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

2021 ◽  
Vol 118 (51) ◽  
pp. e2026362118
Author(s):  
Ajeet K. Sharma ◽  
Johannes Venezian ◽  
Ayala Shiber ◽  
Günter Kramer ◽  
Bernd Bukau ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Half Life ◽  
Synergistic Effects ◽  
Cellular Protein ◽  
Start Codon ◽  
Translational Efficiency ◽  
Control Factors ◽  
The Impact ◽  
Mrna Half Life

The presence of a single cluster of nonoptimal codons was found to decrease a transcript’s half-life through the interaction of the ribosome-associated quality control machinery with stalled ribosomes in Saccharomyces cerevisiae. The impact of multiple nonoptimal codon clusters on a transcript’s half-life, however, is unknown. Using a kinetic model, we predict that inserting a second nonoptimal cluster near the 5′ end can lead to synergistic effects that increase a messenger RNA’s (mRNA’s) half-life in S. cerevisiae. Specifically, the 5′ end cluster suppresses the formation of ribosome queues, reducing the interaction of ribosome-associated quality control factors with stalled ribosomes. We experimentally validate this prediction by introducing two nonoptimal clusters into three different genes and find that their mRNA half-life increases up to fourfold. The model also predicts that in the presence of two clusters, the cluster closest to the 5′ end is the primary determinant of mRNA half-life. These results suggest the “translational ramp,” in which nonoptimal codons are located near the start codon and increase translational efficiency, may have the additional biological benefit of allowing downstream slow-codon clusters to be present without decreasing mRNA half-life. These results indicate that codon usage bias plays a more nuanced role in controlling cellular protein levels than previously thought.

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

2018 ◽  
Author(s):  
Ajeet K. Sharma ◽  
Edward P. O’Brien
Keyword(s):  
Half Life ◽  
Opposite Effect ◽  
Synergistic Effects ◽  
Cellular Protein ◽  
Protein Levels ◽  
Dead Box ◽  
Naturally Occurring ◽  
Optimal Codon ◽  
Primary Determinant ◽  
Mrna Half Life

AbstractThe 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 non-optimal 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.

scholarly journals The Impact of Leadered and Leaderless Gene Structures on Translation Efficiency, Transcript Stability, and Predicted Transcription Rates in Mycobacterium smegmatis

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Tien G. Nguyen ◽  
Diego A. Vargas-Blanco ◽  
Louis A. Roberts ◽  
Scarlet S. Shell
Keyword(s):  
Gene Expression ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Half Life ◽  
Untranslated Regions ◽  
Translation Efficiency ◽  
Production Rates ◽  
Content Type ◽  
The Impact ◽  
Mrna Half Life

ABSTRACT Regulation of gene expression is critical for Mycobacterium tuberculosis to tolerate stressors encountered during infection and for nonpathogenic mycobacteria such as Mycobacterium smegmatis to survive environmental stressors. Unlike better-studied models, mycobacteria express ∼14% of their genes as leaderless transcripts. However, the impacts of leaderless transcript structures on mRNA half-life and translation efficiency in mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5′ untranslated regions (UTRs) to mRNA half-life and translation efficiency are similarly unknown. In M. tuberculosis and M. smegmatis, the essential sigma factor, SigA, is encoded by a transcript with a relatively short half-life. We hypothesized that the long 5′ UTR of sigA causes this instability. To test this, we constructed fluorescence reporters and measured protein abundance, mRNA abundance, and mRNA half-life and calculated relative transcript production rates. The sigA 5′ UTR conferred an increased transcript production rate, shorter mRNA half-life, and decreased apparent translation rate compared to a synthetic 5′ UTR commonly used in mycobacterial expression plasmids. Leaderless transcripts appeared to be translated with similar efficiency as those with the sigA 5′ UTR but had lower predicted transcript production rates. A global comparison of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in protein/mRNA ratios for leadered and leaderless transcripts, suggesting that variability in translation efficiency is largely driven by factors other than leader status. Our data are also discussed in light of an alternative model that leads to different conclusions and suggests leaderless transcripts may indeed be translated less efficiently. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, is a major public health problem killing 1.5 million people globally each year. During infection, M. tuberculosis must alter its gene expression patterns to adapt to the stress conditions it encounters. Understanding how M. tuberculosis regulates gene expression may provide clues for ways to interfere with the bacterium’s survival. Gene expression encompasses transcription, mRNA degradation, and translation. Here, we used Mycobacterium smegmatis as a model organism to study how 5′ untranslated regions affect these three facets of gene expression in multiple ways. We furthermore provide insight into the expression of leaderless mRNAs, which lack 5′ untranslated regions and are unusually prevalent in mycobacteria.

