scholarly journals RsmV a small non-coding regulatory RNA in Pseudomonas aeruginosa that sequesters RsmA and RsmF from target mRNAs

2018 ◽  
Author(s):  
Kayley H. Janssen ◽  
Manisha R. Diaz ◽  
Cindy J. Gode ◽  
Matthew C. Wolfgang ◽  
Timothy L. Yahr
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Secretion System ◽  
Transcriptional Level ◽  
Type Vi Secretion System ◽  
Control Of Gene Expression ◽  
Target Mrnas

ABSTRACTThe Gram-negative opportunistic pathogen Pseudomonas aeruginosa has distinct genetic programs that favor either acute or chronic virulence gene expression. Acute virulence is associated with twitching and swimming motility, expression of a type III secretion system (T3SS), and the absence of alginate, Psl, or Pel polysaccharide production. Traits associated with chronic infection include growth as a biofilm, reduced motility, and expression of a type VI secretion system (T6SS). The Rsm post-transcriptional regulatory system plays an important role in the inverse control of phenotypes associated with acute and chronic virulence. RsmA and RsmF are RNA-binding proteins that interact with target mRNAs to control gene expression at the post-transcriptional level. Previous work found that RsmA activity is controlled by at least three small, non-coding regulatory RNAs (RsmW, RsmY, and RsmZ). In this study, we took an in-silico approach to identify additional sRNAs that might function in the sequestration of RsmA and/or RsmF and identified RsmV, a 192 nt transcript with four predicted RsmA/RsmF consensus binding sites. RsmV is capable of sequestering RsmA and RsmF in vivo to activate translation of tssA1, a component of the T6SS, and to inhibit T3SS gene expression. Each of the predicted RsmA/RsmF consensus binding sites contribute to RsmV activity. Electrophoretic mobility shifts assays show that RsmF binds RsmV with >10-fold higher affinity than RsmY and RsmZ. Gene expression studies revealed that the temporal expression pattern of RsmV differs from RsmW, RsmY, and RsmZ. These findings suggest that each sRNA may play distinct roles in controlling RsmA and RsmF activity.IMPORTANCEThe role of RsmF in post-transcriptional control of gene expression remains enigmatic. While numerous rsmA-dependent phenotypes are more pronounced in an rsmAF double mutant, deletion of rsmF alone has only modest effects. Understanding mechanisms that control RsmF activity will provide insight into additional roles for RsmF. In the current study we identify RsmV as an sRNA that controls RsmA and RsmF activity, and show that RsmV, RsmW, RsmY, and RsmZ are differentially expressed during growth.

scholarly journals RsmV, a Small Noncoding Regulatory RNA in Pseudomonas aeruginosa That Sequesters RsmA and RsmF from Target mRNAs

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Kayley H. Janssen ◽  
Manisha R. Diaz ◽  
Cindy J. Gode ◽  
Matthew C. Wolfgang ◽  
Timothy L. Yahr
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Binding Sites ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Secretion System ◽  
Fine Tuning ◽  
Content Type ◽  
Target Mrnas

ABSTRACT The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has distinct genetic programs that favor either acute or chronic virulence gene expression. Acute virulence is associated with twitching and swimming motility, expression of a type III secretion system (T3SS), and the absence of alginate, Psl, or Pel polysaccharide production. Traits associated with chronic infection include growth as a biofilm, reduced motility, and expression of a type VI secretion system (T6SS). The Rsm posttranscriptional regulatory system plays important roles in the inverse control of phenotypes associated with acute and chronic virulence. RsmA and RsmF are RNA-binding proteins that interact with target mRNAs to control gene expression at the posttranscriptional level. Previous work found that RsmA activity is controlled by at least three small, noncoding regulatory RNAs (RsmW, RsmY, and RsmZ). In this study, we took an in silico approach to identify additional small RNAs (sRNAs) that might function in the sequestration of RsmA and/or RsmF (RsmA/RsmF) and identified RsmV, a 192-nucleotide (nt) transcript with four predicted RsmA/RsmF consensus binding sites. RsmV is capable of sequestering RsmA and RsmF in vivo to activate translation of tssA1, a component of the T6SS, and to inhibit T3SS gene expression. Each of the predicted RsmA/RsmF consensus binding sites contributes to RsmV activity. Electrophoretic mobility shifts assays show that RsmF binds RsmV with >10-fold higher affinity than RsmY and RsmZ. Gene expression studies revealed that the temporal expression pattern of RsmV differs from those of RsmW, RsmY, and RsmZ. These findings suggest that each sRNA may play a distinct role in controlling RsmA and RsmF activity. IMPORTANCE The members of the CsrA/RsmA family of RNA-binding proteins play important roles in posttranscriptional control of gene expression. The activity of CsrA/RsmA proteins is controlled by small noncoding RNAs that function as decoys to sequester CsrA/RsmA from target mRNAs. Pseudomonas aeruginosa has two CsrA family proteins (RsmA and RsmF) and at least four sequestering sRNAs (RsmV [identified in this study], RsmW, RsmY, and RsmZ) that control RsmA/RsmF activity. RsmY and RsmZ are the primary sRNAs that sequester RsmA/RsmF, and RsmV and RsmW appear to play smaller roles. Differences in the temporal and absolute expression levels of the sRNAs and in their binding affinities for RsmA/RsmF may provide a mechanism of fine-tuning the output of the Rsm system in response to environmental cues.

