scholarly journals Transcriptome-wide in vivo mapping of cleavage sites for the compact cyanobacterial ribonuclease E reveals insights into its function and substrate recognition

2021 ◽  
Author(s):  
Ute A. Hoffmann ◽  
Florian Heyl ◽  
Said N. Rogh ◽  
Thomas Wallner ◽  
Rolf Backofen ◽  
...  
Keyword(s):  
Temperature Shift ◽  
Temperature Sensitive ◽  
Cleavage Sites ◽  
Rnase E ◽  
Ribonuclease E ◽  
Regulatory Processes ◽  
Transcriptional Regulatory ◽  
Acting Mechanism ◽  
Post Transcriptional Regulation

Ribonucleases are crucial enzymes in RNA metabolism and post-transcriptional regulatory processes in bacteria. Cyanobacteria encode the two essential ribonucleases RNase E and RNase J. Cyanobacterial RNase E is shorter than homologues in other groups of bacteria and lacks both the chloroplast-specific N-terminal extension as well as the C-terminal domain typical for RNase E of enterobacteria. In order to investigate the function of RNase E in the model cyanobacterium Synechocystis sp. PCC 6803, we engineered a temperature-sensitive RNase E mutant by introducing two site-specific mutations, I65F and spontaneously occurring V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1,472 RNase-E-dependent cleavage sites. We inferred a dominating cleavage signature consisting of an adenine at the -3 and a uridine at the +2 position within a single-stranded segment of the RNA. The data identified putative RNase-E-dependent instances of operon discoordination, mRNAs likely regulated jointly by RNase E and an sRNA, potential 3' end-derived sRNAs and a dual-acting mechanism for the glutamine riboswitch. Our findings substantiate the pivotal role of RNase E in post-transcriptional regulation and suggest the redundant or concerted action of RNase E and RNase J in cyanobacteria.

scholarly journals Maturation of UTR-Derived sRNAs Is Modulated during Adaptation to Different Growth Conditions

2021 ◽  
Vol 22 (22) ◽  
pp. 12260
Author(s):  
Daniel-Timon Spanka ◽  
Gabriele Klug
Keyword(s):  
Growth Conditions ◽  
Global Scale ◽  
Rnase E ◽  
Bacterial Gene ◽  
Ribonuclease E ◽  
Mrna Targets ◽  
Small Regulatory Rnas ◽  
The Stability ◽  
Ngs Data

Small regulatory RNAs play a major role in bacterial gene regulation by binding their target mRNAs, which mostly influences the stability or translation of the target. Expression levels of sRNAs are often regulated by their own promoters, but recent reports have highlighted the presence and importance of sRNAs that are derived from mRNA 3′ untranslated regions (UTRs). In this study, we investigated the maturation of 5′ and 3′ UTR-derived sRNAs on a global scale in the facultative phototrophic alphaproteobacterium Rhodobacter sphaeroides. Including some already known UTR-derived sRNAs like UpsM or CcsR1-4, 14 sRNAs are predicted to be located in 5′ UTRs and 16 in 3′ UTRs. The involvement of different ribonucleases during maturation was predicted by a differential RNA 5′/3′ end analysis based on RNA next generation sequencing (NGS) data from the respective deletion strains. The results were validated in vivo and underline the importance of polynucleotide phosphorylase (PNPase) and ribonuclease E (RNase E) during processing and maturation. The abundances of some UTR-derived sRNAs changed when cultures were exposed to external stress conditions, such as oxidative stress and also during different growth phases. Promoter fusions revealed that this effect cannot be solely attributed to an altered transcription rate. Moreover, the RNase E dependent cleavage of several UTR-derived sRNAs varied significantly during the early stationary phase and under iron depletion conditions. We conclude that an alteration of ribonucleolytic processing influences the levels of UTR-derived sRNAs, and may thus indirectly affect their mRNA targets.

