scholarly journals Absolute measurements of mRNA translation inC. crescentusreveal important fitness costs of vitamin B12scavenging

2019 ◽  
Author(s):  
James R. Aretakis ◽  
Alisa Gega ◽  
Jared M. Schrader
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Program Analysis ◽  
Caulobacter Crescentus ◽  
Mrna Translation ◽  
Cellular Protein ◽  
Ribosome Profiling ◽  
Growth Media ◽  
Cell Cycle Gene ◽  
Absolute Measurements

AbstractCaulobacter crescentusis a model for the bacterial cell cycle which culminates in asymmetric cell division, yet little is known about the absolute levels of protein synthesis of the cellular parts needed to complete the cell cycle. Here we utilize ribosome profiling to provide absolute measurements of mRNA translation of theC. crescentusgenome, providing an important resource for the complete elucidation of the cell cycle gene-regulatory program. Analysis of protein synthesis rates revealed ∼4.5% of cellular protein synthesis are for genes related to vitamin B12import (btuB) and B12independent methionine biosynthesis (metE) when grown in common growth media lacking B12. While its facultative B12lifestyle provides a fitness advantage in the absence of B12, we find that it provides lower fitness of the cells in the presence of B12, potentially explaining why manyCaulobacterspecies have lost themetEgene and become obligates for B12.

scholarly journals Absolute Measurements of mRNA Translation inCaulobacter crescentusReveal Important Fitness Costs of Vitamin B12Scavenging

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
James R. Aretakis ◽  
Alisa Gega ◽  
Jared M. Schrader
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Cell Division ◽  
Asymmetric Cell Division ◽  
Caulobacter Crescentus ◽  
Mrna Translation ◽  
Genetic Network ◽  
Ribosome Profiling ◽  
Content Type ◽  
Bacterial Cell Cycle

ABSTRACTCaulobacter crescentusis a model for the bacterial cell cycle which culminates in asymmetric cell division, yet little is known about the absolute levels of protein synthesis of the cellular parts needed to complete the cell cycle. Here we utilize ribosome profiling to provide absolute measurements of mRNA translation inC. crescentus, providing an important resource with quantitative genome-wide measurements of protein output across individual genes. Analysis of protein synthesis rates revealed ∼4.5% of cellular protein synthesis is for genes related to vitamin B12import (btuB) and B12-independent methionine biosynthesis (metE) when grown in common growth media lacking B12. While its facultative B12lifestyle provides a fitness advantage in the absence of B12, we find that it provides a fitness disadvantage of the cells in the presence of B12, potentially explaining why manyCaulobacterspecies have lost themetEgene and become obligates for B12.IMPORTANCECaulobacter crescentusis a model system of the bacterial cell cycle culminating in asymmetric cell division, with each daughter cell inheriting a distinct set of proteins. While a genetic network of master transcription factors coordinates the cell cycle timing of transcription for nearly 20% ofCaulobactergenes, we lack knowledge of how many of each protein “part” encoded in the genome are synthesized. Therefore, to determine the absolute production rates across the genome, we performed ribosome profiling, providing, for the first time, a quantitative resource with measurements of each protein “part” needed to generate daughter cells. This resource furthers the goal of a systems-level understanding of the genetic network controlling asymmetric cell division. To highlight the utility of this data set, we probe the protein synthesis cost of a B12utilization pathway and provide new insights intoCaulobacter’s adaptation to its natural environments.

scholarly journals Upregulation of 5′-terminal oligopyrimidine mRNA translation upon loss of the ARF tumor suppressor

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kyle A. Cottrell ◽  
Ryan C. Chiou ◽  
Jason D. Weber
Keyword(s):  
Protein Synthesis ◽  
Tumor Cells ◽  
Ribosomal Proteins ◽  
Human Cancer ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Translational Efficiency ◽  
Gene Encoding ◽  
Terminal Oligopyrimidine ◽  
Ribosome Export

AbstractTumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How the numerous mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5′-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 largely phenocopies ARF loss, with increased protein synthesis and expression of 5′-TOP encoded proteins. The 5′-TOP regulators eIF4G1 and LARP1 are upregulated in Arf- and p53-null cells.

scholarly journals Genetic removal of p70 S6K1 corrects coding sequence length-dependent alterations in mRNA translation in fragile X syndrome mice

2020 ◽  
Author(s):  
Sameer Aryal ◽  
Francesco Longo ◽  
Eric Klann
Keyword(s):  
Protein Synthesis ◽  
Fragile X Syndrome ◽  
Fragile X ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Sequence Length ◽  
Rna Seq ◽  
Double Knockout ◽  
Coding Sequence ◽  
Mental Retardation Protein

