Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
Oncogene. 2014 Apr 3;33(14):1818-27. doi: 10.1038/onc.2013.133. Epub 2013 Apr 22.
RRP1B (ribosomal RNA processing 1 homolog B) was first identified as a metastasis susceptibility gene in breast cancer through its ability to modulate gene expression in a manner that can be used to accurately predict prognosis in breast cancer. However, the mechanism(s) by which RRP1B modulates gene expression is currently unclear. Many RRP1B binding candidates are involved in alternative splicing, a mechanism of gene expression regulation that is increasingly recognized to be involved in cancer progression and metastasis. One such target is SRSF1 (serine/arginine-rich splicing factor 1) (SF2/ASF, splicing factor 2/alternative splicing factor), an essential splicing regulator that also functions as an oncoprotein. Earlier studies demonstrated that splicing and transcription occur concurrently and are coupled processes. Given that RRP1B regulates transcriptional activity, we hypothesized that RRP1B also regulates the expression of alternative mRNA isoforms through its interaction with SRSF1. Interaction between RRP1B and SRSF1 was verified by coimmunoprecipitation and coimmunofluorescence. Treatment of cells with transcriptional inhibitors significantly increased this interaction, demonstrating that the association of these two proteins is transcriptionally regulated. To assess the role of RRP1B in the regulation of alternative isoform expression, RNA-sequencing data were generated from control and Rrp1b-knockdown cells. Knockdown of Rrp1b induced a significant change in isoform expression in over 600 genes compared with control cell lines. This was verified by quantitative reverse-transcription PCR using isoform-specific primers. Pathway enrichment analyses identified cell cycle and checkpoint regulation to be those most affected by Rrp1b knockdown. These data suggest that RRP1B suppresses metastatic progression by altering the transcriptome through its interaction with splicing regulators such as SRSF1.
RRP1B（核糖体 RNA 加工 1 同源物 B）最初是通过其调节基因表达的能力被鉴定为乳腺癌的转移易感性基因，这种能力可以准确预测乳腺癌的预后。然而，RRP1B 调节基因表达的机制目前尚不清楚。许多 RRP1B 结合候选物参与可变剪接，这是一种基因表达调控机制，越来越被认为与癌症进展和转移有关。其中一个靶标是 SRSF1（丝氨酸/精氨酸丰富剪接因子 1）（SF2/ASF，剪接因子 2/选择性剪接因子），它是一种必需的剪接调节因子，也是一种癌蛋白。早期的研究表明，剪接和转录同时发生，是偶联的过程。鉴于 RRP1B 调节转录活性，我们假设 RRP1B 还通过与 SRSF1 的相互作用来调节可变 mRNA 异构体的表达。通过共免疫沉淀和共免疫荧光验证了 RRP1B 和 SRSF1 之间的相互作用。用转录抑制剂处理细胞显著增加了这种相互作用，表明这两种蛋白质的结合受转录调控。为了评估 RRP1B 在调节可变异构体表达中的作用，从对照和 Rrp1b 敲低细胞中生成了 RNA-seq 数据。与对照细胞系相比，Rrp1b 敲低导致超过 600 个基因的异构体表达发生显著变化。这通过使用异构体特异性引物的定量逆转录 PCR 得到了验证。途径富集分析表明，细胞周期和检查点调节是受 Rrp1b 敲低影响最大的途径。这些数据表明，RRP1B 通过与 SRSF1 等剪接调节剂相互作用，改变转录组，从而抑制转移进展。
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