scholarly journals Global identification of RsmA/N binding sites in Pseudomonas aeruginosa by in vivo UV CLIP-seq

2020 ◽  
Author(s):  
Kotaro Chihara ◽  
Lars Barquist ◽  
Kenichi Takasugi ◽  
Naohiro Noda ◽  
Satoshi Tsuneda
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Binding Sites ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Chronic Infections ◽  
Uv Crosslinking ◽  
Rna Interaction

ABSTRACTPosttranscriptional regulation of gene expression in bacteria is performed by a complex and hierarchical signaling cascade. Pseudomonas aeruginosa harbors two redundant RNA-binding proteins RsmA/RsmN (RsmA/N), which play a critical role in balancing acute and chronic infections. However, in vivo binding sites on target transcripts and the overall impact on the physiology remains unclear. In this study, we applied in vivo UV crosslinking immunoprecipitation followed by RNA-sequencing (UV CLIP-seq) to detect RsmA/N binding sites at single-nucleotide resolution and mapped more than 500 peaks to approximately 400 genes directly bound by RsmA/N in P. aeruginosa. This also demonstrated the ANGGA sequence in apical loops skewed towards 5’UTRs as a consensus motif for RsmA/N binding. Genetic analysis combined with CLIP-seq results identified previously unrecognized RsmA/N targets involved in LPS modification. Moreover, the small non-coding RNAs RsmY/RsmZ, which sequester RsmA/N away from target mRNAs, are positively regulated by the RsmA/N-mediated translational repression of hptB, encoding a histidine phosphotransfer protein, and cafA, encoding a cytoplasmic axial filament protein, thus providing a possible mechanistic explanation for homeostasis of the Rsm system. Our findings present the global RsmA/N-RNA interaction network that exerts pleiotropic effects on gene expression in P. aeruginosa.IMPORTANCEThe ubiquitous bacterium Pseudomonas aeruginosa is notorious as an opportunistic pathogen causing life-threatening acute and chronic infections in immunocompromised patients. P. aeruginosa infection processes are governed by two major gene regulatory systems, namely, the GacA/GacS (GacAS) two-component system and the RNA-binding proteins RsmA/RsmN (RsmA/N). RsmA/N basically function as a translational repressor or activator directly by competing with the ribosome. In this study, we identified hundreds of RsmA/N regulatory target RNAs and the consensus motifs for RsmA/N bindings by UV crosslinking in vivo. Moreover, our CLIP-seq revealed that RsmA/N posttranscriptionally regulate cell wall organization and exert feedback control on GacAS-RsmA/N systems. Many genes including small regulatory RNAs identified in this study are attractive targets for further elucidating the regulatory mechanisms of RsmA/N in P. aeruginosa.

scholarly journals Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

2021 ◽  
Vol 4 (1) ◽  
pp. 22
Author(s):  
Mrinmoyee Majumder ◽  
Viswanathan Palanisamy
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Control Of Gene Expression ◽  
Regulatory Pathways ◽  
Advantages And Disadvantages ◽  
Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

A Potential Therapeutic Target RNA-binding Protein, Arid5a for the Treatment of Inflammatory Disease Associated with Aberrant Cytokine Expression

2018 ◽  
Vol 24 (16) ◽  
pp. 1766-1771 ◽  
Author(s):  
Kazuya Masuda ◽  
Tadamitsu Kishimoto
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Severe Sepsis ◽  
Inflammatory Cytokines ◽  
Binding Proteins ◽  
Rna Binding ◽  
Cytokine Production ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Transcriptional Level

