A study on alternative splicing regulation of RALY and SRSF4

Abstract

Part I. Identification of RALY as a new alternative splicing regulator RALY (hnRNP protein that is associated with lethal yellow) is a member of hnRNP (heterologous nuclear ribonucleoprotein) family, the RNA-binding proteins with heterogeneous RNA binding motifs. RALY is overexpressed in several cancer types. RALY was shown to regulate transcription by targeting poly U sequences. It was demonstrated that RALY exists in the spliceosome complex, however, its role in RNA splicing are not understood yet. Here, I applied RNA-seq and RASL-seq technology to show that RALY globally regulate splicing. Whereas RNA-seq demonstrated regulation of 2,779 skipped exon, 241 alternative 5` splice site, 318 alternative 3` splice site, 300 mutually exclusive exons and 511 retained intron, RASL-seq showed regulations of 650 cassette exon, 24 alternative 5` splice site, 19 alternative 3` splice site, 14 multi-exon skipping, 4 mutually exclusive exon, 6 alternative transcription initiation, 5 alternative transcription termination and 1 alternative 5` cassette exon by RALY. Gene ontology analysis shows that the RALY-regulated alternative splicing events are mainly enriched in the functions of RNA splicing, mRNA processing, mRNA metabolic process and apoptosis. More over the alternative splicing events by RALY is not correlated with and transcription events by RALY. In conclusion, RALY is a regulating protein of global alternative splicing.

Part II. Binding of SRSF4 to a novel enhancer modulates splicing of exon 6 of Fas pre-mRNA Alternative splicing of exon 6 in Fas pre-mRNA generates a membrane bound pro-apoptotic isoform or soluble anti-apoptotic isoform. SRSF4 is a member of Arginine-Serine rich (SR) protein family. Here we demonstrate that increased SRSF4 expression stimulates exon 6 inclusion, and that reduced SRSF4 expression promotes exon 6 exclusion. We also show that weaker but not stronger 5’ splice-site strength of exon 6 abolishes the SRSF4 effects on exon 6 splicing. In addition, SRSF4 stimulates intron 5 but not intron 6 splicing in the alternative exon 6 splicing. Furthermore, we identified a novel enhancer on exon 6, on which SRSF4 interacts functionally and physically. Our results illustrate a novel regulatory mechanism of Fas pre-mRNA splicing.