A chemical kinetic basis for measuring translation initiation and elongation rates from ribosome profiling data

Sharma, Ajeet K.; Sormanni, Pietro; Ahmed, Nabeel; Ciryam, Prajwal; Friedrich, Ulrike; Krämer, Günter; O’Brien, Edward P.

doi:10.1371/journal.pcbi.1007070

Cited by 56 publications

(93 citation statements)

References 93 publications

(250 reference statements)

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“…Individual codon RDs obtained from the consensus profiles displayed broad distributions similar to what had been reported previously (39)(40)(41)(42). This observation suggested substantial variation of the speeds at which individual codons are translated.…”

Section: Differentially Translated (Dt) Sequences Are Raresupporting

confidence: 83%

“…However, in part this may also stem from persisting challenges in the interpretation of Ribo-seq data that are often characterized by high intrinsic variability. For instance, the inference of individual codon translation rates from Ribo-seq data commonly results in wide distributions rather then narrowly defined individual values (39)(40)(41)(42). This renders the mechanistic modelling of translation, even though strong progress could be made (25,41,(43)(44)(45), challenging.…”

Section: Introductionmentioning

confidence: 99%

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Inferring translational heterogeneity from ribosome profiling data

Bordignon

Pechmann

2019

Preprint

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Translation of messenger RNAs into proteins by the ribosome is the most important step of protein biosynthesis. Accordingly, translation is tightly controlled and heavily regulated to maintain cellular homeostasis. Ribosome profiling (Ribo-seq) has revolutionized the study of translation by revealing many of its underlying mechanisms. However, equally many aspects of translation remain mysterious, in part also due to persisting challenges in the interpretation of data obtained from Ribo-seq experiments. Here, we show that some of the variability observed in Ribo-seq data has biological origins and reflects programmed heterogeneity of translation. To systematically identify sequences that are differentially translated (DT) across mRNAs beyond what can be attributed to experimental variability, we performed a comparative analysis of Ribo-seq data from Saccharomyces cerevisiae and derived a consensus ribosome density profile that reflects consistent signals in individual experiments. Remarkably, the thus identified DT sequences link to mechanisms known to regulate translation elongation and are enriched in genes important for protein and organelle biosynthesis. Our results thus highlight examples of translational heterogeneity that are encoded in the genomic sequences and tuned to optimizing cellular homeostasis. More generally, our work highlights the power of Ribo-seq to understand the complexities of translation regulation.

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Section: Differentially Translated (Dt) Sequences Are Raresupporting

confidence: 83%

Section: Introductionmentioning

confidence: 99%

Inferring translational heterogeneity from ribosome profiling data

Bordignon

Pechmann

2019

Preprint

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“…To rule out the latter possibility, we estimated Cap-dependent and IRES-mediated elongation rates by measuring the time it takes ribosomes to run-off the biosensor after translation initiation is blocked by Harringtonine. 40 Consistent with ribosomal run-off, Harringtonine led to a steady decay in the nascent chain signals within translation sites ( Figure 4 and Figure S4A-B). This decay was not due to photobleaching because it was not observed in untreated control cells ( Figure S4C The similarity of these rates and run-off times demonstrates elongation is not responsible for the lower number of IRES-mediated translation sites compared to Cap-dependent translation sites.…”

Section: Elongation Does Not Alter the Translation Efficiency Of The mentioning

confidence: 56%

Quantifying the spatiotemporal dynamics of IRES versus Cap translation with single-molecule resolution in living cells

