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 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.

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 Na,K-ATPase β1-Subunit Increases the Translation Efficiency of the α1-Subunit in MSV-MDCK Cells

2004 ◽  
Vol 15 (7) ◽  
pp. 3224-3232 ◽  
Author(s):  
Sigrid A. Rajasekaran ◽  
Jegan Gopal ◽  
Dianna Willis ◽  
Cromwell Espineda ◽  
Jeffery L. Twiss ◽  
...  
Keyword(s):  
Half Life ◽  
Mammalian Cells ◽  
Mdck Cells ◽  
Short Pulse ◽  
Ectopic Expression ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Translation Rate ◽  
Subunit Protein ◽  
Subunit Mrna

The Na,K-ATPase consists of an α- and β-subunit. Moloney sarcoma virus-transformed MDCK cells (MSV-MDCK) express low levels of Na,K-ATPase β1-subunit. Ectopic expression of Na,K-ATPase β1-subunit in these cells increased the protein levels of the α1-subunit of Na,K-ATPase. This increase was not due to altered transcription of the α1-subunit gene or half-life of the α1-subunit protein because both α1-subunit mRNA levels and half-life of the α1-subunit protein were comparable in MSV-MDCK and β1-subunit expressing MSV-MDCK cells. However, short pulse labeling revealed that the initial translation rate of the α1-subunit in β1-subunit expressing MSV-MDCK cells was six- to sevenfold higher compared with MSV-MDCK cells. The increased translation was specific to α1-subunit because translation rates of occludin and β-catenin, membrane and cytosolic proteins, respectively, were not altered. In vitro cotranslation/translocation experiments using rabbit reticulocyte lysate and rough microsomes revealed that the α1-subunit mRNA is more efficiently translated in the presence of β1-subunit. Furthermore, sucrose density gradient analysis revealed significantly more α1-subunit transcript associated with the polysomal fraction in β1-subunit expressing MSV-MDCK cells compared with MSV-MDCK cells, indicating that in mammalian cells the Na,K-ATPase β1-subunit is involved in facilitating the translation of the α1-subunit mRNA in the endoplasmic reticulum.

scholarly journals Buffering of transcription rate by mRNA half-life is a conserved feature of Rett syndrome models

2021 ◽  
Author(s):  
Deivid Carvalho Rodrigues ◽  
Marat Mufteev ◽  
Kyoko E Yuki ◽  
Ashrut Narula ◽  
Wei Wei ◽  
...  
Keyword(s):  
Rett Syndrome ◽  
Half Life ◽  
Rna Binding ◽  
Neurodevelopmental Disorder ◽  
Rna Binding Protein ◽  
Transcription Rate ◽  
Mrna Levels ◽  
Life Changes ◽  
Limited Evidence ◽  
Mrna Half Life

Models of MECP2 dysfunction in Rett syndrome (RTT) assume that transcription rate changes directly correlate with altered steady-state mRNA levels. However, limited evidence suggests that transcription rate changes are buffered by poorly understood compensatory post-transcriptional mechanisms. Here we measure transcription rate and mRNA half-life changes in RTT patient neurons using RATE-seq, and reinterpret nuclear and whole-cell RNAseq from Mecp2 mice. Genes are dysregulated by changing transcription rate only or half-life only and are buffered when both are changed. We utilized classifier models to understand the direction of transcription rate changes based on gene-body DNA sequence, and combined frequencies of three dinucleotides were better predictors than contributions by CA and CG. MicroRNA and RNA-Binding Protein (RBP) motifs were enriched in 3ʹUTRs of genes with half-life changes. Motifs for nuclear localized RBPs were enriched on buffered genes with increased transcription rate. Our findings identify post-transcriptional mechanisms in humans and mice that alter half-life only or buffer transcription rate changes when a transcriptional modulator gene is mutated in a neurodevelopmental disorder.

scholarly journals The transcription factor SlHY5 regulates the ripening of tomato fruit at both the transcriptional and translational levels

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Weihao Wang ◽  
Peiwen Wang ◽  
Xiaojing Li ◽  
Yuying Wang ◽  
Shiping Tian ◽  
...  
Keyword(s):  
Fruit Ripening ◽  
Nutritional Quality ◽  
Ribosomal Proteins ◽  
Anthocyanin Biosynthesis ◽  
Tomato Fruit ◽  
Critical Role ◽  
Regulation Of Gene Expression ◽  
Protein Translation ◽  
Transcriptional Level ◽  
Translation Efficiency

