Characterization of ribosomal stalling sequences and their recognition by the RQT quality control factors

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Ribosomal stalling is a hallmark of quality control mechanisms that have evolved to prevent damage from potentially toxic nascent proteins and rescue of ribosomes stalled on aberrant mRNAs. Besides aberrant mRNA, ribosomal stalling can also be caused by specific non-optimal codon pairs and polybasic encoding sequences of mRNA. The proposed research project aims at discovering the molecular mechanisms, which cause mRNA sequence-induced ribosomal stalling and, subsequently, control initiation of ribosome-associated quality control (RQC) on such sequence elements. Three complementary aims will be followed to address pivotal questions of (i) what the specific structural effects of stalling-inducing sequences on ribosomes are, (ii) how the stalled ribosomes are recognized and (iii) how the RQC response is triggered. In all cases, single-particle cryo-electron microscopy (cryo-EM) will be employed to obtain detailed mechanistic insight into these processes. First, we will use an established artificial mRNA reporter system containing confirmed stalling-inducing sequences. We will modify this system to encode a range of different polybasic and tandem codon stalling-inducing sequences in order to isolate specifically stalled ribosomes. Cryo-EM will be used to reveal specific structural features of stalled ribosomes such as potential perturbations of the peptidyl-transferase center. Next, we will isolate and structurally characterize a novel RQC-triggering factor Hel2 in complex with stalled ribosomes obtained in the previous step. This will be done either by isolation from yeast cells or by in vitro reconstitution. This approach is expected to reveal which common structural features of the specifically stalled ribosomes are recognized by Hel2 in order to trigger the RQC response.Finally, we plan to obtain ribosomes in complex with the novel RQC triggering (RQT) complex (Slh1-Cue3-Ykr023w) in presence or absence of Hel2. Here, we will use a similar approach as for ribosome-Hel2 complexes and use the native pulldown protocol to purify ribosome-RQT complexes to produce suitable cryo-EM samples. In parallel, we will purify the individual components from prokaryotic or eukaryotic expression systems (E. coli, S. cerevisiae, insect or human cell culture) and reconstitute them with stalled ribosomes.These combined approaches will allow us to gain important insights into the mechanisms of ribosomal stalling and its recognition by the RQC-triggering machinery. This project will, thus, provide a detailed mechanistic basis of the stalling process caused by polybasic and tandem codon mRNA stalling sequences. Revealing how Hel2 interacts with the ribosome will shed light on its role in triggering RQC by non-proteasomal ubiquitination. The subsequent engagement of the RQT complex is entirely enigmatic so far with respect to its mechanistic and functional basis, and we therefore aim to provide a first glimpse at its structural role in the quality control pathway.