Alternative splicing and transcriptional regulatory mechanism of SF3B1, SRSF2 and SRSF6

Abstract

Part I. Alternative splicing and transcriptional regulatory mechanism of SF3B1 Among the proteins constituting the spliceosome, SF3B1 (Splicing factor 3B subunit 1) is most frequently mutated in various cancers, myelodysplastic syndrome, and chronic lymphocytic leukemia, resulting in abnormal alternative splicing. While the function of SF3B1 in splicing and changes in alternative splicing (AS) due to its mutation are well known, the AS regulatory function of wild-type SF3B1 is not fully understood. Here, RNA-sequencing (RNA-seq) was performed on SF3B1 knockdown (KD) cells to analyze changes in genome-wide AS events, and through this, changes in numerous skipped exons, some changes in alternative 5' splice site, alternative 3' splice site, mutually exclusive exons, and retained intron events were identified. Among them, changes in survival motor neuron (SMN) exon 7 splicing were confirmed when SF3B1 was KD or overexpressed in HCT116, SH-SY5Y, HEK293T, and SMA patient cells. In addition, the interaction with 65 kDa U2 snRNA auxiliary factor (U2AF65) was shown to be important in the function of controlling the AS of SMN. Mutations in SMN exon 7 polypyrimidine tract (PPT) that regulate affinity with U2AF65 demonstrated the importance of the interaction between U2AF65 and PPT in the function of SF3B1. SF3B1 interacts RNA-independently with the nucleosome. In addition to AS, RNA-seq results suggest that the expression levels of numerous genes were altered by SF3B1 KD. Among them, I found that the expression of methyltransferase-like 3 (METTL3), a writer of N6-methyladenosine (m6A), was significantly decreased by SF3B1 KD, leading to a decrease in m6A-marked mRNA. I performed actinomycin D-mediated nascent RNA analysis to demonstrate that reduced METTL3 expression by SF3B1 KD was not mediated by mRNA decay but transcription. Co-immunoprecipitation (Co-IP) analysis indicated that SF3B1 could directly interact with RNA polymerase II (RNAPII) in an RNA-independent manner, and further modulate RNAPII occupancy on the METTL3 gene. In addition, histone modification on the METTL3 promoter was also altered by SF3B1 KD. The above results indicate that SF3B1 plays important roles in AS and transcription.

Part II. Relative strength of 5' splice-site strength defines functions of SRSF2 and SRSF6 in alternative splicing of Bcl-x pre-mRNA Bcl-x, a member of the Bcl-2 family, plays a key role in apoptosis. Alternative splicing of Bcl-x pre-mRNA through alternative 5' splice-site selection produces an anti-apoptotic mRNA isoform that includes exon 2b and a pro-apoptotic Bcl-x mRNA isoform that excludes exon 2b. Here I used Bcl-x minigene and identified Serine/arginine-rich splicing factor 2 (SRSF2) and Serine/arginine-rich splicing factor 6 (SRSF6) as two regulatory factors of 5' splice-site selection of Bcl-x pre-mRNA. I selected binding clusters closer to 5' splice-sites from multiple potential binding sites of SRSF2 and SRSF6 to perform loss of functions analysis through site-directed mutagenesis. My results demonstrated that these mutations did not abolish regulatory functions of SRSF2 or SRSF6, indicating that a single binding motif or a cluster was not a functional target of these proteins in Bcl-x pre-mRNA splicing. Random deletion mutagenesis did not disrupt the role of SRSF2 and SRSF6. Importantly, mutagenesis of 5' splice-site to a conserved or a weaker score demonstrated that the weaker strength of the target 5' splice-site or higher strength of the other 5' splice-site strength limited the role of SRSF2 and SRSF6 in 5' splice-site activation.