AbstractLight plays a critical role in plant growth and development, but the mechanisms through which light regulates fruit ripening and nutritional quality in horticultural crops remain largely unknown. Here, we found that ELONGATED HYPOCOTYL 5 (HY5), a master regulator in the light signaling pathway, is required for normal fruit ripening in tomato (Solanum lycopersicum). Loss of function of tomato HY5 (SlHY5) impairs pigment accumulation and ethylene biosynthesis. Transcriptome profiling identified 2948 differentially expressed genes, which included 1424 downregulated and 1524 upregulated genes, in the Slhy5 mutants. In addition, genes involved in carotenoid and anthocyanin biosynthesis and ethylene signaling were revealed as direct targets of SlHY5 by chromatin immunoprecipitation. Surprisingly, the expression of a large proportion of genes encoding ribosomal proteins was downregulated in the Slhy5 mutants, and this downregulation pattern was accompanied by a decrease in the abundance of ribosomal proteins. Further analysis demonstrated that SlHY5 affected the translation efficiency of numerous ripening-related genes. These data indicate that SlHY5 regulates fruit ripening both at the transcriptional level by targeting specific molecular pathways and at the translational level by affecting the protein translation machinery. Our findings unravel the regulatory mechanisms of SlHY5 in controlling fruit ripening and nutritional quality and uncover the multifaceted regulation of gene expression by transcription factors.

scholarly journals m5U54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs

2021 ◽  
Vol 22 (6) ◽  
pp. 2941
Author(s):  
Marisa Pereira ◽  
Diana R. Ribeiro ◽  
Miguel M. Pinheiro ◽  
Margarida Ferreira ◽  
Stefanie Kellner ◽  
...  
Keyword(s):  
Stress Response ◽  
Small Rnas ◽  
Mammalian Cells ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Translation Regulation ◽  
Translation Efficiency ◽  
Down Regulation ◽  
Gene Expression Control ◽  
The Impact

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5′tRNA-derived stress-induced RNAs (5′tiRNAs), namely 5′tiRNA-GlyGCC and 5′tiRNA-GluCTC, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.

Speciation and changes in male gene expression in Drosophila

Genome ◽  
2020 ◽  
pp. 1-11
Author(s):  
Bahar Patlar ◽  
Alberto Civetta
Keyword(s):  
Gene Expression ◽  
Reproductive Isolation ◽  
Potential Role ◽  
Regulation Of Gene Expression ◽  
Drosophila Model ◽  
Depth Analysis ◽  
The Impact ◽  
Plastic Responses ◽  
Male Gene

It has long been acknowledged that changes in the regulation of gene expression may account for major organismal differences. However, we still do not fully understand how changes in gene expression evolve and how do such changes influence organisms’ differences. We are even less aware of the impact such changes might have in restricting gene flow between species. Here, we focus on studies of gene expression and speciation in the Drosophila model. We review studies that have identified gene interactions in post-mating reproductive isolation and speciation, particularly those that modulate male gene expression. We also address studies that have experimentally manipulated changes in gene expression to test their effect in post-mating reproductive isolation. We highlight the need for a more in-depth analysis of the role of selection causing disrupted gene expression of such candidate genes in sterile/inviable hybrids. Moreover, we discuss the relevance to incorporate more routinely assays that simultaneously evaluate the potential effects of environmental factors and genetic background in modulating plastic responses in male genes and their potential role in speciation.

The KH-type splicing regulatory protein (KSRP) regulates type III interferon expression post-transcriptionally

2019 ◽  
Vol 476 (2) ◽  
pp. 333-352 ◽  
Author(s):  
Lisa Schmidtke ◽  
Katharina Schrick ◽  
Sabrina Saurin ◽  
Rudolf Käfer ◽  
Fabian Gather ◽  
...  
Keyword(s):  
Half Life ◽  
Rna Binding ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Rna Binding Protein ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Type Iii ◽  
Type Iii Ifn ◽  
Mrna Half Life

