Ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates the production of secondary siRNAs in plants

Abstract A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway—Ribosome-associated Quality Control (RQC)—by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF’s role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.

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Ribosome profiling in Streptococcus pneumoniae reveals the role of methylation of 23S rRNA nucleotide G748 on ribosome stalling

10.1101/2020.12.21.423859 ◽

2020 ◽

Author(s):

Tatsuma Shoji ◽

Akiko Takaya ◽

Yoko Kusuya ◽

Hiroki Takahashi ◽

Hiroto Kawashima

Keyword(s):

Streptococcus Pneumoniae ◽

Ribosome Profiling ◽

23S Rrna ◽

Ribosome Stalling ◽

70S Ribosome ◽

Exit Tunnel ◽

Species Specific ◽

Peptidyltransferase Center ◽

First Time

2.Abstract(1) BackgroundMany nucleotides in 23S rRNA are methylated post-transcriptionally by methyltransferases and cluster around the peptidyltransferase center (PTC) and the nascent peptidyl exit tunnel (NPET) located in 50S subunit of 70S ribosome. Biochemical interactions between a nascent peptide and the tunnel may stall ribosome movement and affect expression levels of the protein. However, no studies have shown a role for NPET on ribosome stalling using an NPET mutant.(2) ResultsA ribosome profiling assay in Streptococcus pneumoniae demonstrates for the first time that an NPET mutant exhibits completely different ribosome occupancy compared to wild-type. We demonstrate, using RNA footprinting, that changes in ribosome occupancy correlate with changes in ribosome stalling. Further, statistical analysis shows that short peptide sequences that cause ribosome stalling are species-specific and evolutionarily selected. NPET structure is required to realize these specie-specific ribosome stalling.(3) ConclusionsResults support the role of NPET on ribosome stalling. NPET structure is required to realize the species-specific and evolutionary conserved ribosome stalling. These findings clarify the role of NPET structure on the translation process.

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The landscape of translational stall sites in bacteria revealed by monosome and disome profiling

RNA ◽

10.1261/rna.078188.120 ◽

2021 ◽

pp. rna.078188.120

Author(s):

Tomoya Fujita ◽

Takeshi Yokoyama ◽

Mikako Shirouzu ◽

Hideki Taguchi ◽

Takuhiro Ito ◽

...

Keyword(s):

Electron Microscopy ◽

Low Frequency ◽

Ribosome Profiling ◽

Reporter Assay ◽

Ribosome Stalling ◽

Peptidyl Transfer ◽

Cryo Electron Microscopy ◽

Nascent Chain ◽

Biochemical Validation ◽

Trna Sequencing

Ribosome pauses are associated with various cotranslational events and determine the fate of mRNAs and proteins. Thus, the identification of precise pause sites across the transcriptome is desirable; however, the landscape of ribosome pauses in bacteria remains ambiguous. Here, we harness monosome and disome (or collided ribosome) profiling strategies to survey ribosome pause sites in Escherichia coli. Compared to eukaryotes, ribosome collisions in bacteria showed remarkable differences: a low frequency of disomes at stop codons, collisions occurring immediately after 70S assembly on start codons, and shorter queues of ribosomes trailing upstream. The pause sites corresponded with the biochemical validation by integrated nascent chain profiling (iNP) to detect polypeptidyl-tRNA, an elongation intermediate. Moreover, the subset of those sites showed puromycin resistance, presenting slow peptidyl transfer. Among the identified sites, the ribosome pause at Asn586 of ycbZ was validated by biochemical reporter assay, tRNA sequencing (tRNA-Seq), and cryo-electron microscopy (cryo-EM) experiments. Our results provide a useful resource for ribosome stalling sites in bacteria

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Isolation of Ribosome Stalling Motifs from Random Libraries

Regulatory Nascent Polypeptides ◽

10.1007/978-4-431-55052-5_13 ◽

2014 ◽

pp. 225-240

Author(s):

Christopher J. Woolstenhulme ◽

Allen R. Buskirk

Keyword(s):

Ribosome Stalling

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Role for ribosome-associated quality control in sampling proteins for MHC class I-mediated antigen presentation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914401117 ◽

2020 ◽

Vol 117 (8) ◽

pp. 4099-4108 ◽

Cited By ~ 8

Author(s):

Débora Broch Trentini ◽

Matteo Pecoraro ◽

Shivani Tiwary ◽

Jürgen Cox ◽

Matthias Mann ◽

...

Keyword(s):

Quality Control ◽

Antigen Presentation ◽

Mammalian Cells ◽

Adaptive Immune System ◽

Messenger Rnas ◽

Mhc I ◽

Adaptive Immune ◽

Ribosome Stalling ◽

Endogenous Substrates ◽

Insight Into

Mammalian cells present a fingerprint of their proteome to the adaptive immune system through the display of endogenous peptides on MHC-I complexes. MHC-I−bound peptides originate from protein degradation by the proteasome, suggesting that stably folded, long-lived proteins could evade monitoring. Here, we investigate the role in antigen presentation of the ribosome-associated quality control (RQC) pathway for the degradation of nascent polypeptides that are encoded by defective messenger RNAs and undergo stalling at the ribosome during translation. We find that degradation of model proteins by RQC results in efficient MHC-I presentation, independent of their intrinsic folding properties. Quantitative profiling of MHC-I peptides in wild-type and RQC-deficient cells by mass spectrometry showed that RQC substantially contributes to the composition of the immunopeptidome. Our results also identify endogenous substrates of the RQC pathway in human cells and provide insight into common principles causing ribosome stalling under physiological conditions.

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