scholarly journals The impact of leadered and leaderless gene structures on translation efficiency, transcript stability, and predicted transcription rates in Mycobacterium smegmatis

2019 ◽  
Author(s):  
Tien G. Nguyen ◽  
Diego A. Vargas-Blanco ◽  
Louis A. Roberts ◽  
Scarlet S. Shell
Keyword(s):  
Mycobacterium Smegmatis ◽  
Half Life ◽  
Regulation Of Gene Expression ◽  
Transcript Abundance ◽  
Transcription Rate ◽  
Translation Efficiency ◽  
Translation Rate ◽  
Global Comparison ◽  
The Impact ◽  
Mrna Half Life

ABSTRACTRegulation of gene expression is critical for the pathogen Mycobacterium tuberculosis to tolerate stressors encountered during infection, and for non-pathogenic mycobacteria such as Mycobacterium smegmatis to survive stressors encountered in the environment. Unlike better studied models, mycobacteria express ∼14% of their genes as leaderless transcripts. However, the impacts of leaderless transcript structures on mRNA half-life and translation efficiency in mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5’ UTRs to mRNA half-life and translation efficiency are similarly unknown. In both M. tuberculosis and M. smegmatis, the essential sigma factor, SigA, is encoded by an unstable transcript with a relatively short half-life. We hypothesized that sigA’s long 5’ UTR caused this instability. To test this, we constructed fluorescence reporters and then measured protein abundance, mRNA abundance, and mRNA half-life. From these data we also calculated relative transcription rates. We found that the sigA 5’ UTR confers an increased transcription rate, a shorter mRNA half-life, and a decreased translation rate compared to a synthetic 5’ UTR commonly used in mycobacterial expression plasmids. Leaderless transcripts produced less protein compared to any of the leadered transcripts. However, translation rates were similar to those of transcripts with the sigA 5’ UTR, and the protein levels were instead explained by lower transcript abundance. A global comparison of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in protein:mRNA ratios for natural leadered and leaderless transcripts, consistent with the idea that variability in translation efficiency among mycobacterial genes is largely driven by factors other than leader status. The variability in mRNA half-life and predicted transcription rate among our constructs could not be explained by their different translation efficiencies, indicating that other factors are responsible for these properties and highlighting the myriad and complex roles played by 5’ UTRs and other sequences downstream of transcription start sites.

scholarly journals Oxidized RNA Bodies compartmentalize translation quality control in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
James S. Dhaliwal ◽  
Cristina Panozzo ◽  
Lionel Benard ◽  
William Zerges
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Chain Complex ◽  
Processing Bodies ◽  
Rna Granules ◽  
Translation Quality ◽  
Control Factors ◽  
Cytoplasmic Rna ◽  
Nascent Chain

ABSTRACTCytoplasmic RNA granules compartmentalize phases of the translation cycle. We previously reported on the localization of oxidized RNA in human cells to cytoplasmic foci called oxidized RNA bodies (ORBs). Oxidized mRNAs are substrates of translation quality control, wherein defective mRNAs and nascent polypeptides are released from stalled ribosomes and targeted for degradation. Therefore, we asked whether ORBs compartmentalize translation quality control. Here, we identify ORBs in Saccharomyces cerevisiae and characterize them using fluorescence microscopy and proteomics. ORBs are RNA granules that are distinct from processing bodies and stress granules. Several lines of evidence support a role of ORBs in the compartmentalization of central steps in the translation quality control pathways No-Go mRNA decay and ribosome quality control. Active translation is required by both translation quality control and ORBs. ORBs contain two substrates of translation quality control: oxidized RNA and a stalled mRNA-ribosome-nascent chain complex. Translation quality control factors localize to ORBs. Translation quality control mutants have altered ORB number per cell, size, or both. Therefore, ORBs are an intracellular hub of translational quality control.

scholarly journals Effect of Translational Signals on mRNA Decay in Bacillus subtilis

2003 ◽  
Vol 185 (18) ◽  
pp. 5372-5379 ◽  
Author(s):  
Josh S. Sharp ◽  
David H. Bechhofer
Keyword(s):  
Bacillus Subtilis ◽  
Mrna Stability ◽  
Half Life ◽  
Ternary Complex ◽  
Mrna Decay ◽  
Peptide Bond ◽  
Start Codon ◽  
Transcription Terminator ◽  
Ribosome Binding ◽  
Mrna Half Life