Koch

Aguilera

Morisaki

et al. 2020

Preprint

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Viruses use IRES sequences within their RNA to hijack translation machinery and thereby rapidly replicate in host cells. While this process has been extensively studied in bulk assays, the dynamics of hijacking at the single-molecule level remain unexplored in living cells.To achieve this, we developed a bicistronic biosensor encoding complementary repeat epitopes in two ORFs, one translated in a Cap-dependent manner and the other translated in an IRESmediated manner. Using a pair of complementary probes that bind the epitopes cotranslationally, our biosensor lights up in different colors depending on which ORF is being translated. In combination with single-molecule tracking and computational modeling, we measured the relative kinetics of Cap versus IRES translation and show: (1) Two nonoverlapping ORFs can be simultaneously translated within a single mRNA; (2) EMCV IRESmediated translation sites recruit ribosomes less efficiently than Cap-dependent translation sites but are otherwise nearly indistinguishable, having similar mobilities, sizes, spatial distributions, and ribosomal initiation and elongation rates; (3) Both Cap-dependent and IRES-mediated ribosomes tend to stretch out translation sites; (4) Although the IRES recruits two to three times fewer ribosomes than the Cap in normal conditions, the balance shifts dramatically in favor of the IRES during oxidative and ER stresses that mimic viral infection; and (5) Translation of the IRES is enhanced by translation of the Cap, demonstrating upstream translation can positively impact the downstream translation of a non-overlapping ORF. With the ability to simultaneously quantify two distinct translation mechanisms in physiologically relevant live-cell environments, we anticipate bicistronic biosensors like the one we developed here will become powerful new tools to dissect both canonical and non-canonical translation dynamics with single-molecule precision.( | )) < 60 were accepted). The resulting parameter sets were then used to estimate standard deviations of the parameters as shown in Table 1. Computational Implementation and CodesAll simulations were performed on the W. M. Keck High Performance Computing Cluster at Colorado State University. All codes and required data will be available in the following repository:https://github.com/MunskyGroup/CAP_IRES.

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“…(23). In the results presented we vary the translation-initiation rate α from 0.1 to 0.3 => (21,24,25). We set ./01 and '2345 to 10 =B and 5×10 =E => , respectively, as these rate parameters give the PGK1 mRNA half-lives similar to the ones measured in Ref.…”

Section: Methodsmentioning

confidence: 93%

“…We selected YDR158W, YPL061W and YLR109W S. cerevisiae transcripts to study the effects of multiple slow codon clusters on mRNA half-life. These transcripts have a relatively higher translation-initiation rate (25) as well as higher-than-average fractions of optimal codons that makes them more likely to have significant ribosome-traffic jams upon introducing non-optimal codons. We created four synonymous variants of each of these transcripts by replacing optimal codons with non-optimal ones (Table S1 and supporting file 1).…”

Section: Construction Of Pgk1 Variantsmentioning

confidence: 99%

Combinations of slow translating codon clusters can increase mRNA half-life

Sharma

O’Brien

2018

Preprint

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The introduction of a single, non-optimal codon cluster into a transcript was found to cause ribosomes to queue upstream of it and decrease the transcript's half-life through the action of the dead box protein Dhh1p, which interacts with stalled ribosomes and promotes transcript degradation. Naturally occurring transcripts often contain multiple slow codon clusters, but the influence of these combinations of clusters on a transcript's half-life is unknown. Using a kinetic model that describes translation and mRNA degradation, we find that the introduction of a second slow codon cluster into a transcript near the 5¢-end can have the opposite effect than that of the first cluster by increasing the mRNA's half-life. We experimentally validate this finding by showing that S. cerevisiae transcripts that only have a slow codon cluster towards the 3¢-end of the coding sequence have shorter half-lives than transcripts with nonoptimal clusters at both 3¢ and 5¢ -ends. The origin of this increase in half-life arises from the 5¢-end cluster suppressing the formation of ribosome queues upstream of the second cluster, thereby decreasing the opportunities for translation-dependent degradation machinery, such as the Dhh1p protein, to interact with stalled ribosomes. We also find in the model that in the presence of two slow codon clusters the cluster closest to 5¢-end is the primary determinant of mRNA half-life. These results identify two of the rules governing the influence of slow codon clusters on mRNA half-life, demonstrate that multiple slow codon clusters can have synergistic effects, and indicate that codon usage bias can play a more nuanced role in controlling cellular protein levels than previously thought. * Corresponding

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A chemical kinetic basis for measuring translation initiation and elongation rates from ribosome profiling data

Cited by 56 publications

References 93 publications

Inferring translational heterogeneity from ribosome profiling data

Inferring translational heterogeneity from ribosome profiling data

Quantifying the spatiotemporal dynamics of IRES versus Cap translation with single-molecule resolution in living cells

Combinations of slow translating codon clusters can increase mRNA half-life

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