scholarly journals A fluorescence-based genetic screen reveals diverse mechanisms silencing small RNA signaling in E. coli

2021 ◽  
Author(s):  
Jiandong Chen ◽  
Leann To ◽  
Francois de Mets ◽  
Xing Luo ◽  
Nadim Majdalani ◽  
...  
Keyword(s):  
Small Rna ◽  
Regulatory Networks ◽  
Iron Homeostasis ◽  
Mrna Translation ◽  
Genetic Screen ◽  
Phenotypic Traits ◽  
E Coli ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Screen System

As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation as well as virulence. sRNAs can alter target mRNA translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify new regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in E. coli. The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX and McaS, previously found to act through Hfq competition, RNase T, a 3′-5′ exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two new specific regulators were identified. AspX, a novel 3′ end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria.

scholarly journals A fluorescence-based genetic screen reveals diverse mechanisms silencing small RNA signaling in E. coli

2021 ◽  
Vol 118 (27) ◽  
pp. e2106964118
Author(s):  
Jiandong Chen ◽  
Leann To ◽  
Francois de Mets ◽  
Xing Luo ◽  
Nadim Majdalani ◽  
...  
Keyword(s):  
Small Rna ◽  
Messenger Rna ◽  
Regulatory Networks ◽  
Iron Homeostasis ◽  
Mrna Translation ◽  
Genetic Screen ◽  
Phenotypic Traits ◽  
E Coli ◽  
Small Regulatory Rnas ◽  
Screen System

As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation, as well as virulence. sRNAs can alter target messenger RNA (mRNA) translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in Escherichia coli. The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX, and McaS, previously found to act through Hfq competition, RNase T, a 3′ to 5′ exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two specific regulators were identified. AspX, a 3′end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria.

scholarly journals The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0127468 ◽  
Author(s):  
Hongtao Hu ◽  
Aaron M. Rashotte ◽  
Narendra K. Singh ◽  
David B. Weaver ◽  
Leslie R. Goertzen ◽  
...  
Keyword(s):  
Small Rna ◽  
In Silico ◽  
Regulatory Networks ◽  
In Silico Analysis ◽  
Gossypium Arboreum ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Silico Analysis

scholarly journals Extensive Analysis of miRNA Trimming and Tailing Indicates that AGO1 Has a Complex Role in miRNA Turnover

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 267
Author(s):  
Axel J. Giudicatti ◽  
Ariel H. Tomassi ◽  
Pablo A. Manavella ◽  
Agustin L. Arce
Keyword(s):  
Small Rna ◽  
Stress Responses ◽  
Cellular Localization ◽  
Mirna Biogenesis ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Extensive Analysis ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Argonaute 1

MicroRNAs are small regulatory RNAs involved in several processes in plants ranging from development and stress responses to defense against pathogens. In order to accomplish their molecular functions, miRNAs are methylated and loaded into one ARGONAUTE (AGO) protein, commonly known as AGO1, to stabilize and protect the molecule and to assemble a functional RNA-induced silencing complex (RISC). A specific machinery controls miRNA turnover to ensure the silencing release of targeted-genes in given circumstances. The trimming and tailing of miRNAs are fundamental modifications related to their turnover and, hence, to their action. In order to gain a better understanding of these modifications, we analyzed Arabidopsis thaliana small RNA sequencing data from a diversity of mutants, related to miRNA biogenesis, action, and turnover, and from different cellular fractions and immunoprecipitations. Besides confirming the effects of known players in these pathways, we found increased trimming and tailing in miRNA biogenesis mutants. More importantly, our analysis allowed us to reveal the importance of ARGONAUTE 1 (AGO1) loading, slicing activity, and cellular localization in trimming and tailing of miRNAs.

scholarly journals Stem-loops direct precise processing of 3′ UTR-derived small RNA MicL

2018 ◽  
Author(s):  
Taylor B Updegrove ◽  
Andrew B Kouse ◽  
Katarzyna J Bandyra ◽  
Gisela Storz
Keyword(s):  
Small Rna ◽  
Cleavage Site ◽  
Differential Sensitivity ◽  
Rnase E ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Derivatives Of

