5-Aza-Cytidine Enhances Terminal Polyadenylation Site Usage for Full-Length Transcripts in Cells

As an inhibitor of DNA methyltransferases (DNMTs) and an anti-cancer drug, 5-aza-cytidine (5-azaC)'s many effects on gene expression remain unclear. Here, we show that 5-azaC treatment of cultured GH₃ pituitary tumor cells increases relative usage of genomic terminal exons (GTEs) across the transcriptome. This effect is largely achieved by shifting mRNA polyadenylation from proximal poly(A) sites to GTEs, which harbor a more optimal consensus motif of poly(A) signals. Consistent with this shift, 5-azaC upregulates the mRNA anti-termination factors Scaf4 and Scaf8 while downregulating the early termination enhancer E2f2. In MOLM-13 leukemia cells, 5-azaC similarly promotes the production of full-length transcripts and regulates alternative polyadenylation factors, some of which are in the same direction as observed in GH₃ cells. Moreover, PCF11, a factor known to promote proximal poly(A) site usage, is upregulated in both cell lines, suggesting a homeostatic response by these cells to counteract transcript lengthening during 5-azaC treatment. Together, these findings uncover a previously unknown effect of 5-azaC on gene expression: directional promotion of terminal polyadenylation site usage, driving a transcriptome-wide switch from shortened to full-length mRNAs in tumor or cancer cells and consequently altering the alternative usage of multiple 3' exons.