Transcript shortening through alternative polyadenylation promotes gene expression during fracture healing

Maturation of the 3¢ end of almost all eukaryotic messenger RNAs (mRNAs) requires cleavage and polyadenylation. Most mammalian mRNAs are polyadenylated at different sites within the last exon, generating alternative polyadenylation (APA) isoforms that have the same coding region but distinct 3¢ untranslated regions (UTRs). The 3¢UTR contains motifs that regulate mRNA metabolism; thus, changing the 3¢UTR length via APA can significantly impact gene expression. Endochondral ossification is a central process in bone healing, and the impact of APA on gene expression during this process is unknown. Here, we report widespread utilization of APA that impacts multiple pathways with established roles in bone healing. Importantly, progression of endochondral ossification is typified by global 3¢UTR shortening that is coupled with an increased abundance of shortened transcripts as compared to all other transcripts, underscoring the role of APA in promoting gene expression during endochondral bone formation. Our mechanistic studies of genes that undergo APA in the fracture callus uncover an intricate regulatory network in which APA boosts the expression of collagen, type I, alpha 1 (Col1a1) and Col1a2 genes, which encode the 2 subunits of the abundantly expressed protein collagen 1. APA does so via shortening the 3¢UTRs of Col1a1 and Col1a2 mRNAs, which removes the binding sites of miR-29a-3p that otherwise potently triggered the degradation of both transcripts. Taken together, our study takes the lead in characterizing crucial roles of APA in tailoring the 3¢UTR landscape and regulating gene expression during fracture healing. 16-week-old, male C57BL/6J mice were subjected to mid-diaphyseal tibial fractures. At days 14 and 21 post-fracture, callus tissues were isolated and snap frozen in liquid nitrogen, and RNA was purified and subjected to RNA sequencing. Three biological replicates of each time point were analyzed.