NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs

ABSTRACT RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia coli. Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB, negatively regulated the fusion. An insertion in the rep helicase and one upstream of dnaK decreased expression of the fusion. Multicopy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the gene for the response regulator OmpR; the second contained mipA, encoding a murein hydrolase. The involvement of MipA and OmpR in cell surface synthesis suggested that the rybB promoter might be dependent on σE. The sequence upstream of the +1 of rybB contains a consensus σE promoter. The activity of rybB-lacZ was increased in cells lacking the RseA anti-sigma factor and when σE was overproduced from a heterologous promoter. The activity of rybB-lacZ and the detection of RybB were totally abolished in an rpoE-null strain. In vitro, σE efficiently transcribes from this promoter. Both a rybB mutation and an hfq mutation significantly increased expression of both rybB-lacZ and rpoE-lacZ fusions, consistent with negative regulation of the σE response by RybB and other small RNAs. Based on the plasmid screens, NsrR, a repressor sensitive to nitric oxide, was also found to negatively regulate σE-dependent promoters in an RseA-independent fashion.

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172 IDENTIFICATION OF MICRORNAS IN BOVINE OVARY

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab172 ◽

2009 ◽

Vol 21 (1) ◽

pp. 185

Author(s):

M. M. Hossain ◽

M. Hoelker ◽

C. Phatsara ◽

E. Tholen ◽

K. Schellander ◽

...

Keyword(s):

Small Rna ◽

Hormonal Regulation ◽

Small Rnas ◽

Noncoding Rna ◽

Bovine Genome ◽

Functional Characterization ◽

Sequence Length ◽

P Value ◽

Template Switching ◽

Bovine Ovary

Tightly regulated expression and interaction of a multitude of genes for ovarian folliculogenesis leading to successful oocyte development could be regulated by recently identified new class of small RNAs of ~22 nt (i.e. microRNAs), which are already proved as one of the vital transcriptional regulators in different biological processes including development. But their presence and expression in bovine ovary has not yet been determined. Here, we have attempted to identify miRNAs in bovine ovary by small RNA-cDNA library construction through 5 ligation independent cloning. For this purpose, total RNA enriched with small RNA was isolated from ovary and size fractionated (18 to 24 nt) by denaturing PAGE. Extracted RNA was first 3′ linkered and after template switching by RT, the second 3′ linkering of the first strand cDNA was performed. These linkered small RNA-cDNAs were then amplified with linker-specific primers consisting of BAN I restriction sites, concatemerized by serial ligation, cloned into TOPO TA vector, and transformed into TOP 10 chemically competent cells. After screening, colonies were picked and sequenced. Bioinformatic analysis was done according to the published criteria for the small RNAs. From 233 clones a total of 479 reads were identified. Frequency of sequence length found in the library was 26.8% for ≤18 nt, 55.1% for 19 to 22 nt, and 18.1% for ≥23 nt. The total 479 sequences identified in the library represent 35% miRNAs, 12% mRNA, 12.1% rRNA, 5.6% tRNA, 4.2% repeat associated siRNA, 3.8% non-repeat-associated siRNA, 4% tiny noncoding RNA, 1% small nuclear RNA, and 16% sequences not matched to bovine genome. All 171 miR sequences comprised 79 distinct miRNAs, of which 45 miRNAs already annotated in miRBase for bovine and the other 34 miRNAs are new discoveries. Of the 34 newly identified miRNAs, 12 are described in other species but not yet in bovine. Most of the miRNAs cloned into multiple times, where let-7a cloned for 10, let-7b for 28, let-7c for 13, miR-21 for 4, miR-23b for 11, miR-24 for 7, miR-27a for 6, miR-126 for 4, and miR-143 for 11 times. Based on best hit score, P-value and free energy by online target prediction, some of the bta-miR identified in the library (let-7b, 15b, 18a, 23b, 101, 125b, 126, 140, 145, 199a) are found to target hundreds of genes related to follicular development, ovulation and hormonal regulation. Further functional characterization of some selected miRNAs including expression profiling and in situ localization in follicles of different size and cycles may supplement the results of this study and will enable us to gain insight into their relation to female fertility.