AbstractIncreasing numbers of 3′UTR-derived small, regulatory RNAs (sRNAs) are being discovered in bacteria, most generated by cleavage from longer transcripts. The enzyme required for these cleavages has been reported to be RNase E, the major endoribonuclease in enterica bacteria. Previous studies investigating RNase E have come to a range of different conclusions regarding the determinants for RNase E processing. To understand the sequence and structure determinants for the precise processing of the 3′ UTR-derived sRNAs, we examined the cleavage of multiple mutant and chimeric derivatives of the 3′ UTR-derived MicL sRNA in vivo and in vitro. Our results revealed that tandem stem-loops 3′ to the cleavage site define optimal, correctly-positioned cleavage of MicL and likely other similar sRNAs. Moreover, our assays of MicL, ArcZ and CpxQ showed that sRNAs exhibit differential sensitivity to RNase E, likely a consequence of a hierarchy of sRNA features recognized by the endonuclease.

scholarly journals TheprrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

2014 ◽  
Vol 83 (3) ◽  
pp. 863-875 ◽  
Author(s):  
Alexandria A. Reinhart ◽  
Daniel A. Powell ◽  
Angela T. Nguyen ◽  
Maura O'Neill ◽  
Louise Djapgne ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Iron Homeostasis ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Genes Encoding ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Heme Regulation ◽  
Heme Binding Protein

Pseudomonas aeruginosais an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part ofP. aeruginosa'siron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem inP. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identifiedphuS, encoding a heme binding protein involved in heme acquisition, andvreR, encoding a previously identified regulator ofP. aeruginosavirulence genes, as novel targets ofprrF-mediated heme regulation. Finally, we showed that theprrFlocus encoding the PrrF and PrrH sRNAs is required forP. aeruginosavirulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2deletion mutant protects against future challenge with wild-typeP. aeruginosa. Combined, these data demonstrate that theprrF-encoded sRNAs are critical regulators ofP. aeruginosavirulence.

scholarly journals The Etiological Agent of Lyme Disease, Borrelia burgdorferi, Appears To Contain Only a Few Small RNA Molecules

2004 ◽  
Vol 186 (24) ◽  
pp. 8472-8477 ◽  
Author(s):  
Yngve Östberg ◽  
Ignas Bunikis ◽  
Sven Bergström ◽  
Jörgen Johansson
Keyword(s):  
Borrelia Burgdorferi ◽  
Small Rna ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Rnase E ◽  
Regulatory Pathways ◽  
Rna Molecules ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas

ABSTRACT Small regulatory RNAs (sRNAs) have recently been shown to be the main controllers of several regulatory pathways. The function of sRNAs depends in many cases on the RNA-binding protein Hfq, especially for sRNAs with an antisense function. In this study, the genome of Borrelia burgdorferi was subjected to different searches for sRNAs, including direct homology and comparative genomics searches and ortholog- and annotation-based search strategies. Two new sRNAs were found, one of which showed complementarity to the rpoS region, which it possibly controls by an antisense mechanism. The role of the other sRNA is unknown, although observed complementarities against particular mRNA sequences suggest an antisense mechanism. We suggest that the low level of sRNAs observed in B. burgdorferi is at least partly due to the presumed lack of both functional Hfq protein and RNase E activity.

scholarly journals A small RNA is linking CRISPR-Cas and zinc transport

2021 ◽  
Author(s):  
Pascal Märkle ◽  
Lisa-Katharina Maier ◽  
Sandra Maaß ◽  
Claudia Hirschfeld ◽  
Jürgen Bartel ◽  
...  
Keyword(s):  
Small Rna ◽  
Direct Interaction ◽  
Deletion Strain ◽  
Zinc Transport ◽  
Wild Type ◽  
Mobility Shift ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Electrophoretic Mobility Shift Assays ◽  
Cas Proteins

AbstractThe function and mode of action of small regulatory RNAs is currently still understudied in archaea. In the halophilic archaeon H. volcanii a plethora of sRNAs have been identified, however, in-depth functional analysis is missing for most of them. We selected a small RNA (s479) from H. volcanii for detailed characterization. The sRNA gene is encoded between a CRISPR RNA locus and the Cas protein gene cluster, the s479 deletion strain is viable and was characterized in detail. Transcriptome studies of wild type Haloferax cells and the deletion mutant revealed up-regulation of six genes in the deletion strain, showing that the sRNA has a clearly defined function. Three of the six up-regulated genes encode potential zinc transporter proteins (ZnuA1, ZnuB1, ZnuC1) suggesting involvement of s479 in regulation of zinc transport. Upregulation of these genes in the deletion strain was confirmed by northern blot and proteome analyses. Furthermore, electrophoretic mobility shift assays demonstrate a direct interaction of s479 with the target znuC1 mRNA. Proteome comparison of wild type and deletion strains further expanded the regulon of s479 deeply rooting this sRNA within the metabolism of H. volcanii especially the regulation of transporter abundance. Interestingly, s479 is not only encoded next to CRISPR-cas genes but the mature s479 contains a crRNA-like 5’ handle and experiments with Cas protein deletion strains indicate maturation by Cas6 and interaction with Cas proteins. Together this might suggest that the CRISPR-Cas system is involved in s479 function.

scholarly journals ThePseudomonas aeruginosaPrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of theantRmRNA

2018 ◽  
Vol 200 (10) ◽  
Author(s):  
Louise Djapgne ◽  
Subrata Panja ◽  
Luke K. Brewer ◽  
Jonathan H. Gans ◽  
Maureen A. Kane ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Iron Homeostasis ◽  
Molecular Mechanisms ◽  
Biological Activities ◽  
Content Type ◽  
Downstream Effects ◽  
Infection Models ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Translational Start

ABSTRACTPseudomonas aeruginosais an opportunistic Gram-negative pathogen that requires iron for growth and virulence. Under low-iron conditions,P. aeruginosatranscribes two highly identical (95%) small regulatory RNAs (sRNAs), PrrF1 and PrrF2, which are required for virulence in acute murine lung infection models. The PrrF sRNAs promote the production of 2-akyl-4(1H)-quinolone metabolites (AQs) that mediate a range of biological activities, including quorum sensing and polymicrobial interactions. Here, we show that the PrrF1 and PrrF2 sRNAs promote AQ production by redundantly inhibiting translation ofantR, which encodes a transcriptional activator of the anthranilate degradation genes. A combination of genetic and biophysical analyses was used to define the sequence requirements for PrrF regulation ofantR, demonstrating that the PrrF sRNAs interact with theantR5′ untranslated region (UTR) at sequences overlapping the translational start site of this mRNA. TheP. aeruginosaHfq protein interacted with UA-rich sequences in both PrrF sRNAs (Kd[dissociation constant] = 50 nM and 70 nM). Hfq bound with lower affinity to theantRmRNA (0.3 μM), and PrrF was able to bind toantRmRNA in the absence of Hfq. Nevertheless, Hfq increased the rate of PrrF annealing to theantRUTR by 10-fold. These studies provide a mechanistic description of how the PrrF1 and PrrF2 sRNAs mediate virulence traits, such as AQ production, inP. aeruginosa.IMPORTANCEThe iron-responsive PrrF sRNAs play a central role in regulatingP. aeruginosairon homeostasis and pathogenesis, yet the molecular mechanisms by which PrrF regulates gene expression are largely unknown. In this study, we used genetic and biophysical analyses to define the interactions of the PrrF sRNAs with Hfq, an RNA annealer, and theantRmRNA, which has downstream effects on quorum sensing and virulence factor production. These studies provide a comprehensive mechanistic analysis of how the PrrF sRNAs regulate virulence trait production through a key mRNA target inP. aeruginosa.

scholarly journals Assembling the Current Pieces: The Puzzle of RNA-Mediated Regulation in Staphylococcus aureus

2021 ◽  
Vol 12 ◽  
Author(s):  
Laura Barrientos ◽  
Noémie Mercier ◽  
David Lalaouna ◽  
Isabelle Caldelari
Keyword(s):  
Staphylococcus Aureus ◽  
Stress Responses ◽  
Regulatory Networks ◽  
High Throughput Sequencing ◽  
Bacterial Physiology ◽  
Sequencing Technologies ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Infection Processes

The success of the major opportunistic human Staphylococcus aureus relies on the production of numerous virulence factors, which allow rapid colonization and dissemination in any tissues. Indeed, regulation of its virulence is multifactorial, and based on the production of transcriptional factors, two-component systems (TCS) and small regulatory RNAs (sRNAs). Advances in high-throughput sequencing technologies have unveiled the existence of hundreds of potential RNAs with regulatory functions, but only a fraction of which have been validated in vivo. These discoveries have modified our thinking and understanding of bacterial physiology and virulence fitness by placing sRNAs, alongside transcriptional regulators, at the center of complex and intertwined regulatory networks that allow S. aureus to rapidly adapt to the environmental cues present at infection sites. In this review, we describe the recently acquired knowledge of characterized regulatory RNAs in S. aureus that are associated with metal starvation, nutrient availability, stress responses and virulence. These findings highlight the importance of sRNAs for the comprehension of S. aureus infection processes while raising questions about the interplay between these key regulators and the pathways they control.

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