scholarly journals RNase E Enzymes from Rhodobacter capsulatus and Escherichia coli Differ in Context- and Sequence-Dependent In Vivo Cleavage within the Polycistronicpuf mRNA

1999 ◽  
Vol 181 (24) ◽  
pp. 7621-7625 ◽  
Author(s):  
Claudia Heck ◽  
Elena Evguenieva-Hackenberg ◽  
Angelika Balzer ◽  
Gabriele Klug
Keyword(s):  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Cleavage Sites ◽  
Rnase E ◽  
Endonucleolytic Cleavage ◽  
Sequence Dependent

ABSTRACT The 5′ pufQ mRNA segment and the pufLMXmRNA segment of Rhodobacter capsulatus exhibit different stabilities. Degradation of both mRNA segments is initiated by RNase E-mediated endonucleolytic cleavage. While RhodobacterRNase E does not discriminate between the different sequences present around the cleavage sites within pufQ and pufL,Escherichia coli RNase E shows preference for the sequence harboring more A and U residues.

Conditional defect in mRNA 3' end processing caused by a mutation in the gene for poly(A) polymerase

1992 ◽  
Vol 12 (7) ◽  
pp. 3297-3304
Author(s):  
D Patel ◽  
J S Butler
Keyword(s):  
Temperature Shift ◽  
Temperature Sensitive ◽  
Single Mutation ◽  
Mrna Levels ◽  
Endonucleolytic Cleavage ◽  
Normal Site ◽  
Apparent Loss ◽  
And Function

Maturation of most eukaryotic mRNA 3' ends requires endonucleolytic cleavage and polyadenylation of precursor mRNAs. To further understand the mechanism and function of mRNA 3' end processing, we identified a temperature-sensitive mutant of Saccharomyces cerevisiae defective for polyadenylation. Genetic analysis showed that the polyadenylation defect and the temperature sensitivity for growth result from a single mutation. Biochemical analysis of extracts from this mutant shows that the polyadenylation defect occurs at a step following normal site-specific cleavage of a pre-mRNA at its polyadenylation site. Molecular cloning and characterization of the wild-type allele of the mutated gene revealed that it (PAP1) encodes a previously characterized poly(A) polymerase with unknown RNA substrate specificity. Analysis of mRNA levels and structure in vivo indicate that shift of growing, mutant cells to the nonpermissive temperature results in the production of poly(A)-deficient mRNAs which appear to end at their normal cleavage sites. Interestingly, measurement of the rate of protein synthesis after the temperature shift shows that translation continues long after the apparent loss of polyadenylated mRNA. Our characterization of the pap1-1 defect implicates this gene as essential for mRNA 3' end formation in S. cerevisiae.

scholarly journals Additional layer of regulation via convergent gene orientation in yeasts

2019 ◽  
Author(s):  
Jules Gilet ◽  
Romain Conte ◽  
Claire Torchet ◽  
Lionel Benard ◽  
Ingrid Lafontaine
Keyword(s):  
Transcriptional Regulation ◽  
Additional Layer ◽  
Gene Orientation ◽  
Messenger Ribonucleoprotein ◽  
Binding Preference ◽  
Transcriptional Regulatory ◽  
Additional Level ◽  
Duplex Formation ◽  
Post Transcriptional Regulation

AbstractConvergent gene pairs can produce transcripts with complementary sequences. We had shown that mRNA duplexes form in vivo in Saccharomyces cerevisiae via interactions of their 3′-ends and can lead to post-transcriptional regulatory events. Here we show that mRNA duplex formation is restricted to convergent genes separated by short intergenic distance, independently of their 3′-UTR length. We disclose an enrichment in genes involved in biological processes related to stress among these convergent genes. They are markedly conserved in convergent orientation in budding yeasts, meaning that this mode of post-transcriptional regulation could be shared in these organisms, conferring an additional level for modulating stress response. We thus investigated the mechanistic advantages potentially conferred by 3′-UTR mRNA interactions. Analysis of genome-wide transcriptome data revealed that Pat1 and Lsm1 factors, having 3′-UTR binding preference and participating to the remodeling of messenger ribonucleoprotein particles, bind differently these mRNA duplexes in comparison to canonical mRNAs. Functionally, the translational repression upon stress also appears limited on mRNA duplexes. We thus propose that mRNA duplex formation modulates the regulation of mRNA expression by limiting their access to translational repressors. Our results thus show that post-transcriptional regulation is an additional factor that determine the order of coding genes.