AbstractLoss of the fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS). FMRP is widely thought to repress protein synthesis, but its translational targets and modes of control remain in dispute. We previously showed that genetic removal of p70 S6 kinase 1 (S6K1) corrects altered protein synthesis as well as synaptic and behavioral phenotypes in FXS mice. In this study, we examined the gene-specificity of altered mRNA translation in FXS and the mechanism of rescue with genetic reduction of S6K1 by carrying out ribosome profiling and RNA-Seq on cortical lysates from wild-type, FXS, S6K1 knockout, and double knockout mice. We observed reduced ribosome footprint abundance in the majority of differentially translated genes in the cortices of FXS mice. We used molecular assays to discover evidence that the reduction in ribosome footprint abundance reflects an increased rate of ribosome translocation, which is captured as a decrease in the number of translating ribosomes at steady state, and is normalized by inhibition of S6K1. We also found that genetic removal of S6K1 prevented a positive-to-negative gradation of alterations in translation efficiencies (RF/mRNA) with coding sequence length across mRNAs in FXS mouse cortices. Our findings reveal the identities of dysregulated mRNAs and a molecular mechanism by which reduction of S6K1 prevents altered translation in FXS.

scholarly journals ARF suppresses 5’-terminal oligopyrimidine mRNA translation

2020 ◽  
Author(s):  
Kyle A. Cottrell ◽  
Ryan C. Chiou ◽  
Jason D. Weber
Keyword(s):  
Protein Synthesis ◽  
Tumor Cells ◽  
Ribosomal Proteins ◽  
Human Cancer ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Translational Efficiency ◽  
Gene Encoding ◽  
Terminal Oligopyrimidine ◽  
Ribosome Export

AbstractTumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How many of the mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5’-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 caused a similar increase in 5’-TOP mRNA translation. The 5’-TOP regulators mTORC1, eIF4G1 and LARP1 are dysregulated in ARF and p53 null cells.

scholarly journals Inferring efficiency of translation initiation and elongation from ribosome profiling

2020 ◽  
Vol 48 (17) ◽  
pp. 9478-9490
Author(s):  
Juraj Szavits-Nossan ◽  
Luca Ciandrini
Keyword(s):  
Protein Synthesis ◽  
Mathematical Modelling ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Elongation Rate ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Recent Advances ◽  
Two Stages

Abstract One of the main goals of ribosome profiling is to quantify the rate of protein synthesis at the level of translation. Here, we develop a method for inferring translation elongation kinetics from ribosome profiling data using recent advances in mathematical modelling of mRNA translation. Our method distinguishes between the elongation rate intrinsic to the ribosome’s stepping cycle and the actual elongation rate that takes into account ribosome interference. This distinction allows us to quantify the extent of ribosomal collisions along the transcript and identify individual codons where ribosomal collisions are likely. When examining ribosome profiling in yeast, we observe that translation initiation and elongation are close to their optima and traffic is minimized at the beginning of the transcript to favour ribosome recruitment. However, we find many individual sites of congestion along the mRNAs where the probability of ribosome interference can reach $50\%$. Our work provides new measures of translation initiation and elongation efficiencies, emphasizing the importance of rating these two stages of translation separately.

scholarly journals Structural Basis for Competitive Inhibition of eIF4G-Mnk1 Interaction by the Adenovirus 100-Kilodalton Protein

2004 ◽  
Vol 78 (14) ◽  
pp. 7707-7716 ◽  
Author(s):  
Rafael Cuesta ◽  
Qiaoran Xi ◽  
Robert J. Schneider
Keyword(s):  
Protein Synthesis ◽  
Competitive Inhibition ◽  
Late Phase ◽  
Mrna Translation ◽  
Cellular Protein ◽  
Structural Basis ◽  
Binding Motif ◽  
Amino Acid Peptide ◽  
Cellular Mrnas ◽  
Cellular Protein Synthesis

