Taolan Zhao1,2, Yan-Ming Chen3,4,5, Yu Li6,7, Jia Wang3,4,5, Siyu Chen3,4,5, Ning Gao8, Wenfeng Qian9,10,11. 1. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, 100101, China. tlzhao@genetics.ac.cn. 2. Key Laboratory of Genetic Network Biology, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, 100101, China. tlzhao@genetics.ac.cn. 3. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, 100101, China. 4. Key Laboratory of Genetic Network Biology, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, 100101, China. 5. University of Chinese Academy of Sciences, Beijing, 100049, China. 6. Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, School of Life Science, Tsinghua University, Beijing, 100084, China. 7. State Key Laboratory of Membrane Biology, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing, 100871, China. 8. State Key Laboratory of Membrane Biology, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing, 100871, China. gaon@pku.edu.cn. 9. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, 100101, China. wfqian@genetics.ac.cn. 10. Key Laboratory of Genetic Network Biology, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, 100101, China. wfqian@genetics.ac.cn. 11. University of Chinese Academy of Sciences, Beijing, 100049, China. wfqian@genetics.ac.cn.
Abstract
BACKGROUND: The folding of proteins is challenging in the highly crowded and sticky environment of a cell. Regulation of translation elongation may play a crucial role in ensuring the correct folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes by ribo-seq. However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation. RESULTS: In this study, we performed disome-seq to sequence mRNA fragments protected by two stacked ribosomes, a product of translational pauses during which the 5'-elongating ribosome collides with the 3'-paused one. We detected widespread ribosome collisions that are related to slow ribosome release when stop codons are at the A-site, slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and slow leaving of polylysine from the exit tunnel of ribosomes. The structure of disomes obtained by cryo-electron microscopy suggests a different conformation from the substrate of the ribosome-associated protein quality control pathway. Collisions occurred more frequently in the gap regions between α-helices, where a translational pause can prevent the folding interference from the downstream peptides. Paused or collided ribosomes are associated with specific chaperones, which can aid in the cotranslational folding of the nascent peptides. CONCLUSIONS: Therefore, cells use regulated ribosome collisions to ensure protein homeostasis.
BACKGROUND: The folding of proteins is challenging in the highly crowded and sticky environment of a cell. Regulation of translation elongation may play a crucial role in ensuring the correct folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes by ribo-seq. However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation. r> RESULTS: In this study, we performeddisome-seq to sequence mRNA fragments protected by two stackedribosomes, a product of translational pauses during which the 5'-elongating ribosome collides with the 3'-paused one. We detected widespreadribosome collisions that are related to slow ribosome release when stop codons are at the A-site, slow peptide bond formation from proline, glycine, asparagine, andcysteine when they are at the P-site, and slow leaving of polylysine from the exit tunnel of ribosomes. The structure of disomes obtained by cryo-electron microscopy suggests a different conformation from the substrate of the ribosome-associated protein quality control pathway. Collisions occurred more frequently in the gap regions between α-helices, where a translational pause can prevent the folding interference from the downstream peptides. Paused or collidedribosomes are associated with specific chaperones, which can aid in the cotranslational folding of the nascentpeptides. r> CONCLUSIONS: Therefore, cells use regulatedribosome collisions to ensure protein homeostasis.
Entities:
Keywords:
Cotranslational protein folding; Disome structure; Disome-seq; Protein homeostasis; Ribosome collision; Ribosome release; Ribosome-associated chaperones; Translation elongation; Translational pause
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