Abstract Type III interferons (IFNs) are the latest members of the IFN family. They play an important role in immune defense mechanisms, especially in antiviral responses at mucosal sites. Moreover, they control inflammatory reactions by modulating neutrophil and dendritic cell functions. Therefore, it is important to identify cellular mechanisms involved in the control of type III IFN expression. All IFN family members contain AU-rich elements (AREs) in the 3′-untranslated regions (3′-UTR) of their mRNAs that determine mRNA half-life and consequently the expressional level of these cytokines. mRNA stability is controlled by different proteins binding to these AREs leading to either stabilization or destabilization of the respective target mRNA. The KH-type splicing regulatory protein KSRP (also named KHSRP) is an important negative regulator of ARE-containing mRNAs. Here, we identify the interferon lambda 3 (IFNL3) mRNA as a new KSRP target by pull-down and immunoprecipitation experiments, as well as luciferase reporter gene assays. We characterize the KSRP-binding site in the IFNL3 3′-UTR and demonstrate that KSRP regulates the mRNA half-life of the IFNL3 transcript. In addition, we detect enhanced expression of IFNL3 mRNA in KSRP−/− mice, establishing a negative regulatory function of KSRP in type III IFN expression also in vivo. Besides KSRP the RNA-binding protein AUF1 (AU-rich element RNA-binding protein 1) also seems to be involved in the regulation of type III IFN mRNA expression.

scholarly journals Retrograde Control of Cytosolic Translation Targets Synthesis of Plastid Localized Proteins and Nuclear Responses for Efficient Light Acclimation

2021 ◽  
Author(s):  
Marten Moore ◽  
Aaron Smith ◽  
Corinna Wesemann ◽  
Sonja Schmidtpott ◽  
Melanie Wegener ◽  
...  
Keyword(s):  
Transcriptional Response ◽  
Transcript Abundance ◽  
Protein Translation ◽  
Retrograde Signaling ◽  
Just In Time ◽  
Protein Accumulation ◽  
Translation Efficiency ◽  
Multiple Components ◽  
Associated Proteins ◽  
Nuclear Responses

AbstractCanonical retrograde signaling is the transmission of information from organelles to the nucleus. Discrepancies between protein accumulation and transcript abundance in response to oxidative stress were suggestive of protein translation responding to retrograde signaling. Here we uncover multiple components of a translation-dependent retrograde signaling pathway that impact translation efficiency and gene expression, including the kinases, MPK6 and the SnRK1 subunit, AKIN10. Global ribosome foot-printing demonstrated rapid differential loading of 939 of transcripts from polyribosomes within 10 min after transfer from Low to High-light. Translationally regulated transcripts shared motifs in their 5’-UTR that act as binding sites for RBPs such as GAPC. The Stress Associated Proteins 2 and 3 carry such motifs in their UTRs and interact with the calcium sensor Calmodulin-like 49, relocating to the nucleus to co-regulate a translation-dependent transcriptional response. Translation dependent retrograde signaling bifurcates into a direct translational circuit and a translation-reliant nuclear circuit synchronizing translation, nuclear and anterograde response pathways, which may serve as a just in time-provision of needed proteins to the plastids.

scholarly journals A psychrometric model to assess the biological decay of the SARS-CoV-2 virus in aerosols

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11024
Author(s):  
Clive B. Beggs ◽  
Eldad J. Avital
Keyword(s):  
Air Temperature ◽  
Vapour Pressure ◽  
Half Life ◽  
Analytical Study ◽  
Decay Constant ◽  
Meteorological Data ◽  
Environmental Parameters ◽  
Published Data ◽  
Reasonable Accuracy ◽  
The Impact

There is increasing evidence that the 2020 COVID-19 pandemic has been influenced by variations in air temperature and humidity. However, the impact that these environmental parameters have on survival of the SARS-CoV-2 virus has not been fully characterised. Therefore, an analytical study was undertaken using published data to develop a psychrometric model to assess the biological decay rate of the virus in aerosols. This revealed that it is possible to describe with reasonable accuracy (R2 = 0.718, p < 0.001) the biological decay constant for the SARS-CoV-2 virus using a regression model with enthalpy, vapour pressure and specific volume as predictors. Applying this to historical meteorological data from London, Paris and Milan over the pandemic period, produced results which indicate that the average half-life of the virus in aerosols outdoors was in the region 13–22 times longer in March 2020, when the outbreak was accelerating, than it was in August 2020 when epidemic in Europe was at its nadir. However, indoors, this variation is likely to be much less. As such, this suggests that changes in virus survivability due the variations in the psychrometric qualities of the air might influence the transmission of SARS-CoV-2.

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