RBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. To accomplish this, we downregulated RBM10 in human cell lines, using small interfering RNAs, then monitored alternative splicing, using a reverse transcription-PCR screening platform. RBM10 knockdown (KD) provoked alterations in splicing events in 10–20% of the pre-mRNAs, most of which had not been previously identified as RBM10 targets. Hierarchical clustering of the genes affected by RBM10 KD revealed good conservation of alternative exon inclusion or exclusion across cell lines. Pathway annotation showed RAS signaling to be most affected by RBM10 KD. Of particular interest was the finding that splicing of SMN pre-mRNA, encoding the survival of motor neuron (SMN) protein, was influenced by RBM10 KD. Inhibition of RBM10 resulted in preferential expression of the full-length, exon 7 retaining, SMN transcript in four cancer cell lines and one normal skin fibroblast cell line. SMN protein is expressed from two genes, SMN1 and SMN2, but the SMN1 gene is homozygously disrupted in people with spinal muscular atrophy; as a consequence, all of the SMN that is expressed in people with this disease is from the SMN2 gene. Expression analyses using primary fibroblasts from control, carrier and spinal muscle atrophy donors demonstrated that RBM10 KD resulted in preferential expression of the full-length, exon 7 retaining, SMN2 transcript. At the protein level, upregulation of the full-length SMN2 was also observed. Re-expression of RBM10, in a stable RBM10 KD cancer cell line, correlated with a reversion of the KD effect, demonstrating specificity. Our work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion.

中文翻译：

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剪接阵列揭示了新颖的RBM10靶标，包括SMN2 pre-mRNA。

RBM10是一种RNA结合蛋白，参与消息稳定和选择性剪接调控。本文所述研究的目的是鉴定RBM10调节的剪接的新靶标。为此，我们使用小的干扰RNA下调了人类细胞系中的RBM10，然后使用逆转录PCR筛选平台监测了选择性剪接。RBM10敲低（KD）引起了10-20％的pre-mRNA剪接事件的改变，其中大多数以前未被鉴定为RBM10靶标。受RBM10 KD影响的基因的层次聚类揭示了交替外显子包含或跨细胞系排斥的良好保守性。路径注释显示RAS信号受RBM10 KD影响最大。特别令人感兴趣的是发现SMN pre-mRNA的剪接，RBM10 KD影响了编码运动神经元（SMN）蛋白存活的编码。RBM10的抑制导致全长，保留第7外显子，SMN转录本在四种癌细胞系和一种正常皮肤成纤维细胞系中优先表达。SMN蛋白由SMN1和SMN2两个基因表达，但在患有脊髓性肌萎缩症的人中，SMN1基因被纯合破坏。结果，患有这种疾病的人表达的所有SMN都来自SMN2基因。使用来自对照，载体和脊髓肌肉萎缩供体的原代成纤维细胞进行表达分析表明，RBM10 KD导致全长外显子7保留SMN2转录本的优先表达。在蛋白质水平上，也观察到全长SMN2的上调。在稳定的RBM10 KD癌细胞系中重新表达RBM10，与KD效应的逆转相关，证明了特异性。我们的工作不仅扩大了RBM10的pre-mRNA靶标的数量，而且将RBM10确定为SMN2替代包涵体的新型调节剂。