Anti-PCBP1 monoclonal antibody, clone FQS22166

Alternative splicing generates distinct mRNA isoforms and is crucial for proteome diversity in eukaryotes. The RNA-binding protein (RBP) U2AF2 is central to splicing decisions, as it recognizes 3' splice sites and recruits the spliceosome. We establish "in vitro iCLIP" experiments, in which recombinant RBPs are incubated with long transcripts, to study how U2AF2 recognizes RNA sequences and how this is modulated by trans-acting RBPs. We measure U2AF2 affinities at hundreds of binding sites and compare in vitro and in vivo binding landscapes by mathematical modeling. We find that trans-acting RBPs extensively regulate U2AF2 binding in vivo, including enhanced recruitment to 3' splice sites and clearance of introns. Using machine learning, we identify and experimentally validate novel trans-acting RBPs (including FUBP1, CELF6, and PCBP1) that modulate U2AF2 binding and affect splicing outcomes. Our study offers a blueprint for the high-throughput characterization of in vitro mRNP assembly and in vivo splicing regulation.

The expression of legumain which has been shown overexpressed in patients with metastatic gastric cancer is positively correlated to both disease progression and outcome, and negatively correlated to microRNA (miR)-3978 expression. The RNA-binding protein, poly r(C) binding protein 1 (PCBP1) was the most downregulated protein in the metastatic tissue specimens. Quantitative real-time PCR showed that PCBP1 expression is transcriptionally downregulated in peritoneal metastasis tissues. RNA immunoprecipitation experiments showed that PCBP1 and miR-3978 are sequestered in normal peritoneal tissue, but the complex is disrupted following metastatic progression. PCBP1 expression mimicked miR-3978 expression across gastric cancer patients. Finally, replenishment of PCBP1 or miR-3978 expression in the peritoneal metastasis cell line MKN45 decreased legumain protein expression and chemosensitized the cells to treatment with docetaxel. However, replenishment of one and concomitant depletion of the other failed to induce chemosensitivity to docetaxel. Replenishment of miR-3978 also resulted in induction of PCBP1 protein expression, potentially indicating that miR-3978 expression might downregulate a negative regulator targeting PCBP1. Our current study reveals PCBP1 as an additional biomarker in peritoneal metastasis. PCBP1 and miR-3978 expression were correlated and suggests a potential interplay of differential miRNA biogenesis and RNA binding protein during metastatic progression.