The RNA-binding landscape of RBM10 and its role in alternative splicing regulation in models of mouse early development

Mutations in the RNA-binding protein, RBM10, result in a human syndromic form of cleft palate, termed TARP syndrome. A role for RBM10 in alternative splicing regulation has been previously demonstrated in human cell lines. To uncover the cellular functions of RBM10 in a cell line that is relevant to the phenotype observed in TARP syndrome, we used iCLIP to identify its endogenous RNA targets in a mouse embryonic mandibular cell line. We observed that RBM10 binds to pre-mRNAs with significant enrichment in intronic regions, in agreement with a role for this protein in pre-mRNA splicing. In addition to protein-coding transcripts, RBM10 also binds to a variety of cellular RNAs, including non-coding RNAs, such as spliceosomal small nuclear RNAs, U2 and U12. RNA-seq was used to investigate changes in gene expression and alternative splicing in RBM10 KO mouse mandibular cells and also in mouse ES cells. We uncovered a role for RBM10 in the regulation of alternative splicing of common transcripts in both cell lines but also identified cell-type specific events. Importantly, those pre-mRNAs that display changes in alternative splicing also contain RBM10 iCLIP tags, suggesting a direct role of RBM10 in these events. Finally, we show that depletion of RBM10 in mouse ES cells leads to proliferation defects and to gross alterations in their differentiation potential. These results demonstrate a role for RBM10 in the regulation of alternative splicing in two cell models of mouse early development and suggests that mutations in RBM10 could lead to splicing changes that affect normal palate development and cause human disease.

RNA结合蛋白（RNA-binding protein）RBM10的突变会引发一种人类综合征性腭裂，即TARP综合征。此前已有研究在人类细胞系中证实，RBM10具备可变剪接（alternative splicing）调控功能。为阐明RBM10在与TARP综合征表型相关的细胞系中的细胞生物学功能，我们借助iCLIP（单核苷酸分辨率紫外交联免疫沉淀，individual-nucleotide resolution UV crosslinking and immunoprecipitation）技术在小鼠胚胎下颌细胞系中鉴定了其内源RNA结合靶点。我们发现，RBM10可结合前体mRNA（pre-mRNA），且在内含子区域呈现显著富集，这与该蛋白参与前体mRNA剪接的功能相符。除编码蛋白的转录本外，RBM10还可结合多种细胞RNA，其中包括剪接体小核RNA（spliceosomal small nuclear RNA）U2与U12等非编码RNA（non-coding RNA）。我们采用RNA测序（RNA-seq）技术，分别在RBM10敲除（knockout, KO）小鼠下颌细胞与小鼠胚胎干细胞（embryonic stem cell, ES cell）中检测基因表达与可变剪接的变化情况。本研究不仅证实了RBM10对两种细胞系中共有的转录本的可变剪接具有调控作用，同时还鉴定出了细胞类型特异性的剪接事件。值得注意的是，那些发生可变剪接改变的前体mRNA同时也携带RBM10的iCLIP标签，这提示RBM10在这些剪接事件中发挥直接调控作用。最后，我们证实小鼠胚胎干细胞中RBM10的敲低会引发增殖缺陷，并导致其分化潜能发生显著改变。上述研究结果证实，RBM10在两种小鼠早期发育细胞模型中均可调控可变剪接；同时也提示，RBM10的突变可能通过引发剪接异常，干扰正常腭部发育进程，进而诱发人类疾病。