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Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training

RNA ◽

10.1261/rna.2123811 ◽

2010 ◽

Vol 17 (1) ◽

pp. 166-181 ◽

Cited By ~ 44

Author(s):

N. R. Smalheiser ◽

G. Lugli ◽

J. Thimmapuram ◽

E. H. Cook ◽

J. Larson

Keyword(s):

Discrimination Training ◽

Small Rnas ◽

Noncoding Rna ◽

Adult Mouse ◽

Olfactory Discrimination ◽

Mouse Hippocampus

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Characterization of 475 novel, putative small RNAs (sRNAs) in Carbon-starved Salmonella enterica serovar typhimurium

10.1101/2021.01.11.426214 ◽

2021 ◽

Author(s):

Dominika M Houserova ◽

Donavon J Dahmer ◽

Shivam V Amin ◽

Valeria M King ◽

Emmaline C Barnhill ◽

...

Keyword(s):

Salmonella Enterica ◽

Small Rnas ◽

Noncoding Rna ◽

Foodborne Pathogen ◽

Northern Blotting ◽

Protein Coding ◽

Mrna Targets ◽

Stress Survival ◽

Serovar Typhimurium

An increasingly apparent role of noncoding RNA (ncRNAs) is to coordinate gene expression when bacteria faces various environmental stressors. Salmonella enterica, a well-studied foodborne pathogen, is known for its ability to survive in and adapt to various environmental challenges, making it a difficult pathogen to eliminate, as well as an important model for examining ncRNA contributions to cellular stress survival. A mounting body of evidence implicates small RNAs (sRNAs) as key drivers of Salmonella stress response. Generally thought to be 50-500 nucleotides in length and to occur mainly in intergenic regions, sRNAs regulate protein expression through base pairing with mRNA targets. Through employing a refined definition of sRNAs that allows for shorter sequences and for sRNA loci to overlap with annotated protein-coding gene loci, we have identified 475 previously unannotated sRNAs that are significantly differentially expressed during Carbon starvation (C-starvation). Northern blotting and quantitative RT-PCRs confirm the expressions and identities of several of these novel sRNAs. Additionally, our computational analyses find the majority to be highly conserved and structurally-related to known sRNAs. Importantly, we show that deletion of one of the dynamic sRNAs, sRNA4130247, significantly impairs the Salmonella C-starvation response (CSR), confirming its involvement (and suggesting the involvements of many other sRNAs identified in this work) in the Salmonella CSR. Strikingly, the 475 novel sRNAs identified in this study more than double the number of Salmonella enterica serovar Typhimurium SL1344 sRNAs described to date, increasing the total number of annotated Salmonella sRNAs from 396 to 871. In conclusion, the work presented here provides the first-ever characterization of intragenic sRNAs in Salmonella, experimentally confirms that sRNAs dynamically expressed during the CSR are directly involved in stress survival, and strongly suggests that sRNA loci likely outnumber those of protein-coding genes in Salmonella.

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Regulatory Noncoding Small RNAs Are Diverse and Abundant in an Extremophilic Microbial Community

mSystems ◽

10.1128/msystems.00584-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 3

Author(s):

Diego R. Gelsinger ◽

Gherman Uritskiy ◽

Rahul Reddy ◽

Adam Munn ◽

Katie Farney ◽

...

Keyword(s):