scholarly journals Dynamics of transcriptional and post-transcriptional regulation

2020 ◽  
Author(s):  
Mattia Furlan ◽  
Stefano de Pretis ◽  
Mattia Pelizzola
Keyword(s):  
Gene Expression ◽  
Gene Expression Regulation ◽  
Metabolic Labeling ◽  
Degradation Kinetic ◽  
Regulatory Processes ◽  
Transcriptional Regulatory ◽  
Kinetic Rates ◽  
Complex Scenario ◽  
Post Transcriptional Regulation ◽  
Transcriptional Output

Abstract Despite gene expression programs being notoriously complex, RNA abundance is usually assumed as a proxy for transcriptional activity. Recently developed approaches, able to disentangle transcriptional and post-transcriptional regulatory processes, have revealed a more complex scenario. It is now possible to work out how synthesis, processing and degradation kinetic rates collectively determine the abundance of each gene’s RNA. It has become clear that the same transcriptional output can correspond to different combinations of the kinetic rates. This underscores the fact that markedly different modes of gene expression regulation exist, each with profound effects on a gene’s ability to modulate its own expression. This review describes the development of the experimental and computational approaches, including RNA metabolic labeling and mathematical modeling, that have been disclosing the mechanisms underlying complex transcriptional programs. Current limitations and future perspectives in the field are also discussed.

scholarly journals Conditional defect in mRNA 3' end processing caused by a mutation in the gene for poly(A) polymerase.

1992 ◽  
Vol 12 (7) ◽  
pp. 3297-3304 ◽  
Author(s):  
D Patel ◽  
J S Butler
Keyword(s):  
Temperature Shift ◽  
Temperature Sensitive ◽  
Single Mutation ◽  
Mrna Levels ◽  
Endonucleolytic Cleavage ◽  
Normal Site ◽  
Apparent Loss ◽  
And Function

Maturation of most eukaryotic mRNA 3' ends requires endonucleolytic cleavage and polyadenylation of precursor mRNAs. To further understand the mechanism and function of mRNA 3' end processing, we identified a temperature-sensitive mutant of Saccharomyces cerevisiae defective for polyadenylation. Genetic analysis showed that the polyadenylation defect and the temperature sensitivity for growth result from a single mutation. Biochemical analysis of extracts from this mutant shows that the polyadenylation defect occurs at a step following normal site-specific cleavage of a pre-mRNA at its polyadenylation site. Molecular cloning and characterization of the wild-type allele of the mutated gene revealed that it (PAP1) encodes a previously characterized poly(A) polymerase with unknown RNA substrate specificity. Analysis of mRNA levels and structure in vivo indicate that shift of growing, mutant cells to the nonpermissive temperature results in the production of poly(A)-deficient mRNAs which appear to end at their normal cleavage sites. Interestingly, measurement of the rate of protein synthesis after the temperature shift shows that translation continues long after the apparent loss of polyadenylated mRNA. Our characterization of the pap1-1 defect implicates this gene as essential for mRNA 3' end formation in S. cerevisiae.

scholarly journals ISOLATION, GENETIC MAPPING AND SOME CHARACTERIZATION OF A MUTATION IN ESCHERICHIA COLI THAT AFFECTS THE PROCESSING OF RIBONUCLEIC ACID

Genetics ◽  
1978 ◽  
Vol 90 (4) ◽  
pp. 659-671
Author(s):  
David Apirion
Keyword(s):  
Escherichia Coli ◽  
Temperature Sensitivity ◽  
Single Point ◽  
5S Rrna ◽  
23S Rrna ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Rnase E ◽  
Nonpermissive Temperature ◽  
Ribonuclease E

