Bcl-2-associated transcription factor 1 (BCLAF1) is known to be involved in multiple biological processes. Although several splice variants of BCLAF1 have been identified, little is known about how BCLAF1 splicing is regulated or the contribution of alternative splicing to its developmental functions. Here we find that inclusion of alternative exon5a was significantly increased in colorectal cancer (CRC) samples. Knockdown of the BCLAF1 protein isoform resulting from exon5a inclusion inhibited growth and that its overexpression increased tumorigenic potential. We also found that the splicing factor SRSF10 stimulates inclusion of exon5a and has growth-inducing activity. Importantly, the upregulation of SRSF10 expression observed in clinical CRC samples parallels the increased inclusion of BCLAF1 exon5a, both of which are associated with higher tumour grade. These findings identify SRSF10 as a key regulator of BCLAF1 pre-mRNA splicing and the maintenance of oncogenic features in human colon cancer cells.

Aberrant RNA splicing is recognized to contribute to cancer pathogenesis, but the underlying mechanisms remain mainly obscure. Here, we report that the splicing factor SRSF2 is upregulated frequently in human hepatocellular carcinoma (HCC), where this event is associated with poor prognosis in patients. RNA-seq and other molecular analyses were used to identify SRSF2-regulated alternative splicing events. SRSF2 binding within an alternative exon was associated with its inclusion in the RNA, whereas SRSF2 binding in a flanking constitutive exon was associated with exclusion of the alternative exon. Notably, cancer-associated splice variants upregulated by SRSF2 in clinical specimens of HCC were found to be crucial for pathogenesis and progression in hepatoma cells, where SRSF2 expression increased cell proliferation and tumorigenic potential by controlling expression of these variants. Our findings identify SRSF2 as a key regulator of RNA splicing dysregulation in cancer, with possible clinical implications as a candidate prognostic factor in patients with HCC. Cancer Res; 77(5); 1168–78. ©2017 AACR.

Splicing factor SRSF10 is known to function as a sequence-specific splicing activator. Here, we used RNA-seq coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Motif analysis revealed that SRSF10 binding to cassette exons was associated with exon inclusion, whereas the binding of SRSF10 within downstream constitutive exons was associated with exon exclusion. This positional effect was further demonstrated by the mutagenesis of potential SRSF10 binding motifs in two minigene constructs. Functionally, many of SRSF10-verified alternative exons are linked to pathways of stress and apoptosis. Consistent with this observation, cells depleted of SRSF10 expression were far more susceptible to endoplasmic reticulum stress-induced apoptosis than control cells. Importantly, reconstituted SRSF10 in knockout cells recovered wild-type splicing patterns and considerably rescued the stress-related defects. Together, our results provide mechanistic insight into SRSF10-regulated alternative splicing events in vivo and demonstrate that SRSF10 plays a crucial role in cell survival under stress conditions.

Dysregulated alternative splicing events have been implicated in many types of cancer, but the underlying molecular mechanisms remain unclear. Here, we observe that the splicing factor SRSF1 regulates DBF4B exon6 splicing by specifically binding and promoting its inclusion. Knockdown of the exon6-containing isoform (DBF4B-FL) significantly inhibits the tumorigenic potential of colon cancer cells in vitro and in mice, and SRSF1 inactivation phenocopies DBF4B-FL depletion. DBF4B-FL and SRSF1 are required for cancer cell proliferation and for the maintenance of genomic stability. Overexpression of DBF4B-FL can protect against DNA damage induced by SRSF1 knockdown and rescues growth defects in SRSF1-depleted cells. Increased DBF4B exon6 inclusion parallels SRSF1 upregulation in clinical colorectal cancer samples. Taken together, our findings identify SRSF1 as a key regulator of DBF4B pre-mRNA splicing dysregulation in colon cancer, with possible clinical implications as candidate prognostic factors in cancer patients.

During adipocyte differentiation, significant alternative splicing changes occur in association with the adipogenic process. However, little is known about roles played by splicing factors in this process. We observed that mice deficient for the splicing factor SRSF10 exhibit severely impaired development of subcutaneous white adipose tissue (WAT) as a result of defects in adipogenic differentiation. To identify splicing events responsible for this, transcriptome sequencing (RNA-seq) analysis was performed using embryonic fibroblast cells. Several SRSF10-affected splicing events that are implicated in adipogenesis have been identified. Notably, lipin1, known as an important regulator during adipogenesis, was further investigated. While lipin1β is mainly involved in lipogenesis, its alternatively spliced isoform lipin1α, generated through the skipping of exon 7, is primarily required for initial adipocyte differentiation. Skipping of exon 7 is controlled by an SRSF10-regulated cis element located in the constitutive exon 8. The activity of this element depends on the binding of SRSF10 and correlates with the relative abundance of lipin1α mRNA. A series of experiments demonstrated that SRSF10 controls the production of lipin1α and thus promotes adipocyte differentiation. Indeed, lipin1α expression could rescue SRSF10-mediated adipogenic defects. Taken together, our results identify SRSF10 as an essential regulator for adipocyte differentiation and also provide new insights into splicing control by SRSF10 in lipin1 pre-mRNA splicing.

RNA-sequencing (RNA-seq) can generate millions of reads to provide clues for analyzing novel or abnormal alternative splicing (AS) events in cells. However, current methods for exploring AS events are still far from being satisfactory. Here, we present Comprehensive AS Hunting (CASH), which constructs comprehensive splice sites including known and novel AS sites in cells, and identifies differentially AS events between cells. We illuminated the versatility of CASH on RNA-seq data from a wide range of species and also on simulated in silico data, validated the advantages of CASH over other AS predictors and exhibited novel differentially AS events. Moreover, we used CASH to identify SRSF10-regulated AS events and investigated the evolution of SRSF10-regulated splicing. The results showed that SRSF10-regulated splicing events are highly evolvable from chickens, mice to humans. However, SRSF10-regulated splicing model was observed to be immutable, in which SRSF10 binding to cassette exon promotes exon inclusion while binding to downstream exon induces exon skipping. Altogether, CASH can significantly improve the detection of AS events and facilitate the study of AS regulation and function in cells; the SRSF10 data first demonstrate a flexibility of SRSF10 with their regulated splicing events but an immutability of SRSF10-regulated splicing model to produce opposite AS outcomes in vertebrates.