Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control

Marie-Luise Winz,Juri Rappsilber,Lauri Peil,David Tollervey,Tomasz W Turowski

doi:10.1038/s41467-019-08382-z

Abstract

Ribosome-associated quality control (RQC) pathways monitor and respond to ribosome stalling. Using in vivo UV-crosslinking and mass spectrometry, we identified a C-terminal region in Hel2/Rqt1 as an RNA binding domain. Complementary crosslinking and sequencing data for Hel2 revealed binding to 18S rRNA and translated mRNAs. Hel2 preferentially bound mRNAs upstream and downstream of the stop codon. C-terminal truncation of Hel2 abolished the major 18S crosslink and polysome association, and altered mRNA binding. HEL2 deletion caused loss of RQC and, we report here, no-go decay (NGD), with comparable effects for Hel2 truncation including the RNA-binding site. Asc1 acts upstream of Hel2 in RQC and asc1∆ impaired Hel2 binding to 18S and mRNA. In conclusion: Hel2 is recruited or stabilized on translating 40S ribosomal subunits by interactions with 18S rRNA and Asc1. This 18S interaction is required for Hel2 function in RQC and NGD. Hel2 probably interacts with mRNA during translation termination.

Highlights

Background peaksU494, minorC1490-A1492, major U1362, minor RDN37-1 ETS-1 ITS-1 ITS-2 ETS-2 c Head domain 3′ minor Crosslinked
Regions of RNA-binding proteins that make direct contact with RNA can be identified by ultraviolet (UV) crosslinking followed by mass spectrometry (MS), to detect and characterize the covalent nucleotide–peptide conjugate[30]
In the recently reported identification of RNA-associated peptides technique[31], RNA–protein crosslinking sites can be identified with amino acid resolution from all RNA classes

Summary

Results

The unstructured C-terminus of Hel[2] contacts RNA. Inspection of the sequence of Hel[2] did not reveal any evident RNA-binding motif or interaction domain. Regions of RNA-binding proteins that make direct contact with RNA can be identified by ultraviolet (UV) crosslinking followed by mass spectrometry (MS), to detect and characterize the covalent nucleotide–peptide conjugate[30]. In the recently reported identification of RNA-associated peptides (iRAP) technique[31], RNA–protein crosslinking sites can be identified with amino acid resolution from all RNA classes. Following initial protein digestion with LysC, RNA–protein a RNA UV. Hel[2], including the region around the crosslinked site, is not highly conserved between Hel[2] and hZNF598, possibly related to altered ribosomal protein substrate specificity

Background peaks

Discussion

Methods