ABSTRACT Temperature-sensitive mutants were isolated from an rnc (RNase III-) strain of Escherichia coli, and their rRNA metabolism was analyzed on 3% polyacrylamide gels. One of these mutants was unable to produce 23S and 5S rRNAs at the nonpermissive temperature. When an rnc  + allele was introduced to this strain, it remained temperature sensitive. At the nonpermissive temperature, this strain could then produce 23S rRNA but was unable to make normal levels of 5S rRNA. In matings and transduction experiments, the defect in rRNA metabolism and temperature sensitivity behaved as a syndrome caused by a single point mutation, which was mapped at min 23.5 on the E. coli chromosome. This mutation probably affects an enzyme, ribonuclease E (RNase E), which introduces a cut in the nascent rRNA transcript between the 23S and the 5S rRNA cistrons. The mutation rne is recessive with respect to temperature sensitivity and the pattern of rRNA. Revertants able to grow at 43° and with normal metabolism of rRNA were isolated; genetic analysis showed that they do not contain the original rne mutation, suggesting that they were true revertants. By combining the rne mutation with an rnc mutation, double rnc rne strains were synthesized, which behaved very similarly to the original rnc strain from which the rne mutation was isolated. Such strains have RNA metabolism that is similar to that of rnc strains at permissive temperatures, but at the nonpermissive temperature they fail to synthesize p23, m23 and 5S rRNAs. Thus, the experiments reported here, together with previous studies, suggest the existence of a new processing ribonuclease activity in Escherichia coli, which is called ribonuclease E.

scholarly journals RBM45 Promoted HCC Growth and Metastasis by Stabilizing BCL2 and Twist1 mRNA

2021 ◽  
Author(s):  
Lili Han ◽  
Lin Wang ◽  
Fei Wu
Keyword(s):  
Rna Binding ◽  
Binding Motif ◽  
Potential Therapeutic Target ◽  
Transcriptional Regulatory ◽  
Rna Immunoprecipitation ◽  
Post Transcriptional Regulation ◽  
Rip Assay

Abstract The post-transcriptional of mRNA expression involved in the hepatocellular carcinoma (HCC) pathogenesis and progression need to be further explored. RBPs, the main undertaker of post-transcriptional regulatory process, has been shown to impact HCC carcinogenesis and progression. However, the role of RBP, RNA-binding motif 45 (RBM45) in hepatocarcinogenesis and its interaction with its potential target mRNA remains entirely unknown. The expression of RBM45 was significantly increased in HCC and was associated with poor clinicopathological features and clinical outcome of HCC patients. RBM45 promoted HCC cells growth, invasion, migration and EMT in vitro and in vivo. Mechanistically, RNA immunoprecipitation sequencing (RIP-seq) approach was utilized to screen the important differentially expressed RBM45 genes in HCC. Furthermore, RIP assay, pull-down assay and mRNA decay assay were carried out to uncover the effect of RBM45 on its downstream genes. And the results revealed that RBM45 mediated the stabilization of BCL2 and Twist1 mRNA via respectively binding to their 3`UTR. Further assay results suggested RBM45 promoted HCC growth and metastasis upon BCL2 and Twist1. In short, we unveiled a novel role of RBM45 in promoting hepatocarcinogenesis via the post-transcriptional regulation of BCL2 and Twist1 expression. The results proposes that RBM45 may serve as a potential therapeutic target for HCC.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

scholarly journals Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

2021 ◽  
Author(s):  
Li-Nan Wang ◽  
Xiang-Lei Peng ◽  
Min Xu ◽  
Yuan-Bo Zheng ◽  
Yue-Ying Jiao ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Gene Deletion ◽  
Human Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
T7 Promoter ◽  
Vaccine Candidates ◽  
Rsv Infection ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

Sign in / Sign up

Export Citation Format

Share Document