Regulation of alternative polyadenylation isoforms of Timp2 is an effector event of RAS signaling in cell transformation

Abstract

Alternative polyadenylation (APA) generates mRNA isoforms with different lengths of the 3 untranslated region (3 UTR). The tissue inhibitor of metalloproteinase 2 (TIMP2) plays a key role in extracellular matrix remodeling under various developmental and disease conditions. Both human and mouse genes encoding TIMP2 contain two highly conserved 3UTR APA sites, leading to mRNA isoforms that differ substantially in 3UTR size. APA of Timp2 is one of the most significantly regulated events in multiple cell differentiation lineages. Here we show that Timp2 APA is highly regulated in transformation of NIH3T3 cells by the oncogene HRASG12V. Perturbations of isoform expression with long 3UTR isoform-specific knockdown or genomic removal of the alternative UTR (aUTR) region indicate that the long 3UTR isoform contributes to the secreted Timp2 protein much more than the short 3UTR isoform. The short and long 3UTR isoforms differ in subcellular localization to endoplasmic reticulum (ER). Strikingly, Timp2 aUTR enhances secreted protein expression but no effect on intracellular proteins in reporter assays. Furthermore, downregulation of Timp2 long isoform mitigates gene expression changes elicited by HRASG12V. Together, our data indicate that regulation of Timp2 protein expression through APA isoform changes is an integral part of RAS-mediated cell transformation and 3UTR isoforms of Timp2 can have distinct impacts on expression of secreted vs. intracellular proteins.