Alternative splicing regulation of ALKBH5 and identification of cancer specific alternative splicing

Abstract

Alternative splicing is the process that generates more than one mRNA isoform from a single gene, and it plays a major role in expanding human protein diversity. Dysregulation of alternative splicing is a hallmark of cancer and a potential target for novel anti-cancer therapeutics. N6-methyladenosine (m6A) is the most prevalent RNA modification that regulates several biological processes such as development and cancer. ALKBH5 is an m6A demethylase and plays important roles in spermatogenesis and cancer development. Although ALKBH5 has been shown to be essential for the correct splicing of longer 3’ UTR mRNAs in spermatogenesis, the regulatory role of ALKBH5 in alternative splicing is not well understood. Here, I performed RNA-Seq with ALKBH5 knockdown (KD) cells and bioinformatical alternative splicing analysis with rMATS and found that ALKBH5 KD altered alternative splicing events including skipped exon (SE), retained intron (RI), mutually exclusive exons (MXE), alternative 5’ splice site (A5SS), and alternative 3’ splice site (A3SS). The high validation rate of RNA-Seq results was then confirmed through RT-PCR analysis. I next performed m6A-Seq with ALKBH5 KD cells to ask whether ALKBH5-mediated m6A alteration regulates alternative splicing of ALKBH5. My results showed that m6A contents were mainly altered in exons and 3’ UTR but there was no correlation between altered m6A contents and alternative splicing by ALKBH5 KD. To investigate the alternative splicing regulatory mechanisms of ALKBH5, I applied FAS mini-gene, as the cassette exon skipping of endogenous FAS was promoted by ALKBH5 KD, and oppositely, FAS cassette exon inclusion was promoted by overexpression of ALKBH5. I found that deletion of the SRSF1 binding motif (GGGGUG) from FAS exon 6 disrupted ALKBH5 activity on FAS alternative splicing, and ALKBH5 could directly interact with this motif. In addition, co-IP approach demonstrated that ALKBH5 showed much stronger binding ability with SRSF1 in the absence of RNA than in the presence of RNA. These results indicated a possibility that ALKBH5 interfered binding affinity of SRSF1 to regulate alternative splicing. Also, I performed RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) to identify altered alternative splicing programs occurring in breast cancer progression and metastasis. I found significantly altered alternative splicing events and the high accuracy of RASL-seq results by RT-PCR validation. By Gene Ontology (GO) analysis, I confirmed aberrant alternative splicing genes enriched in several biological processes related to tumor progression and metastasis such as cell cycle, cell-cell adhesion, and Wnt signaling. Among aberrant alternative splicing events in metastasis, I identified DCUN1D5, a gene for which skipping of exon 4 in its pre-mRNA introduces a premature termination codon (PTC), thus generating an unstable transcript degraded by nonsense-mediated mRNA decay (NMD). Significantly, distinct breast cancer subtypes showed different DCUN1D5 isoform ratio with metastatic breast cancer expressing the highest level of the NMD-insensitive DCUN1D5 mRNA, thus showing high DCUN1D5 expression levels. Collectively, my results revealed an m6A-independent alternative splicing regulation of ALKBH5 and novel alternative splicing errors occurring in human breast cancer tumorigenesis and metastasis, which open new possibility for the treatment of breast cancer.