Alternative poly-adenylation modulates α1-antitrypsin expression in chronic obstructive pulmonary disease

Abstract

α1-anti-trypsin (A1AT), encoded by SERPINA1, is a neutrophil elastase inhibitor that controls the inflammatory response in the lung. Severe A1AT deficiency increases risk for Chronic Obstructive Pulmonary Disease (COPD), however, the role of A1AT in COPD in non-deficient individuals is not well known. We identify a 2.1-fold increase (p = 2.5x10^-6) in the use of a distal poly-adenylation site in primary lung tissue RNA-seq in 82 COPD cases when compared to 64 controls and replicate this in an independent study of 376 COPD and 267 controls. This alternative polyadenylation event involves two sites, a proximal and distal site, 61 and 1683 nucleotides downstream of the A1AT stop codon. To characterize this event, we measured the distal ratio in human primary tissue short read RNA-seq data and corroborated our results with long read RNA-seq data. Integrating these results with 3’ end RNA-seq and nanoluciferase reporter assay experiments we show that use of the distal site yields mRNA transcripts with over 50-fold decreased translation efficiency and A1AT expression. We identified seven RNA binding proteins using enhanced CrossLinking and ImmunoPrecipitation precipitation (eCLIP) with one or more binding sites in the SERPINA1 3’ UTR. We combined these data with measurements of the distal ratio in shRNA knockdown experiments, nuclear and cytoplasmic fractionation, and chemical RNA structure probing. We identify Quaking Homolog (QKI) as a modulator of SERPINA1 mRNA translation and confirm the role of QKI in SERPINA1 translation with luciferase reporter assays. Analysis of single-cell RNA-seq showed differences in the distribution of the SERPINA1 distal ratio among hepatocytes, macrophages, αβ-Tcells and plasma cells in the liver. Alveolar Type 1,2, dendritic cells and macrophages also vary in their distal ratio in the lung. Our work reveals a complex post-transcriptional mechanism that regulates alternative polyadenylation and A1AT expression in COPD. In certain cases, the molecular mechanism underlying a human genetic disease association occurs at the level of the RNA transcript. The SERPINA1 gene is transcribed into multiple RNA isoforms that differ in their non-coding regions and influence production of the A1AT protein, which is associated with Chronic Obstructive Pulmonary Disease (COPD). Here we report a newly discovered alternative polyadenylation event as an underlying regulatory mechanism that influences A1AT protein expression. In the SERPINA1 mRNA there are two polyadenylation sites in the 3’ untranslated region (3’UTR), resulting in a short and a long transcript. We find that the longer 3’UTR strongly represses protein production, and is expressed at a higher level in individuals with COPD, in two independent studies of lung tissue. The inhibitory mechanism in the long transcript is likely caused by an interaction with the RNA binding protein Quaking, which is also differentially expressed in individuals with COPD. This work demonstrates how post-transcriptional regulation can affect disease and also identifies a potential target in the SERPINA1 3’ UTR for RNA targeted therapeutics.