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

Lela Lackey,Abigail Hatfield,Auyon J Ghosh,Craig P Hersh,John Platig,Edwin K Silverman,Silvia B V Ramos,Aaztli Coria,Phil Grayeski,Peter Castaldi,Victor E Ortega,Alain Laederach,Vijay Shankar,Brian D Hobbs,Zhonghui Xu,Michael H Cho,Mihaela Zavolan

doi:10.1371/journal.pgen.1009912

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.

Highlights

The α1-anti-trypsin (A1AT) protein functions primarily as a neutrophil elastase inhibitor and controls the inflammatory response in the lung [1,2]
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)
We find that the longer 3’UnTranslated Region (UTR) strongly represses protein production, and is expressed at a higher level in individuals with COPD, in two independent studies of lung tissue

Summary

Introduction

The α1-anti-trypsin (A1AT) protein functions primarily as a neutrophil elastase inhibitor and controls the inflammatory response in the lung [1,2]. Recent deep resequencing of the SERPINA1 locus in the SubPopulations and InteRmediate Outcome Measures In COPD Study (SPIROMICS) identified additional variation mapping to the 5’ UTR of the gene associated with lowered A1AT serum levels and functional small airway disease [6]. These results support that post-transcriptional regulation of expression is an important and understudied component of A1AT function and lung physiology

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