Gene Regulation ◽

Microbial Community ◽

Small Rnas ◽

Noncoding Rna ◽

Field Experiments ◽

Natural World ◽

Community Level ◽

Model Organisms ◽

Rain Event ◽

Expression Levels

ABSTRACT Regulatory small RNAs (sRNAs) play large-scale and essential roles in many cellular processes across all domains of life. Microbial sRNAs have been extensively studied in model organisms, but very little is known about the dynamics of sRNA synthesis and their roles in the natural environment. In this study, we discovered hundreds of intergenic (itsRNAs) and antisense (asRNAs) sRNAs expressed in an extremophilic microbial community inhabiting halite nodules (salt rocks) in the Atacama Desert. For this, we built SnapT, a new sRNA annotation pipeline that can be applied to any microbial community. We found asRNAs with expression levels negatively correlated with that of their overlapping putative target and itsRNAs that were conserved and significantly differentially expressed between 2 sampling time points. We demonstrated that we could perform target prediction and correlate expression levels between sRNAs and predicted target mRNAs at the community level. Functions of putative mRNA targets reflected the environmental challenges members of the halite communities were subjected to, including osmotic adjustments to a major rain event and competition for nutrients. IMPORTANCE Microorganisms in the natural world are found in communities, communicating and interacting with each other; therefore, it is essential that microbial regulatory mechanisms, such as gene regulation affected by small RNAs (sRNAs), be investigated at the community level. This work demonstrates that metatranscriptomic field experiments can link environmental variation with changes in RNA pools and have the potential to provide new insights into environmental sensing and responses in natural microbial communities through noncoding RNA-mediated gene regulation.

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Atypical DNA methylation, sRNA size distribution and female gametogenesis correlate with genome compaction in Utricularia gibba

10.1101/2020.12.05.413054 ◽

2020 ◽

Author(s):

Sergio Alan Cervantes-Pérez ◽

Lenin Yong-Villalobos ◽

Nathalia M. V. Florez-Zapata ◽

Araceli Oropeza-Aburto ◽

Félix Rico-Reséndiz ◽

...

Keyword(s):

Dna Methylation ◽

Genome Size ◽

Small Rnas ◽

Noncoding Rna ◽

Noncoding Rnas ◽

Initial Step ◽

Carnivorous Plant ◽

Plant Genome ◽

List Type ◽

Utricularia Gibba

SummaryThe most studied DNA methylation pathway in plants is the RNA Directed DNA Methylation (RdDM), which is a conserved mechanism that involves noncoding-RNAs to control the expansion of intergenic regions. However, little is known about relationship between plant genome size reductions and DNA methylation.Because the compact genome size of the carnivorous plant Utricularia gibba, we investigate in this plant the noncoding-RNA landscape and DNA methylation through a combination of cytological, evolutionary, and genome-wide transcriptomic and methylation approaches.We report an unusual distribution of noncoding RNAs in U. gibba in comparison with other characterized angiosperms, which correlated with a lower level of global genome methylation, as determined by a novel strategy based on long-read DNA sequencing and corroborated by whole-genome bisulfite analysis. Moreover, found that genes involved in the RdDM pathway may not be functionally active in U. gibba, including a truncated DICER-LIKE 3 (DCL3), involved in the production of 24-nt small-RNAs.Our findings suggest that selective pressure to conserve a fully functional RdDM pathway might be reduced in compact genomes and a defective DCL3 correlate with a decreased proportion of 24-nt small-RNAs and developmental alterations in U. gibba, which could represent an initial step in the evolution of apomixis.

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Roles of Regulatory RNAs in Nutritional Control

Annual Review of Nutrition ◽

10.1146/annurev-nutr-122319-035633 ◽

2020 ◽

Vol 40 (1) ◽

pp. 77-104

Author(s):

Elizabeth M. McNeill ◽

Kendal D. Hirschi

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Small Rnas ◽

Noncoding Rna ◽

Small Cell ◽

Altered Expression ◽

Nutritional Interventions ◽

Metabolic Genes ◽

Regulatory Rnas ◽

Dietary Bioactive Compounds

Small RNAs (sRNAs), including microRNAs (miRNAs), are noncoding RNA (ncRNA) molecules involved in gene regulation. sRNAs play important roles in development; however, their significance in nutritional control and as metabolic modulators is still emerging. The mechanisms by which diet impacts metabolic genes through miRNAs remain an important area of inquiry. Recent work has established how miRNAs are transported in body fluids often within exosomes, which are small cell-derived vesicles that function in intercellular communication. The abundance of other recently identified ncRNAs and new insights regarding ncRNAs as dietary bioactive compounds could remodel our understanding about how foods impact gene expression. Although controversial, some groups have shown that dietary RNAs from plants and animals (i.e., milk) are functional in consumers. In the future, regulating sRNAs either directly through dietary delivery or indirectly by altered expression of endogenous sRNA may be part of nutritional interventions for regulating metabolism.