ABSTRACT A 254-nucleotide model mRNA, designated ΔermC mRNA, was used to study the effects of translational signals and ribosome transit on mRNA decay in Bacillus subtilis. ΔermC mRNA features a strong ribosome-binding site (RBS) and a 62-amino-acid-encoding open reading frame, followed by a transcription terminator structure. Inactivation of the RBS or the start codon resulted in a fourfold decrease in the mRNA half-life, demonstrating the importance of ternary complex formation for mRNA stability. Data for the decay of ΔermC mRNAs with stop codons at positions increasingly proximal to the translational start site showed that actual translation—even the formation of the first peptide bond—was not important for stability. The half-life of an untranslated 3.2-kb ΔermC-lacZ fusion RNA was similar to that of a translated ΔermC-lacZ mRNA, indicating that the translation of even a longer RNA was not required for wild-type stability. The data are consistent with a model in which ribosome binding and the formation of the ternary complex interfere with a 5′-end-dependent activity, possibly a 5′-binding endonuclease, which is required for the initiation of mRNA decay. This model is supported by the finding that increasing the distance from the 5′ end to the start codon resulted in a 2.5-fold decrease in the mRNA half-life. These results underscore the importance of the 5′ end to mRNA stability in B. subtilis.

scholarly journals Reciprocal hemizygosity analysis reveals that the Saccharomyces cerevisiae CGI121 gene affects lag time duration in synthetic grape must

2021 ◽  
Author(s):  
Runze Li ◽  
Rebecca C Deed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Low Temperatures ◽  
Lag Time ◽  
Lag Phase ◽  
Phenotypic Traits ◽  
Time Duration ◽  
Phase Duration ◽  
Grape Must ◽  
The Impact ◽  
Reciprocal Hemizygosity Analysis

Abstract It is standard practice to ferment white wines at low temperatures (10-18 °C). However, low temperatures increase fermentation duration and risk of problem ferments, leading to significant costs. The lag duration at fermentation initiation is heavily impacted by temperature; therefore, identification of Saccharomyces cerevisiae genes influencing fermentation kinetics is of interest for winemaking. We selected 28 S. cerevisiae BY4743 single deletants, from a prior list of open reading frames (ORFs) mapped to quantitative trait loci (QTLs) on chromosomes VII and XIII, influencing the duration of fermentative lag time. Five BY4743 deletants, Δapt1, Δcgi121, Δclb6, Δrps17a, and Δvma21, differed significantly in their fermentative lag duration compared to BY4743 in synthetic grape must (SGM) at 15 °C, over 72 h. Fermentation at 12.5 °C for 528 h confirmed the longer lag times of BY4743 Δcgi121, Δrps17a, and Δvma21. These three candidate ORFs were deleted in S. cerevisiae RM11-1a and S288C to perform single reciprocal hemizygosity analysis (RHA). RHA hybrids and single deletants of RM11-1a and S288C were fermented at 12.5 °C in SGM and lag time measurements confirmed that the S288C allele of CGI121 on chromosome XIII, encoding a component of the EKC/KEOPS complex, increased fermentative lag phase duration. Nucleotide sequences of RM11-1a and S288C CGI121 alleles differed by only one synonymous nucleotide, suggesting that intron splicing, codon bias, or positional effects might be responsible for the impact on lag phase duration. This research demonstrates a new role of CGI121 and highlights the applicability of QTL analysis for investigating complex phenotypic traits in yeast.

Cytosolic protein quality control machinery: Interactions of Hsp70 with a network of co-chaperones and substrates

2021 ◽  
pp. 153537022199981
Author(s):  
Chamithi Karunanayake ◽  
Richard C Page
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Structural Features ◽  
Cellular Protein ◽  
Mechanisms Of Action ◽  
Control Mechanisms ◽  
Nucleotide Exchange ◽  
Domain Organization ◽  
Nucleotide Exchange Factors

The chaperone heat shock protein 70 (Hsp70) and its network of co-chaperones serve as a central hub of cellular protein quality control mechanisms. Domain organization in Hsp70 dictates ATPase activity, ATP dependent allosteric regulation, client/substrate binding and release, and interactions with co-chaperones. The protein quality control activities of Hsp70 are classified as foldase, holdase, and disaggregase activities. Co-chaperones directly assisting protein refolding included J domain proteins and nucleotide exchange factors. However, co-chaperones can also be grouped and explored based on which domain of Hsp70 they interact. Here we discuss how the network of cytosolic co-chaperones for Hsp70 contributes to the functions of Hsp70 while closely looking at their structural features. Comparison of domain organization and the structures of co-chaperones enables greater understanding of the interactions, mechanisms of action, and roles played in protein quality control.

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