Background: Infection, tissue damage and aging can cause inflammation with high levels of inflammatory cytokines. Overproduction of inflammatory cytokines often leads to systemic inflammatory response syndrome (SIRS), severe sepsis, and septic shock. However, prominent therapeutic targets have not been found, although the incidence of sepsis is likely to increase annually. Our recent studies indicate that some RNA-binding proteins, which control gene expression of inflammatory cytokines at the post-transcriptional level, may play a critical role in inflammatory diseases such as sepsis. Results: 1) One of the RNA-binding proteins, AT-rich interactive domain-containing 5a (Arid5a) promotes cytokine production through control of mRNA half-lives of pro-inflammatory molecules such as IL-6, STAT3, T-bet, and OX40 in activated macrophages and T cells. Arid5a KO mice are refractory to endotoxin shock, bleomycininduced lung injury, and inflammatory autoimmune disease. 2) Chlorpromazine (CPZ), which is recognized as a psychotic drug, impairs post-transcriptional gene expression of Il6 in LPS-stimulated macrophages: CPZ inhibits the binding activity of Arid5a to the 3’UTR of Il6 mRNA, thereby destabilizing Il6 mRNA possibly through suppression of Arid5a expression. 3) CPZ has strong suppressive effects on cytokine production such as TNF-α in vivo. Mice with treatment of CPZ are resistant to lipopolysaccharide (LPS)-induced shock. Conclusion: Thus, Arid5a contributes to the activation of macrophages and T cells through positive control of mRNA half-lives of inflammatory cytokines and its related molecules, which might lead to cytokine storm. Interestingly, Arid5a was identified from an inhibitory effect of CPZ on IL-6 production in macrophages activated by LPS. Therefore, CPZ derivatives or Arid5a inhibitors may have a potential to suppress severe sepsis through control of post-transcriptional gene expression.

238 RNA BINDING PROTEIN EXPRESSION IN CULTURED EARLY BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 277
Author(s):  
L. A. Favetta ◽  
E. Van de Laar ◽  
W. A. King ◽  
J. LaMarre
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Bovine Embryo ◽  
Rt Pcr ◽  
Control Of Gene Expression ◽  
Morula Stage ◽  
Bovine Oocytes

The control of gene expression in the early embryo requires a highly regulated turnover of specific mRNA, particularly those of maternal origin, as the embryo becomes transcriptionally autonomous. In cattle, the period during which maternal transcripts persist can last 72 to 96 h or longer, suggesting a dynamic, regulated interplay between factors that protect transcripts before this point and those that subsequently facilitate decay. Some decay pathways for specific embryonic transcripts are now known, but many are not. In somatic cells, mRNA decay is often mediated by interactions between defined sequence elements (ARE) in the 3′ untranslated region of important target genes and specific RNA-binding proteins (AUBP) that promote or inhibit decay of the associated transcript. These have not been extensively characterized in embryos. We hypothesized that changes in the pattern of expression of one or several AUBP in the developing bovine embryo would support a role for these proteins in mRNA turnover and the control of gene expression. We, therefore, evaluated the expression of different AUBP (HuR, AUF1, TTP) in bovine oocytes and early embryos in vitro. Bovine oocytes obtained at slaughter were matured, fertilized, and cultured using standard protocols. Oocytes and embryos from different stages were either placed in Trizol for subsequent RNA isolation and RT-PCR analysis or fixed in 4% paraformaldehyde and subsequently processed for immunohistochemical detection of AUBP. Analysis by RT-PCR revealed that AUF1, an mRNA destabilizing protein, was expressed at all stages examined (immature oocyte, mature oocyte, 2 to 4 cells, 8 to 16 cells, morulae, and blastocyst) except in morulae. Another mRNA destabilizing protein, TTP, was expressed at the morula stage only. An mRNA stabilizing factor, HuR, was expressed at all stages except the morula. Immunohistochemical analysis revealed that the pattern of protein expression for AUF1 and TTP essentially mirrored that observed at the RNA level as detected by RT-PCR. Together, these results show that AUBP expression in the early bovine embryo is dynamic, with RNA-binding proteins present at all times during development and changes in expression evident at the morula stage. This suggests that modification of presynthesized (i.e. maternal) AUBP is likely to control mRNA decay during the maternal to embryonic transition (8-cell stage) and that the expression of TTP at the morula stage might mark the onset of embryonic control of mRNA stability. Research was supported by NSERC, OMAFRA, and the Canada Research Chairs Program.

