hnRNPC regulates cancer-specific alternative cleavage and polyadenylation profiles

Alternative cleavage and polyadenylation (APA) can occur at more than half of all human genes, greatly enhancing the cellular repertoire of mRNA isoforms. As these isoforms can have altered stability, localisation and coding potential, deregulation of APA can disrupt gene expression and this has been linked to many diseases including cancer progression. How APA generates cancer-specific isoform profiles and what their physiological consequences are, however, is largely unclear. Here we use a subcellular fractionation approach to determine the nuclear and cytoplasmic APA profiles of successive stages of colon cancer using a cell line-based model. Using this approach, we show that during cancer progression specific APA profiles are established. We identify that overexpression of hnRNPC has a critical role in the establishment of APA profiles characteristic for metastatic colon cancer cells, by regulating poly(A) site selection in a subset of genes that have been implicated in cancer progression including MTHFD1L. Overall design: RNA was extracted from nuclear and cytoplasmic subcellular fractions from two biological replicates of the following cell lines: the non-malignant adult-derived human male colonic epithelial 1CT cell line and the SW480 and SW620 cell lines, which both derive from the same patient. The SW480 cell line was established from a Dukes' type B primary adenocarcinoma of the colon and the SW620 cell line was derived from a lymph node after cancer recurred with widespread metastasis. The 3' ends of extracted RNA were generated into libraries using the QuantSeq 3'mRNA-Seq library kit (Lexogen). Libraries were processed on the Ion Chef platform and subsequently sequenced on the Ion Proton system. Sequences were aligned using the Ion Torrent Server TMAP aligner to genome build hg19. RNA was also extracted from nuclear and cytoplasmic subcellular fractions from two biological replicates in SW620 cells following siRNA knockdown of HNRNPC, ELAVL1, or a control knockdown using scrambled siRNA. The 3' ends of extracted RNA were processed and sequenced as before.

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Fischl H, Neve J, Wang Z, Patel R, Louey A, Tian B, Furger A