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Identification of a Functional Small Noncoding RNA of African Swine Fever Virus

Journal of Virology ◽

10.1128/jvi.01515-20 ◽

2020 ◽

Vol 94 (21) ◽

Author(s):

Laura E. M. Dunn ◽

Alasdair Ivens ◽

Christopher L. Netherton ◽

David A. G. Chapman ◽

Philippa M. Beard

Keyword(s):

Small Rnas ◽

Noncoding Rna ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Hemorrhagic Disease ◽

Small Noncoding Rna ◽

Small Noncoding Rnas ◽

Log Reduction ◽

Complex Interactions

ABSTRACT African swine fever virus (ASFV) causes a lethal hemorrhagic disease of domestic pigs, against which no vaccine is available. ASFV has a large, double-stranded DNA genome that encodes over 150 proteins. Replication takes place predominantly in the cytoplasm of the cell and involves complex interactions with host cellular components, including small noncoding RNAs (sncRNAs). A number of DNA viruses are known to manipulate sncRNA either by encoding their own or disrupting host sncRNA. To investigate the interplay between ASFV and sncRNAs, a study of host and viral small RNAs extracted from ASFV-infected primary porcine macrophages (PAMs) was undertaken. We discovered that ASFV infection had only a modest effect on host miRNAs, with only 6 miRNAs differentially expressed during infection. The data also revealed 3 potential novel small RNAs encoded by ASFV, ASFVsRNA1-3. Further investigation of ASFVsRNA2 detected it in lymphoid tissue from pigs with ASF. Overexpression of ASFVsRNA2 led to an up to 1-log reduction in ASFV growth, indicating that ASFV utilizes a virus-encoded small RNA to disrupt its own replication. IMPORTANCE African swine fever (ASF) poses a major threat to pig populations and food security worldwide. The disease is endemic to Africa and Eastern Europe and is rapidly emerging into Asia, where it has led to the deaths of millions of pigs in the last 12 months. The development of safe and effective vaccines to protect pigs against ASF has been hindered by lack of understanding of the complex interactions between ASFV and the host cell. We focused our work on characterizing the interactions between ASFV and sncRNAs. Although comparatively modest changes to host sncRNA abundances were observed upon ASFV infection, we discovered and characterized a novel functional ASFV-encoded sncRNA. The results from this study add important insights into ASFV host-pathogen interactions. This knowledge may be exploited to develop more effective ASFV vaccines that take advantage of the sncRNA system.

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Two novel aspects of the kinetics of gene expression including miRNAs

Open Physics ◽

10.2478/s11534-013-0190-8 ◽

2013 ◽

Vol 11 (4) ◽

Cited By ~ 1

Author(s):

Vladimir Zhdanov

Keyword(s):

Small Rnas ◽

Noncoding Rna ◽

Regulatory Protein ◽

Experimental Studies ◽

Kinetic Equations ◽

Mean Field ◽

Mrna Translation ◽

The Mean ◽

Non Coding Rnas ◽

Kinetics Of

AbstractIn eukaryotic cells, many genes are transcribed into non-coding RNAs. Small RNAs or, more specifically, microRNAs (miRNAs) form an abundant sub-class of such RNAs. miRNAs are transcribed as long noncoding RNA and then generated via a processing pathway down to the 20–24-nucleotide length. The key ability of miRNAs is to associate with target mRNAs and to suppress their translation and/or facilitate degradation. Using the mean-field kinetic equations and Monte Carlo simulations, we analyze two aspects of this interplay. First, we describe the situation when the formation of mRNA or miRNA is periodically modulated by a transcription factor which itself is not perturbed by these species. Depending on the ratio between the mRNA and miRNA formation rates, the corresponding induced periodic kinetics are shown to be either nearly harmonic or shaped as anti-phase pulses. The second part of the work is related to recent experimental studies indicating that differentiation of stem cells often involves changes in gene transcription into miRNAs and/or the interference between miRNAs, mRNAs and proteins. In particular, the regulatory protein obtained via mRNA translation may suppress the miRNA formation, and the latter may suppress in turn the miRNA-mRNA association and degradation. The corresponding bistable kinetics are described in detail.

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