Abstract
Abstract RRP1B (ribosomal RNA processing 1 homolog B) was first identified as a metastasis susceptibility gene in breast cancer by its association with the altered expression of extracellular matrix genes, which are common factors in metastasis predictive gene expression signatures. A non-synonymous polymorphism in RRP1B (1421C>T; 436P>L) is associated with metastasis-free survival in multiple breast cancer cohorts, representing over 2,000 patients. These data collectively demonstrate the clinical importance of RRP1B in tumor progression in breast cancer. Yet, the mechanism through which RRP1B alters the expression of survival-associated genes is unknown. Previous studies have shown that many RRP1B binding candidates are involved in alternative splicing, a method of gene expression regulation that is increasingly recognized to be involved in cancer progression and metastasis. One such target is SRSF1 (SF2/ASF), an essential splicing regulator that also functions as an oncogene. Since 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 co-immunoprecipitation and co-immunofluorescence. Earlier studies demonstrated that transcription and mRNA splicing are co-dependent in which each process affects the other through either physical interaction or kinetic regulation. Given that RRP1B interacts with both chromatin-associated factors and splicing regulators, we examined whether RRP1B acts as a mediator between the two sets of machinery. Treatment of cells with transcriptional inhibitors significantly increased the interaction between RRP1B and SRSF1, demonstrating that the association of these two proteins is transcriptionally modulated. In addition, immunoprecipitation assays showed that RRP1B interacts with RNA polymerase II. To assess the role of RRP1B in the regulation of alternative isoforms, we generated RNA-sequencing data from control and Rrp1b-knockdown cells. Knockdown of Rrp1b induced a significant change in isoform expression in over 600 genes compared to control cell lines. This was verified by qRT-PCR using isoform-specific primers. Pathway enrichment analyses identified genes involved in cell cycle and checkpoint regulation to be those most affected by Rrp1b knockdown. Also, knockdown of Rrp1b increased in vitro cell invasiveness and in vivo metastasis, confirming the role of RRP1B as a metastasis suppressor. Based on these findings, we propose that RRP1B regulates metastatic progression by altering the transcriptome through its interaction with transcriptional machinery as well as splicing regulators such as SRSF1. To the best of our knowledge, this is the first study to show that a metastasis susceptibility gene is directly involved in regulating alternative isoform expression. Citation Format: Minnkyong Lee, Derek Gildea, Niraj S. Trivedi, NISC Comparative Sequencing Program, Tyra G. Wolfsberg, Nigel P.S. Crawford. RRP1B, a hereditary metastasis modifier, mediates transcription and splicing to regulate mRNA expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4590. doi:10.1158/1538-7445.AM2013-4590
Published Version
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