Altered polyadenylation site usage in SERPINA1 3'UTR in response to cellular stress affects A1AT protein expression

Alternative polyadenylation results in different 3' isoforms of messenger RNA (mRNA) transcripts. Alternative polyadenylation in the 3' untranslated region (3'UTR) can alter RNA localization, stability and translational efficiency. The SERPINA1 mRNA has two distinct 3' UTR isoforms, both of which express the protease inhibitor a-1-antitrypsin (A1AT). A1AT is an acute phase protein that is expressed and secreted from liver hepatocytes and upregulated during inflammation. Low levels of A1AT in the lung contributes to chronic obstructive pulmonary disease, while misfolding of A1AT in the liver contributes to liver cirrhosis. We analyzed the dynamics of alternative polyadenylation during cellular stress by treating the liver cell line HepG2 with the cytokine interleukin 6 (IL-6), ethanol or peroxide. SERPINA1 is transcriptionally upregulated after IL-6 treatment and has altered polyadenylation, resulting in an increase in long 3'UTR isoforms. We find that the long 3'UTR represses endogenous A1AT protein expression even with high levels of SERPINA1 mRNA. SERPINA1 expression and 3' end processing are not affected by ethanol or peroxide. IL-6-induced changes in transcriptome-wide transcriptional regulation suggest changes to the endoplasmic reticulum and in secretory protein processing. Our data suggest that inflammation influences polyA site choice for SERPINA1 transcripts, resulting in reduced A1AT protein expression. Overall design: We treated HepG2 cells with interleukin 6 (IL-6), ethanol or peroxide and performed polyA selection and 3' end specific library preparation (Lexogen QuantSeq Rev). We generated CRISPR mutant lines with a change in the first polyadenylation site of the SERPINA1 gene (APA-mut HepG2 cell line). We performed the same treatments on the APA-mut HepG2 cells.