Dissection of Dom34–Hbs1 reveals independent functions in two RNA quality control pathways

Abstract

Eukaryotic cells have several quality control pathways that rely on translation to detect and degrade defective RNAs. Dom34 and Hbs1 are two proteins that are related to translation termination factors and are involved in no-go decay (NGD) and nonfunctional 18S ribosomal RNA (rRNA) decay (18S NRD) pathways that eliminate RNAs that cause strong ribosomal stalls. Here we present the structure of Hbs1 with and without GDP and a low-resolution model of the Dom34-Hbs1 complex. This complex mimics complexes of the elongation factor and transfer RNA or of the translation termination factors eRF1 and eRF3, supporting the idea that it binds to the ribosomal A-site. We show that nucleotide binding by Hbs1 is essential for NGD and 18S NRD. Mutations in Hbs1 that disrupted the interaction between Dom34 and Hbs1 strongly impaired NGD but had almost no effect on 18S NRD. Hence, NGD and 18S NRD could be genetically uncoupled, suggesting that mRNA and rRNA in a stalled translation complex may not always be degraded simultaneously.

Strategies for the structural analysis of multi-protein complexes: Lessons from the 3D-Repertoire project

“…Each of the Dom34 derivatives was fused to a Strep tag. These vectors were introduced into BL21(DE3) cells, together with a plasmid encoding full-length 6His-tagged Hbs1 (van den Elzen et al, 2010). Extracts were prepared after induction of protein expression and incubated with Streptactin beads.…”

“…The first example concerns the Dom34-Hbs1 complex from S. cerevisiae, which is involved in the degradation of RNA, inducing strong translational stalls of the ribosomes (Doma and Parker, 2006). We have solved the structures of Dom34 and Hbs1 from yeast and the structure of a Dom34 archaeal ortholog was described elsewhere Lee et al, 2007;van den Elzen et al, 2010). However, we could not obtain crystals for the complex.…”

The Hbs1-Dom34 Protein Complex Functions in Non-stop mRNA Decay in Mammalian Cells

“…As described previously, Hbs1 and Dom34 bind directly, and the formation of the Hbs1-Dom34 complex results in a significant conformational change in Dom34, which increases the affinity of the complex for the A site of the ribosome (32,55,56). Thus, in the no-go decay mechanism, Hbs1 and Dom34 are thought to form a complex at the A site of the stalled ribosome.…”

The tRNA Splicing Endonuclease Complex Cleaves the Mitochondria-localized CBP1 mRNA

“…The nonstop decay quality control system rapidly degrades aberrant mRNAs lacking a termination codon (nonstop mRNAs), which are produced mainly by premature polyadenylation within an ORF (1,2). Perturbation of translation elongation by stable RNA secondary structures, rare codons, or positively charged nascent peptides induces both no-go decay (NGD), 4 in which mRNAs in the vicinity of stalled ribosomes undergo endonucleolytic cleavage (3,4), and ribosome quality control, in which the arrested protein product is co-translationally degraded by the proteasome (5)(6)(7)(8)(9)(10)(11)(12).…”