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An RNA structure-mediated, posttranscriptional model of human α-1-antitrypsin expression

  1. Alain Laederacha,b,1
  1. aDepartment of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;
  2. bCurriculum in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;
  3. cDepartment of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;
  4. dLineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;
  5. eDepartment of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;
  6. fDepartment of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
  1. Edited by Rachel Green, Johns Hopkins University, Baltimore, MD, and approved October 19, 2017 (received for review April 20, 2017)

Significance

Protein and mRNA expression are in most cases poorly correlated, which suggests that the posttranscriptional regulatory program of a cell is an important component of gene expression. This regulatory network is still poorly understood, including how RNA structure quantitatively contributes to translational control. We present here a series of structural and functional experiments that together allow us to derive a quantitative, structure-dependent model of translation that accurately predicts translation efficiency in reporter assays and primary human tissue for a complex and medically important protein, α-1-antitrypsin. Our model demonstrates the importance of accurate, experimentally derived RNA structural models partnered with Kozak sequence information to explain protein expression and suggests a strategy by which α-1-antitrypsin expression may be increased in diseased individuals.

Abstract

Chronic obstructive pulmonary disease (COPD) affects over 65 million individuals worldwide, where α-1-antitrypsin deficiency is a major genetic cause of the disease. The α-1-antitrypsin gene, SERPINA1, expresses an exceptional number of mRNA isoforms generated entirely by alternative splicing in the 5′-untranslated region (5′-UTR). Although all SERPINA1 mRNAs encode exactly the same protein, expression levels of the individual mRNAs vary substantially in different human tissues. We hypothesize that these transcripts behave unequally due to a posttranscriptional regulatory program governed by their distinct 5′-UTRs and that this regulation ultimately determines α-1-antitrypsin expression. Using whole-transcript selective 2′-hydroxyl acylation by primer extension (SHAPE) chemical probing, we show that splicing yields distinct local 5′-UTR secondary structures in SERPINA1 transcripts. Splicing in the 5′-UTR also changes the inclusion of long upstream ORFs (uORFs). We demonstrate that disrupting the uORFs results in markedly increased translation efficiencies in luciferase reporter assays. These uORF-dependent changes suggest that α-1-antitrypsin protein expression levels are controlled at the posttranscriptional level. A leaky-scanning model of translation based on Kozak translation initiation sequences alone does not adequately explain our quantitative expression data. However, when we incorporate the experimentally derived RNA structure data, the model accurately predicts translation efficiencies in reporter assays and improves α-1-antitrypsin expression prediction in primary human tissues. Our results reveal that RNA structure governs a complex posttranscriptional regulatory program of α-1-antitrypsin expression. Crucially, these findings describe a mechanism by which genetic alterations in noncoding gene regions may result in α-1-antitrypsin deficiency.

Footnotes

  • ?1To whom correspondence should be addressed. Email: alain{at}unc.edu.

Online Impact

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