Analysis of codon-specific translation by ribosome profiling

Kim, Yeji; Eggers, Cristián; Shvetsova, Ekaterina; Kleemann, Leon; Sin, Olga; Leidel, Sebastian A.

doi:10.1016/bs.mie.2021.06.025

Cited by 6 publications

(10 citation statements)

References 26 publications

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“…For library preparation, we adapted a protocol previously developed for small RNA sequencing (23), and further optimized it for tRNA sequencing (Figure 1B). We ligated the end-repaired tRNA samples to a pre-adenylated adapter, which bears six randomized nucleotides at the 5’-end in order to minimize sequence-preference bias in the ligation step.…”

Section: Resultsmentioning

confidence: 99%

“…Reverse transcription (RT): the adapter-ligated tRNA pellets were redissolved and annealed in a volume of 12 μl to 2.5 pmol RT primer (p-NNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCG C-Spc18-CACTCA-Spc18-TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG) (23) by 2 minutes of incubation at 85 °C followed by 5 minutes of incubation at 25 °C. The reverse transcription was carried out in a reaction volume of 20 μl containing the annealed tRNA-RT primer, 500 nM TGIRT-III (InGex, or kindly provided by A. Lambowitz), 1X Protoscript II buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl 2 ) 40 U Murine RNase Inhibitor (NEB), 5 mM DTT and pre-incubated for 10 minutes at 42 °C.…”

Section: Methodsmentioning

confidence: 99%

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tRNA expression and modification landscapes, and their dynamics during zebrafish embryo development

Rappol,

Waldl,

Chugunova

et al. 2024

Preprint

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tRNA genes exist in multiple copies in the genome of all organisms across the three domains of life. Besides the sequence differences across tRNA copies, extensive post-transcriptional modification adds a further layer to tRNA diversification. Whilst the crucial role of tRNAs as adapter molecules in protein translation is well established, whether all tRNA are actually expressed, and whether the differences across isodecoders play any regulatory role is only recently being uncovered. Here we built upon recent developments in the use of NGS-based methods for RNA modification detection and developed tRAM-seq, an experimental protocol and in silico analysis pipeline to investigate tRNA expression and modification. Using tRAM-seq we analysed the full ensemble of nucleo-cytoplasmic and mitochondrial tRNAs during embryonic development of the model vertebrate zebrafish. We show that the repertoire of tRNAs changes during development, with an apparent major switch in tRNA isodecoder expression and modification profile taking place around the start of gastrulation. Taken together, our findings suggest the existence of a general reprogramming of the expressed tRNA pool, possibly gearing the translational machinery for distinct stages of the delicate and crucial process of embryo development.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

tRNA expression and modification landscapes, and their dynamics during zebrafish embryo development

Rappol,

Waldl,

Chugunova

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…(A) Metagene analyses of read depth around translation initiation (left panel) and termination codons (right panel). Compared are libraries generated with the classical protocol (Ingolia 2010; Kim et al 2021) (shown in green) versus libraries generated with the novel protocol (shown in yellow). Depicted are mean values (solid line) ± 95% confidence interval (shaded area).…”

Section: Resultsmentioning

confidence: 99%

“…All primer sequences are listed in Supplemental Table 1. Comparison with the `classical´ library preparation protocol: HEK293 cells were harvested as described in Kim et al 2021 with the following adaption: the lysis buffer was supplemented with 25U/ml Turbo DNase [ThermoFisher] before use. Ten A260 units of lysate were digested with 1000U RNase I [ThermoFisher].…”

Section: Optimized Protocolmentioning

confidence: 99%

A rapid protocol for ribosome profiling of low input samples

Meindl

Romberger

Lehmann

et al. 2022

Preprint

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Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected by ribosomes from nucleolytic digestion. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of RNA. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.

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“…Ribosome profiling was performed as described with minor modifications (Kim et al, 2021; Sharma et al, 2021). Yeast cells were harvested by vacuum filtration and the pellet snap frozen in liquid nitrogen with droplets of lysis buffer.…”

Section: Methodsmentioning

confidence: 99%

tRNA modifications tune decoding of codon pairs to prevent cellular quality control responses

Wu,

Eggers,

Sin

et al. 2024

Preprint

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AbstracttRNA modifications tune translation rates and codon optimality, thereby optimizing co-translational protein folding, but how codon optimality defects trigger cellular phenotypes remains unclear. Here, we show that ribosomes stall at specific modification-dependent codon pairs, triggering ribosome collisions and inducing a coordinated and hierarchical response of cellular quality control pathways. Ribosome profiling reveals an unexpected functional diversity for wobble-uridine (U34) modifications during decoding. The same modification can have different effects at the A and P sites. Furthermore, modification-dependent stalling codon pairs induce ribosome collisions, triggering ribosome-associated quality control (RQC) to prevent protein aggregation by degrading aberrant nascent peptides and mRNAs. RQC inactivation stimulates the expression of molecular chaperones to remove protein aggregates. Our results show that loss of tRNA modifications primarily disrupts translation rates of suboptimal codon pairs and reveal the coordinated regulation and adaptability of cellular surveillance systems to ensure efficient and accurate protein synthesis and maintain protein homeostasis.

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Analysis of codon-specific translation by ribosome profiling

Cited by 6 publications

References 26 publications

tRNA expression and modification landscapes, and their dynamics during zebrafish embryo development

tRNA expression and modification landscapes, and their dynamics during zebrafish embryo development

A rapid protocol for ribosome profiling of low input samples

tRNA modifications tune decoding of codon pairs to prevent cellular quality control responses

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