ABSTRACT Translation of most cellular mRNAs involves cap binding by the translation initiation complex. Among this complex of proteins are cap-binding protein eIF4E and the eIF4E kinase Mnk1. Cap-dependent mRNA translation generally correlates with Mnk1 phosphorylation of eIF4E when both are bound to eIF4G. During the late phase of adenovirus (Ad) infection translation of cellular mRNA is inhibited, which correlates with displacement of Mnk1 from eIF4G by the viral 100-kDa (100K) protein and dephosphorylation of eIF4E. Here we describe the molecular mechanism for 100K protein displacement of Mnk1 from eIF4G and elucidate a structural basis for eIF4G interaction with Mnk1 and 100K proteins and Ad inhibition of cellular protein synthesis. The eIF4G-binding site is located in an N-terminal 66-amino-acid peptide of 100K which is sufficient to bind eIF4G, displace Mnk1, block eIF4E phosphorylation, and inhibit eIF4F (cap)-dependent cellular mRNA translation. Ad 100K and Mnk1 proteins possess a common eIF4G-binding motif, but 100K protein binds more strongly to eIF4G than does Mnk1. Unlike Mnk1, for which binding to eIF4G is RNA dependent, competitive binding by 100K protein is RNA independent. These data support a model whereby 100K protein blocks cellular protein synthesis by coopting eIF4G and cap-initiation complexes regardless of their association with mRNA and displacing or blocking binding by Mnk1, which occurs only on preassembled complexes, resulting in dephosphorylation of eIF4E.

scholarly journals A systematic view on influenza induced host shutoff

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Adi Bercovich-Kinori ◽  
Julie Tai ◽  
Idit Anna Gelbart ◽  
Alina Shitrit ◽  
Shani Ben-Moshe ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Influenza A ◽  
Mrna Translation ◽  
Cellular Protein ◽  
Ribosome Profiling ◽  
Viral Mrnas ◽  
Viral Propagation ◽  
Mrna Pool ◽  
Common Strategy ◽  
Host Shutoff

Host shutoff is a common strategy used by viruses to repress cellular mRNA translation and concomitantly allow the efficient translation of viral mRNAs. Here we use RNA-sequencing and ribosome profiling to explore the mechanisms that are being utilized by the Influenza A virus (IAV) to induce host shutoff. We show that viral transcripts are not preferentially translated and instead the decline in cellular protein synthesis is mediated by viral takeover on the mRNA pool. Our measurements also uncover strong variability in the levels of cellular transcripts reduction, revealing that short transcripts are less affected by IAV. Interestingly, these mRNAs that are refractory to IAV infection are enriched in cell maintenance processes such as oxidative phosphorylation. Furthermore, we show that the continuous oxidative phosphorylation activity is important for viral propagation. Our results advance our understanding of IAV-induced shutoff, and suggest a mechanism that facilitates the translation of genes with important housekeeping functions.

scholarly journals A toxin-antitoxin system associated transcription factor of Caulobacter crescentus can influence cell cycle-regulated gene expression during the SOS response

2020 ◽  
Author(s):  
Koyel Ghosh ◽  
Kamilla Ankær Brejndal ◽  
Clare L. Kirkpatrick
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Transcription Factor ◽  
Caulobacter Crescentus ◽  
Sos Response ◽  
Cell Cycle Gene ◽  
Regulated Gene Expression ◽  
Cell Cycle Gene Expression

AbstractToxin-antitoxin (TA) systems are widespread in bacterial chromosomes but their functions remain enigmatic. Although many are transcriptionally upregulated by stress conditions, it is unclear what role they play in cellular responses to stress and to what extent the role of a given TA system homologue varies between different bacterial species. In this work we investigate the role of the DNA damage-inducible TA system HigBA of Caulobacter crescentus in the SOS response and discover that in addition to the toxin HigB affecting cell cycle gene expression through inhibition of the master regulator CtrA, HigBA possesses a transcription factor third component, HigC, which both auto-regulates the TA system and acts independently of it. Through HigC, the system exerts downstream effects on antibiotic (ciprofloxacin) resistance and cell cycle gene expression. HigB and HigC had inverse effects on cell cycle gene regulation, with HigB reducing and HigC increasing the expression of CtrA-dependent promoters. Neither HigBA nor HigC had any effect on formation of persister cells in response to ciprofloxacin. Rather, their role in the SOS response appears to be as transcriptional and post-transcriptional regulators of cell cycle-dependent gene expression, transmitting the status of the SOS response as a regulatory input into the cell cycle control network via CtrA.ImportanceAlmost all bacteria respond to DNA damage by upregulating a set of genes that helps them to repair and recover from the damage, known as the SOS response. The set of genes induced during the SOS response varies between species, but frequently includes toxin-antitoxin systems. However, it is unknown what the consequence of inducing these systems is, and whether they provide any benefit to the cells. We show here that the DNA damage-induced TA system HigBA of the asymmetrically dividing bacterium Caulobacter crescentus affects the cell cycle regulation of this bacterium. HigBA also has a transcription factor encoded immediately downstream of it, named here HigC, which controls expression of the TA system and potentially other genes as well. Therefore, this work identifies a new role for TA systems in the DNA damage response, distinct from non-specific stress tolerance mechanisms which had been proposed previously.

Sign in / Sign up

Export Citation Format

Share Document