PT  - JOURNAL ARTICLE
AU  - Andrea Hildebrandt
AU  - Mirko Brüggemann
AU  - Susan Boerner
AU  - Cornelia Rücklé
AU  - Jan Bernhard Heidelberger
AU  - Annabelle Dold
AU  - Anke Busch
AU  - Heike Hänel
AU  - Andrea Voigt
AU  - Stefanie Ebersberger
AU  - Ingo Ebersberger
AU  - Jean-Yves Roignant
AU  - Kathi Zarnack
AU  - Julian König
AU  - Petra Beli
TI  - The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation
AID  - 10.1101/516005
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 516005
4099  - http://biorxiv.org/content/early/2019/01/25/516005.short
4100  - http://biorxiv.org/content/early/2019/01/25/516005.full
AB  - Cells 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 the ribosome-associated quality control (RQC). Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during RQC. We show that MKRN1 interacts with the cytoplasmic poly(A)-binding protein (PABP) and is positioned upstream of poly(A) tails in mRNAs. Ubiquitin remnant profiling uncovers PABP and ribosomal protein RPS10, as well as additional translational regulators as main ubiquitylation substrates of MKRN1. We propose that MKRN1 serves as a first line of poly(A) recognition at the mRNA level to prevent production of erroneous proteins, thus maintaining proteome integrity.AbbreviationsAdenosine(A)Makorin Ring Finger Protein 1(MKRN1)ribosome-associated quality control(RQC)poly(A)-binding protein(PABP)mass spectrometry(MS)affinity purification(AP)stable isotope labelling with amino acids in cell culture(SIALC)false discovery rate(FDR)Gene Ontology(GO)Biological Process(BP)Molecular Function(MF)mRNA ribonucleoprotein particle(mRNP)PABP-interacting motif(PAM2)MKRN1 variant with point mutations in the PAM2 motif(GFP-MKRN1PAM2mut)MKRN1 variant with point mutation in RING domain(GFP-MKRN1RINGmut)individual-nucleotide resolution UV crosslinking and immunoprecipitation(iCLIP)signal-over-background(SOB)nucleotides(nt)4-thiouridine(4SU)Pearson correlation coefficients(r)A-rich stretches(A-stretches)lysine(K)knock down(KD)RNA recognition motif(RRM)Polyethylenimine(PEI)modified RIPA(mRIPA)N-ethylmaleimide(NEM)dithiothreitol(DTT)chloroacetamide(CAA)higher-energy collisional dissociation(HCD)glycine-glycine(GlyGly)strong-cation exchange chromatography(SCX)