SRSF5 regulates alternative splicing of <i>DMTF1</i> pre-mRNA through modulating SF1 binding

Among the three DMTF1 splicing isoforms, DMTF1α acts as a tumour suppressor through promoting p14ARF expression, while DMTF1β exhibits an oncogenic role likely through antagonizing DMTF1α. However, the molecular mechanism underlying alternative splicing of <i>DMTF1</i> pre-mRNA has not been delineated. In the current study, we discovered SRSF5 as a regulatory protein binding to a region located between <i>DMTF1β</i> and <i>α</i> acceptor splice sites to promote <i>DMTF1β</i> and <i>γ</i> splicing. We demonstrated that SRSF5 expression positively correlated with <i>DMTF1β</i>/<i>α</i> ratio in breast cancer samples, and ectopically expressed SRSF5 promoted the splicing of <i>DMTF1β</i> and <i>γ</i>, but not <i>DMTF1α</i>, when testing endogenous <i>DMTF1</i> pre-mRNA and a reporter construct. Upon SRSF5 knockdown, we observed significantly decreased <i>DMTF1β</i> and <i>γ</i> ratios of endogenous transcripts. An RNA sequence just upstream of the <i>α</i> acceptor site contains two adjacent SRSF5 binding elements, one of which overlaps with an SF1 binding site. Our mechanistic studies revealed that SRSF5 binding to both elements in this region could consign SF1 to its distal-binding sites close to the <i>β</i> and <i>γ</i> acceptor sites, favouring splicing of their isoforms. Overall, our study revealed SRSF5 as a key regulator to promote <i>DMTF1β</i> and <i>γ</i> splicing, and consequently reduce <i>DMTF1α</i> splicing. ARF: alternative reading frame, that is, p14ARF, or CDKN2A (cyclin-dependent kinase inhibitor 2A); β-gal: β-galactosidase; CLIP-seq: crosslinking and immunoprecipitation-sequencing; <i>DMTF1</i>: the cyclin D binding myb-like transcription factor 1; ESS/ESE: exonic splicing silencer/enhancer; Ex: exon; FBS: fetal bovine serum; Gluc: Gaussia luciferase; hnRNPs: heterogeneous nuclear ribonucleoproteins; In: intron; ISS/ISE: intronic splicing silencer/enhancer; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PSI: percent-splice-in; qPCR: quantitative real-time PCR; RIP: RNA immunoprecipitation; RNAseq: RNA sequencing; RT: reverse transcription; SF1: splicing factor 1; SR: serine/arginine-rich proteins; SRSF5: serine and arginine-rich splicing factor 5; TCGA: the cancer genome atlas; UCSC: University of California, Santa Cruz. WT: Wild type