High-Resolution Ribosome Profiling Defines Discrete Ribosome Elongation States and Translational Regulation during Cellular Stress

Wu, Colin Chih-Chien; Zinshteyn, Boris; Wehner, Karen A.; Green, Rachel

doi:10.1016/j.molcel.2018.12.009

Cited by 259 publications

(363 citation statements)

References 64 publications

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“…Polysomes were isolated from sucrose cushions for library construction as described previously (Wu et al, 2019). Cell pellets were ground with 1 ml footprint lysis buffer [20 mM Tris-Cl (pH 8.0), 140 mM KCl, 1.5 mM MgCl 2 , 1% Triton X-100, 0.1 mg/ml CHX, 0.1 mg/ml TIG] in a Spex 6870 freezer mill.…”

Section: Preparation Of Ribosome Footprint Libraries and Analysis Of mentioning

confidence: 99%

“…Ribosome profiling datasets have been published previously and deposited under GSE115162 (Wu et al, 2019) https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE115162 Ribosome profiling datasets have been published previously and deposited under GSE115162 (Wu et al, 2019) https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE115162…”

Section: Data Availabilitymentioning

confidence: 99%

“…To further investigate the molecular mechanisms of inhibition underlying the inhibitory codon pairs, we turned to high-resolution ribosome profiling (Wu et al, 2019). We recently reported that ribosome profiling using a cocktail of elongation inhibitors can trap ribosomes in their different functional states, distinguished by the size of ribosome-protected footprints (RPFs).…”

Section: Increased 21-nt Rpfs On Inhibitory Pairs Indicate An Empty Rmentioning

confidence: 99%

“…We recently reported that ribosome profiling using a cocktail of elongation inhibitors can trap ribosomes in their different functional states, distinguished by the size of ribosome-protected footprints (RPFs). For example, when cycloheximide (CHX) and tigecycline (TIG) are added to yeast lysates to prevent ribosomes from translating post-cell lysis, RPFs that are 21 nucleotides (nts) in length correspond to ribosomes in a "classical" or POST state waiting to decode the next aminoacyl-tRNA, while RPFs that are 28 nts in length correspond primarily to ribosomes trapped in a "rotated" or PRE state (Wu et al, 2019). Building on an earlier study that showed an enrichment in ribosome density when the 17 inhibitory codon pairs are aligned (Gamble et al, 2016;Matsuo et al, 2017), we generated libraries using CHX and TIG to better distinguish the functional state of the paused ribosomes.…”

Section: Increased 21-nt Rpfs On Inhibitory Pairs Indicate An Empty Rmentioning

confidence: 99%

“…Lysed cell pellets were diluted to 15 ml in footprint lysis buffer and clarified by centrifugation. Polysomes were isolated from sucrose cushions for library construction as described previously (Wu et al, 2019).…”

Section: Preparation Of Ribosome Footprint Libraries and Analysis Of mentioning

confidence: 99%

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Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

et al. 2019

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Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall-and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

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Section: Preparation Of Ribosome Footprint Libraries and Analysis Of mentioning

confidence: 99%

Section: Data Availabilitymentioning

confidence: 99%

Section: Increased 21-nt Rpfs On Inhibitory Pairs Indicate An Empty Rmentioning

confidence: 99%

Section: Increased 21-nt Rpfs On Inhibitory Pairs Indicate An Empty Rmentioning

confidence: 99%

Section: Preparation Of Ribosome Footprint Libraries and Analysis Of mentioning

confidence: 99%

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Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

et al. 2019

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A Circular RNA Generated from Nebulin (NEB) Gene Splicing Promotes Skeletal Muscle Myogenesis in Cattle as Detected by a Multi‐Omics Approach

Huang,

Li,

Zhong

et al. 2023

Advanced Science

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Cattle and the draught force provided by its skeletal muscle have been integral to agro‐ecosystems of agricultural civilization for millennia. However, relatively little is known about the cattle muscle functional genomics (including protein coding genes, non‐coding RNA, etc.). Circular RNAs (circRNAs), as a new class of non‐coding RNAs, can be effectively translated into detectable peptides, which enlightened us on the importance of circRNAs in cattle muscle physiology function. Here, RNA‐seq, Ribosome profiling (Ribo‐seq), and peptidome data are integrated from cattle skeletal muscle, and detected five encoded peptides from circRNAs. It is further identified and functionally characterize a 907‐amino acids muscle‐specific peptide that is named circNEB‐peptide because derived by the splicing of Nebulin (NEB) gene. This peptide localizes to the nucleus and cytoplasm and directly interacts with SKP1 and TPM1, key factors regulating physiological activities of myoblasts, via ubiquitination and myoblast fusion, respectively. The circNEB‐peptide is found to promote myoblasts proliferation and differentiation in vitro, and induce muscle regeneration in vivo. These findings suggest circNEB‐peptide is an important regulator of skeletal muscle regeneration and underscore the possibility that more encoding polypeptides derived by RNAs currently annotated as non‐coding exist.

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Rfoot‐seq: Transcriptomic RNase Footprinting for Mapping Stable RNA‐Protein Complexes and Rapid Ribosome Profiling

Stroup

2023

Current Protocols

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Ribosome profiling isolates ribosome-protected fragments for sequencing and is a valuable method for studying different aspects of RNA translation. However, conventional protocols require millions of input cells and time-consuming steps to isolate translating ribosome complexes using ultracentrifugation or immunoprecipitation. These limitations have prevented their application to rare physiological samples. To address these technical barriers, we developed an RNase footprinting approach named Rfoot-seq to map stable transcriptomic RNA-protein complexes that allows rapid ribosome profiling using low-input samples (Li, Yang, Stroup, Wang, & Ji, 2022). In this assay, we treat a cell lysate with concentrated RNase without complex crosslinking and retained only RNA footprints associated with stable complexes for sequencing. The footprints in coding regions represent ribosome-protected fragments and can be used to study cytosolic and mitochondrial translation simultaneously. Rfootseq achieves comparable results to conventional ribosome profiling to quantify ribosome occupancy and works robustly for various cultured cells and primary tissue samples. Moreover, Rfoot-seq maps RNA fragments associated with stable non-ribosomal RNA-protein complexes in noncoding domains of small noncoding RNAs and some long noncoding RNAs. Taken together, Rfoot-seq opens an avenue to quantify transcriptomic translation and characterize functional noncoding RNA domains using low-input samples.

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High-Resolution Ribosome Profiling Defines Discrete Ribosome Elongation States and Translational Regulation during Cellular Stress

Abstract: Highlights d High-resolution ribosome profiling resolves distinct ribosome functional states d Codon occupancy of decoding ribosomes is highly anticorrelated with tRNA abundance d Cellular stress conditions trigger distinct paths of elongation regulation

Cited by 259 publications

References 64 publications

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

A Circular RNA Generated from Nebulin (NEB) Gene Splicing Promotes Skeletal Muscle Myogenesis in Cattle as Detected by a Multi‐Omics Approach

Rfoot‐seq: Transcriptomic RNase Footprinting for Mapping Stable RNA‐Protein Complexes and Rapid Ribosome Profiling

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