Biology subjects

Ikeuchi, K.

Publications and source records attributed to Ikeuchi, K..

3 recordsLinked to original sources

bioRxiv2022

Clearing of ribosome collisions by the ribosome quality control trigger complex RQT

After translational stalls, colliding eukaryotic ribosomes are cleared through dissociation into subunits by the ribosome quality control trigger complex, RQT, by an unknown mechanism. Here we show that RQT requires accessible mRNA and the presence of a neighboring ribosome. Cryo-EM of several RQT-ribosome complexes revealed the structural basis of splitting: RQT engages the 40S subunit of the lead ribosome and can switch between two conformations. We propose a mechanistic model in which the Slh1 helicase subunit of RQT applies a pulling force on the mRNA, causing destabilizing conformational changes of the 40S subunit. The collided ribosome functions as a ram or giant wedge, ultimately resulting in subunit dissociation. Our findings provide a first conceptual framework for a helicase driven ribosomal splitting mechanism. One-Sentence SummaryRQT clears collided ribosomes by pulling mRNA to trigger destabilizing conformational transitions for subunit dissociation.

biochemistry↗

bioRxiv2021

Recognition and deubiquitination of free 40S for translational reset by Otu2

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not41,2 and deubiquitinated by the deubiquitination enzyme Otu2 upon ribosomal subunit recycling3. Despite its importance for general efficiency of translation the exact role and structural basis for this specific translational reset are only poorly understood. Here we present biochemical and structural data showing that Otu2 can engage the recycled 40S subunit together with the recycling factors ABCE1 and Tma64 immediately after 60S dissociation for mRNA recycling, and that it dissociates before 48S initiation complex formation. A combined structural analysis of Otu2 and Otu2-40S complexes by X-ray crystallography, AlphaFold2 prediction4 and cryo-EM revealed how Otu2 can specifically be recruited to the 40S, but not to the 80S ribosome, for removal of the eS7-bound ubiquitin moiety. Here, interactions of the largely helical N-terminal domain of Otu2 to sites that are masked and therefore inaccessible in the 80S ribosome are of crucial importance. Collectively, we provide the structural basis for the Otu2 driven deubiquitination step providing a first mechanistic understanding of this translational reset step during ribosome recycling/(re)initiation.

molecular biology↗

bioRxiv2019

The Ccr4-Not complex monitors the translating ribosome for codon optimality

Control of mRNA decay rate is intimately connected to translation elongation but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. Using a combination of cryo-electron microscopy, ribosome profiling and mRNA stability assays we show recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site. This interaction only occurs when the ribosome lacks accommodated A-site tRNA, indicative of low codon optimality. Loss of Not5 results in the inability of the mRNA degradation machinery to sense codon optimality. Our analysis elucidates a physical link between the Ccr4-Not complex and the ribosome providing mechanistic insight into the coupling of decoding efficiency with mRNA stability.

molecular biology↗