3′ Splice Site Sequences of Spinal Muscular Atrophy Related SMN2 Pre-mRNA Include Enhancers for Nearby Exons

Spinal muscular atrophy (SMA) is a human genetic disease which occurs because of the deletion or mutation of SMN1 gene. SMN1 gene encodes the SMN protein which plays a key role in spliceosome assembly. Although human patients contain SMN2, a duplicate of SMN1, splicing of SMN2 produces predominantly exon 7 skipped isoform. In order to understand the functions of splice site sequences on exon 7 and 8, we analyzed the effects of conserved splice site sequences on exon 7 skipping of SMN2 and SMN1 pre-mRNA. We show here that conserved 5′ splice site sequence of exon 7 promoted splicing of nearby exons and subsequently reduced splicing of distant exons. However, to our surprise, conserved 3′ splice site sequence of exon 7 and 8 did not promote splicing of nearby exons. By contrast, the mutation inhibited splicing of nearby exons and subsequently promoted splicing of distant exons. Our study shows that 3′ splice sites of exon 7 and 8 contain enhancer for their splice site selection, in addition to providing cleavage sites

The molecular basis underlying T cell specificity towards citrullinated epitopes presented by HLA-DR4

CD4+ T cells recognising citrullinated self-epitopes presented by HLA-DRB1 bearing the shared susceptibility epitope (SE) are implicated in rheumatoid arthritis (RA). However, the underlying T cell receptor (TCR) determinants of epitope specificity towards distinct citrullinated peptide antigens, including vimentin-64cit59-71 and α-enolase-15cit10-22 remain unclear. Using HLA-DR4-tetramers, we examine the T cell repertoire in HLA-DR4 transgenic mice and observe biased TRAV6 TCR gene usage across these two citrullinated epitopes which matches with TCR bias previously observed towards the fibrinogen β−74cit69-81 epitope. Moreover, shared TRAV26-1 gene usage is evident in four α-enolase-15cit10-22 reactive T cells in three human samples. Crystal structures of mouse TRAV6+ and human TRAV26-1+ TCR-HLA-DR4 complexes presenting vimentin-64cit59-71 and α-enolase-15cit10-22, respectively, show three-way interactions between the TCR, SE, citrulline, and the basis for the biased selection of TRAV genes. Position 2 of the citrullinated epitope is a key determinant underpinning TCR specificity. Accordingly, we provide a molecular basis of TCR specificity towards citrullinated epitopes

PSF contacts exon 7 of SMN2 pre-mRNA to promote exon 7 inclusion

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease and a leading cause of infant mortality. Deletions or mutations in SMN1, a gene that encodes an SMN protein, which regulates assembly/disassembly of U snRNP and is suggested to direct axonal transport of beta-actin mRNA in neurons, are responsible for most cases of SMA disease. However, humans contain a second SMN gene called SMN2. Unlike SMN1, the majority of SMN2 mRNA don\u27t include exon 7. Here we show that increased expression of PSF significantly promotes inclusion of exon 7 in the SMN2 and SMN1 mRNA, whereas reduced expression of PSF promotes exon 7 skipping in various cell lines and in fibroblast cells from SMA patients. In addition, we present evidence showing that PSF interacts with the GAAGGA enhancer in exon 7. We also demonstrate that a mutation in this enhancer abolishes the effects of PSF on exon 7 splicing. Furthermore we show that the RNA target sequences of PSF and tra2beta in exon 7 are partially overlapped. These results lead us to conclude that PSF interacts with an enhancer in exon 7 to promote exon 7 splicing of SMN2 pre-mRNA

Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8

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 pro-apoptotic 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 SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26nt RNA from 5\u27 end of exon 8, a 33nt RNA from 3\u27 end of exon 10 and a 2225nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 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

SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene

The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ron△165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induces a decrease in the splicing of both introns 10 and 11; by contrast, overexpression of SRSF2 induces an increase in the splicing of introns 10 and 11. Through mutation analysis, we show that SRSF2 functionally targets and physically interacts with CGAG sequence on exon 11. In addition, we reveal that the weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed