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Tomoya Maeda, Yuya Tanaka, Masayuki Inui
The Corynebacterium glutamicum R grtA (cgR_2936), grtB (cgR_2934), and grtC (cgR_2933) genes were identified as paralogs encoding glutamine-rich toxic proteins. We also identified a new antisense small RNA AsgR (antisense sRNA for grtA) that overlaps the 3' end of the grtA gene. Single over-expressions of grtA, grtB, and grtC resulted in complete inhibition of Escherichia coli cell growth. This growth was rescued by co-expression of AsgR. Similar effects were observed in C. glutamicum, although the toxicities of these proteins were moderate...
March 14, 2018: Molecular Microbiology
Zhenyi Li, Hongyu Xu, Yue Li, Xiufu Wan, Zhao Ma, Jing Cao, Zhensong Li, Feng He, Yufei Wang, Liqiang Wan, Zongyong Tong, Xianglin Li
The induction of miR399 and miR398 and the inhibition of miR156, miR159, miR160, miR171, miR2111, and miR2643 were observed under Pi deficiency in alfalfa. The miRNA-mediated genes involved in basic metabolic process, root and shoot development, stress response and Pi uptake. Inorganic phosphate (Pi) deficiency is known to be a limiting factor in plant development and growth. However, the underlying miRNAs associated with the Pi deficiency-responsive mechanism in alfalfa are unclear. To elucidate the molecular mechanism at the miRNA level, we constructed four small RNA (sRNA) libraries from the roots and shoots of alfalfa grown under normal or Pi-deficient conditions...
March 12, 2018: Plant Molecular Biology
Brice Felden, Vincent Cattoir
The extensive use of antibiotics has resulted in a situation where multidrug-resistant pathogens have become a severe menace to human health worldwide. A deeper understanding of the principles used by pathogens to adapt, respond and resist against antibiotics will pave the road to drugs with novel mechanisms. For bacteria, antibiotics are clinically-relevant stresses that induce protective responses. The recent implication of regulatory RNAs (sRNAs) into antibiotic response and resistance in many bacterial pathogens suggests that they should be considered as innovative drug targets...
March 12, 2018: Antimicrobial Agents and Chemotherapy
Maciej Dylewski, Monika Ćwiklińska, Katarzyna Potrykus
Small RNA are very important post-transcriptional regulators in both, bacteria and eukaryotes. One of such sRNA is GraL, encoded in the greA leader region and conserved among enteric bacteria. Here, we conducted a bioinformatics search for GraL's targets in trans and validated our findings in vivo by constructing fusions of probable targets with lacZ and measuring their activity when GraL was overexpressed. Only one target's activity (nudE) decreased under those conditions and was thus selected for further analysis...
March 12, 2018: Acta Biochimica Polonica
Tokuko Ujino-Ihara, Saneyoshi Ueno, Kentaro Uchiyama, Norihiro Futamura
Deep sequencing of small RNAs (sRNAs) in developing male strobili of second-generation offspring originating from a nuclear genic male sterile tree of Cryptomeria japonica were performed to characterize sRNA populations in the male strobili at early pollen developmental stages. Comparing to sequences of microRNA (miRNA) families of plant species and sRNAs expressed in the reproductive organs of representative vascular plants, 37 conserved miRNA families were detected, of which eight were ubiquitously expressed in the reproductive organs of land plant species...
2018: PloS One
Karin L Meibom, Elena M Cabello, Rizlan Bernier-Latmani
Shewanella oneidensis produces an extensive electron transfer network that results in metabolic flexibility. A large number of c -type cytochromes are expressed by S. oneidensis and these function as the fundamental electron transport chain proteins. Although several S. oneidensis cytochromes have been well-characterized, little is known about how their expression is regulated. In this study, we investigate the role of the ferric uptake regulator (Fur) and the sRNA RyhB in regulation. Our results demonstrate that loss of Fur leads to diminished growth and an apparent decrease in heme-containing proteins...
2018: Frontiers in Microbiology
Wenfeng Liu, Tatiana Rochat, Claire Toffano-Nioche, Thao Nguyen Le Lam, Philippe Bouloc, Claire Morvan
Bacterial regulatory RNAs have been extensively studied for over a decade, and are progressively being integrated into the complex genetic regulatory network. Transcriptomic arrays, recent deep-sequencing data and bioinformatics suggest that bacterial genomes produce hundreds of regulatory RNAs. However, while some have been authenticated, the existence of the others varies according to strains and growth conditions, and their detection fluctuates with the methodologies used for data acquisition and interpretation...
2018: Frontiers in Microbiology
Diego Rivera Gelsinger, Jocelyne DiRuggiero
Small non-coding RNAs (sRNAs) are ubiquitously found in the three domains of life playing large-scale roles in gene regulation, transposable element silencing and defense against foreign elements. While a substantial body of experimental work has been done to uncover function of sRNAs in Bacteria and Eukarya, the functional roles of sRNAs in Archaea are still poorly understood. Recently, high throughput studies using RNA-sequencing revealed that sRNAs are broadly expressed in the Archaea, comprising thousands of transcripts within the transcriptome during non-challenged and stressed conditions...
March 5, 2018: Genes
Tanmay Dutta, Shubhangi Srivastava
Small RNAs (sRNAs) in bacteria have evolved with diverse mechanisms to balance their target gene expression in response to changes in the environment. Accumulating studies on bacterial regulatory processes firmly established that sRNAs modulate their target gene expression generally at the posttranscriptional level. Identification of large number of sRNAs by advanced technologies, like deep sequencing, tilling microarray, indicates the existence of a plethora of distinctive sRNA-mediated regulatory mechanisms in bacteria...
March 1, 2018: Gene
Annalisa Giampetruzzi, Michela Chiumenti, Angelantonio Minafra, Pasquale Saldarelli
A protocol is described to purify small (s)RNA molecules from tissues of grapevine and other woody plants. The protocol has been specifically developed to analyze sRNA populations by high-throughput sequencing. It has been widely used on species of the genera Prunus and Vitis particularly rich in polyphenols and other enzyme-inhibiting compounds. The high quality of the sRNAs extracted from leaf or phloem tissues makes them suitable for all molecular biology reactions, in particular for next-generation sequencing library preparation...
2018: Methods in Molecular Biology
Stefania Bortoluzzi, Federica Lovisa, Enrico Gaffo, Lara Mussolin
Extracellular vesicles (EVs) secreted from many cell types play important roles in intercellular communication, both as paracrine and endocrine factors, as they can circulate in biological fluids, including plasma. Amid EVs, exosomes are actively secreted vesicles that contain proteins, lipids, soluble factors, and nucleic acids, including microRNAs (miRNAs) and other classes of small RNAs (sRNA). miRNAs are prominent post-transcriptional regulators of gene expression and epigenetic silencers of transcription...
October 6, 2017: High Throughput
Samuel D Stimple, Ashwin Lahiry, Joseph E Taris, David W Wood, Richard A Lease
RNA biology and RNA engineering are subjects of growing interest due to recent advances in our understanding of the diverse cellular functions of RNAs, including their roles as genetic regulators. The noncoding small RNAs (sRNAs) of bacteria are a fundamental basis of regulatory control that can regulate gene expression via antisense base-pairing to one or more target mRNAs. The sRNAs can be customized to generate a range of mRNA translation rates and stabilities. The sRNAs can be applied as a platform for metabolic engineering, to control expression of genes of interest by following relatively straightforward design rules (Kushwaha et al...
2018: Methods in Molecular Biology
Marta Robledo, Ana M Matia-González, Natalia I García-Tomsig, José I Jiménez-Zurdo
The identification of the protein partners of bacterial small noncoding RNAs (sRNAs) is essential to understand the mechanistic principles and functions of riboregulation in prokaryotic cells. Here, we describe an optimized affinity chromatography protocol that enables purification of in vivo formed sRNA-protein complexes in Sinorhizobium meliloti, a genetically tractable nitrogen-fixing plant symbiotic bacterium. The procedure requires the tagging of the desired sRNA with the MS2 aptamer, which is affinity-captured by the MS2-MBP protein conjugated to an amylose resin...
2018: Methods in Molecular Biology
Jonathan Gans, Jonathan Osborne, Juliet Cheng, Louise Djapgne, Amanda G Oglesby-Sherrouse
Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules...
2018: Methods in Molecular Biology
David Partouche, Antoine Malabirade, Thomas Bizien, Marisela Velez, Sylvain Trépout, Sergio Marco, Valeria Militello, Christophe Sandt, Frank Wien, Véronique Arluison
Post-transcriptional control of gene expression by small regulatory noncoding RNA (sRNA) needs protein accomplices to occur. Past research mainly focused on the RNA chaperone Hfq as cofactor. Nevertheless, recent studies indicated that other proteins might be involved in sRNA-based regulations. As some of these proteins have been shown to self-assemble, we describe in this chapter protocols to analyze the nano-assemblies formed. Precisely, we focus our analysis on Escherichia coli Hfq as a model, but the protocols presented here can be applied to analyze any polymer of proteins...
2018: Methods in Molecular Biology
Thierry Bizebard, Véronique Arluison, Ulrich Bockelmann
In recent years, single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful technique to study macromolecular interactions. The chief advantages of smFRET analysis compared to bulk measurements include the possibility to detect sample heterogeneities within a large population of molecules and the facility to measure kinetics without needing the synchronization of intermediate states. As such, the methodology is particularly well adapted to observe and analyze RNA/RNA and RNA/protein interactions involved in small noncoding RNA-mediated gene regulation networks...
2018: Methods in Molecular Biology
Kimberly A Stanek, Cameron Mura
Hfq is a bacterial RNA-binding protein that plays key roles in the post-transcriptional regulation of gene expression. Like other Sm proteins, Hfq assembles into toroidal discs that bind RNAs with varying affinities and degrees of sequence specificity. By simultaneously binding to a regulatory small RNA (sRNA) and an mRNA target, Hfq hexamers facilitate productive RNA∙∙∙RNA interactions; the generic nature of this chaperone-like functionality makes Hfq a hub in many sRNA-based regulatory networks. That Hfq is crucial in diverse cellular pathways-including stress response, quorum sensing, and biofilm formation-has motivated genetic and "RNAomic" studies of its function and physiology (in vivo), as well as biochemical and structural analyses of Hfq∙∙∙RNA interactions (in vitro)...
2018: Methods in Molecular Biology
Florent Busi, Véronique Arluison, Philippe Régnier
Regulation of RNA turnover is of utmost importance for controlling the concentration of transcripts and consequently cellular protein levels. Among the processes controlling RNA decay, small noncoding regulatory RNAs (sRNAs) have recently emerged as major new players. In this chapter, we describe and discuss protocols that can be used to measure sRNA concentration in vivo and to assess sRNA decay rates in Gram-negative bacteria. Precisely, we focus our analyses on the Escherichia coli Gram-negative bacterium as a model...
2018: Methods in Molecular Biology
Eva Maria Sternkopf Lillebæk, Birgitte Haahr Kallipolitis
Small regulatory RNAs (sRNAs) in bacteria often act by base pairing to mRNAs. Direct interactions between an sRNA and its target mRNA can be investigated by electrophoretic mobility shift assay. In this assay, regions engaged in base pairing are analyzed by introducing mutations in one of the RNAs that prevent sRNA-mRNA complex formation, followed by the introduction of complementary mutations in its partner RNA that restore base pairing. Here, we describe the design of a mutational strategy used to analyze the base pairing between two CU-rich regions of the sRNA Rli22 and the AG-rich Shine-Dalgarno region of the mRNA oppA in Listeria monocytogenes...
2018: Methods in Molecular Biology
Cédric Nadiras, Annie Schwartz, Mildred Delaleau, Marc Boudvillain
Besides their well-known posttranscriptional effects on mRNA translation and decay, sRNAs and associated RNA chaperones (e.g., Hfq, CsrA) sometimes regulate gene expression at the transcriptional level. In this case, the sRNA-dependent machinery modulates the activity of the transcription termination factor Rho, a ring-shaped RNA translocase/helicase that dissociates transcription elongation complexes at specific loci of the bacterial genome. Here, we describe biochemical assays to detect Rho-dependent termination signals in genomic regions of interest and to assess the effects of sRNAs and/or associated RNA chaperones on such signals...
2018: Methods in Molecular Biology
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