The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

Andrea Hildebrandt,Andrea Voigt,Ingo Ebersberger,Anica Scholz,Mirko Brüggemann,Jean-Yves Roignant,Annabelle Dold,Heike Hänel,Martin M Möckel,Tobias Schmid,Kathi Zarnack,Susan Boerner,Cornelia Rücklé,Stefanie Ebersberger,Jan B Heidelberger,Anke Busch,Petra Beli,Julian König

doi:10.1186/s13059-019-1814-0

Abstract

BackgroundCells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via ribosome-associated quality control.ResultsHere, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during ribosome-associated quality control. We show that MKRN1 directly binds to the cytoplasmic poly(A)-binding protein (PABPC1) and associates with polysomes. MKRN1 is positioned upstream of poly(A) tails in mRNAs in a PABPC1-dependent manner. Ubiquitin remnant profiling and in vitro ubiquitylation assays uncover PABPC1 and ribosomal protein RPS10 as direct ubiquitylation substrates of MKRN1.ConclusionsWe propose that MKRN1 mediates the recognition of poly(A) tails to prevent the production of erroneous proteins from prematurely polyadenylated transcripts, thereby maintaining proteome integrity.

Highlights

During gene expression, quality control pathways monitor each step to detect aberrant mRNAs and proteins
Makorin Ring Finger Protein 1 (MKRN1) interacts with PABPC1 and other RNA-binding proteins (RBPs) In order to learn about potential functions, we characterized the protein interaction profile of MKRN1 in HEK293T cells
We used affinity purification (AP) coupled to stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS) using GFP-MKRN1wt or GFP as a bait

Summary

Introduction

Quality control pathways monitor each step to detect aberrant mRNAs and proteins. These mechanisms ensure protein homeostasis and are essential to prevent neurodegenerative diseases [1]. A common source of aberrant mRNAs is premature polyadenylation, often in combination with mis-splicing, which results in truncated non-functional protein products [2]. Whereas peptide release and ribosome recycling by the RQC complex are relatively well understood, less is known about the mechanisms that promote poly(A) recognition and initial ribosome stalling. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via ribosomeassociated quality control

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Results

Conclusion