnRNP A1 significantly reduced the expression of c5v6v7v8v9v10c6 and c5v6v8v9v10c6, and promoted the expression of c5v6c6. hnRNP A1 knockdown significantly induced cell death. In addition, hnRNP A1 knockdown induced a decrease in cell invasion in the MDA-MB-231 cells. Our results indicate that the knockdown of hnRNP A1 has a specific function on the splicing of CD44 in breast cancer cells.

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and proapoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRp20 with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRp20 promoted exon 9 skipping. Thus we conclude that SRp20 promotes exon 9 skipping. In order to understand the functional target of SRp20 on caspase-2 premRNA, we performed substitution and deletion mutagenesis on the potential SRp20 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRp20 binding site on exon 8 severely disrupted the effects of SRp20 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRp20 interacts with the potential SRp20 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26 nt RNA from 5’ end of exon 8, a 33 nt RNA from 3’ end of exon 10 and a 2225 nt RNA from intron 9 did not compromise the function of SRp20 on exon 9 splicing. Therefore we conclude that SRp20 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of Caspase-2 pre-mRNA splicing.

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Fas is a transmembrane cell surface protein recognized by Fas ligand (FasL). When FasL binds to Fas, the target cells undergo apoptosis. A soluble Fas molecule that lacks the transmembrane domain is produced from skipping of exon 6 encoding this region in alternative splicing procedure. The soluble Fas molecule has the opposite function of intact Fas molecule, protecting cells from apoptosis. Here we show that knockdown of hnRNP A1 promotes exon 6 skipping of Fas pre-mRNA, whereas overexpression of hnRNP A1 reduces exon 6 skipping. Based on the bioinformatics approach, we have hypothesized that hnRNP A1 functions through interrupting 5′ splice site selection of exon 5 by interacting with its potential binding site close to 5′ splice site of exon 5. Consistent with our hypothesis, we demonstrate that mutations of the hnRNP A1 binding site on exon 5 disrupted the effects of hnRNP A1 on exon 6 inclusion. RNA pull-down assay and then western blot analysis with hnRNP A1 antibody prove that hnRNP A1 contacts the potential binding site RNA sequence on exon 5 but not the mutant sequence. In addition, we show that the mutation of 5′ splice site on exon 5 to a less conserved sequence destructed the effects of hnRNP A1 on exon 6 inclusion. Therefore we conclude that hnRNP A1 interacts with exon 5 to promote distal exon 6 inclusion of Fas pre-mRNA. Our study reveals a novel alternative splicing mechanism of Fas pre-mRNA.