scholarly journals Cip1 and Cip2 Are Novel RNA-Recognition-Motif Proteins That Counteract Csx1 Function during Oxidative Stress

2006 ◽  
Vol 17 (3) ◽  
pp. 1176-1183 ◽  
Author(s):  
Victoria Martín ◽  
Miguel A. Rodríguez-Gabriel ◽  
W. Hayes McDonald ◽  
Stephen Watt ◽  
John R. Yates ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Stress Response ◽  
Protein Identification ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Bzip Transcription Factor ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Control Of Gene Expression

Eukaryotic cells reprogram their global patterns of gene expression in response to stress. Recent studies in Schizosaccharomyces pombe showed that the RNA-binding protein Csx1 plays a central role in controlling gene expression during oxidative stress. It does so by stabilizing atf1+ mRNA, which encodes a subunit of a bZIP transcription factor required for gene expression during oxidative stress. Here, we describe two related proteins, Cip1 and Cip2, that were identified by multidimensional protein identification technology (MudPIT) as proteins that coprecipitate with Csx1. Cip1 and Cip2 are cytoplasmic proteins that have RNA recognition motifs (RRMs). Neither protein is essential for viability, but a cip1Δ cip2Δ strain grows poorly and has altered cellular morphology. Genetic epistasis studies and whole genome expression profiling show that Cip1 and Cip2 exert posttranscriptional control of gene expression in a manner that is counteracted by Csx1. Notably, the sensitivity of csx1Δ cells to oxidative stress and their inability to induce expression of Atf1-dependent genes are partially rescued by cip1Δ and cip2Δ mutations. This study emphasizes the importance of a modulated mRNA stability in the eukaryotic stress response pathways and adds new information to the role of RNA-binding proteins in the oxidative stress response.

scholarly journals A Trypanosoma cruzi Zinc Finger protein that controls expression of epimastigote specific genes and affects metacyclogenesis

2020 ◽  
Author(s):  
Thais Silva Tavares ◽  
Fernanda Lins Brandão Mügge ◽  
Viviane Grazielle-Silva ◽  
Bruna Mattioly Valente ◽  
Wanessa Moreira Goes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Trypanosoma Cruzi ◽  
Zinc Finger ◽  
Rna Binding ◽  
Environmental Changes ◽  
Zinc Finger Protein ◽  
Negative Regulator ◽  
Transcriptional Level ◽  
Control Of Gene Expression ◽  
Genes Encoding

SummaryTrypanosoma cruzi has three biochemically and morphologically distinct developmental stages that are programed to rapidly respond to environmental changes the parasite faces during its life cycle. Unlike other eukaryotes, Trypanosomatid genomes contain protein coding genes that are transcribed into polycistronic pre-mRNAs and control of gene expression relies on mechanisms acting at the post-transcriptional level. Transcriptome analyses comparing epimastigote, trypomastigote and intracellular amastigote stages revealed changes in gene expression that reflect the parasite adaptation to distinct environments. Several genes encoding RNA binding proteins (RBP), known to act as key post-transcriptional regulatory factors, were also differentially expressed. We characterized one T. cruzi RBP (TcZH3H12) that contains a zinc finger domain, and whose transcripts are upregulated in epimastigotes compared to trypomastigotes and amastigotes. TcZC3H12 knockout epimastigotes showed decreased growth rates and increased capacity to differentiate into metacyclic trypomastigotes. Comparative transcriptome analysis revealed a TcZC3H12-dependent expression of epimastigote specific genes encoding amino acid transporters and proteins associated with differentiation (PAD), among others. RNA immunoprecipitation assays showed that transcripts from the PAD family interact with TcZC3H12. Taken together, these findings suggest that TcZC3H12 positively regulates the expression of genes involved in epimastigote proliferation and also acts as a negative regulator of metacyclogenesis.

scholarly journals Substrate Specificity of the TRAMP Nuclear Surveillance Complexes

2020 ◽  
Author(s):  
Clémentine Delan-Forino ◽  
Christos Spanos ◽  
Juri Rappsilber ◽  
David Tollervey
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Binding Protein ◽  
Rna Helicase ◽  
Nuclear Rna ◽  
Rna Exosome

ABSTRACTDuring nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity was apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association showed co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduced Mtr4 recruitment and increased RNA abundance for mRNAs specifically showing high Trf5 binding.

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