RNA-Seq of the Nucleolus Reveals Abundant SNORD44-Derived Small RNAs

SummaryPlant small RNAs regulate key physiological mechanisms through post-transcriptional and transcriptional silencing of gene expression. sRNAs fall into two major categories: those that are reliant on RNA Dependent RNA Polymerases (RDRs) for biogenesis and those that aren’t. Known RDR-dependent sRNAs include phased and repeat-associated short interfering RNAs, while known RDR-independent sRNAs are primarily microRNAs and other hairpin-derived sRNAs. In this study, we produced and analyzed small RNA-seq libraries from rdr1/rdr2/rdr6 triple mutant plants. Only a small fraction of all sRNA loci were RDR1/RDR2/RDR6-independent; most of these were microRNA loci or associated with predicted hairpin precursors. We found 58 previously annotated microRNA loci that were reliant on RDR1, −2, or −6 function, casting doubt on their classification. We also found 38 RDR1/2/6-independent small RNA loci that are not MIRNAs or otherwise hairpin-derived, and did not fit into other known paradigms for small RNA biogenesis. These 38 small RNA-producing loci have novel biogenesis mechanisms, and are frequently located in the vicinity of protein-coding genes. Altogether, our analysis suggest that these 38 loci represent one or more new types of small RNAs in Arabidopsis thaliana.Significance StatementSmall RNAs regulate gene expression in plants and are produced through a variety of previously-described mechanisms. Here, we examine a set of previously undiscovered small RNA-producing loci that are produced by novel mechanisms.

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Removing bias against short sequences enables northern blotting to better complement RNA-seq for the study of small RNAs

10.1101/068031 ◽

2016 ◽

Author(s):

Yun S. Choi ◽

Lanelle O. Edwards ◽

Aubrey DiBello ◽

Antony M. Jose

Keyword(s):

Small Rna ◽

Small Rnas ◽

Degradation Products ◽

Northern Blotting ◽

Rna Seq ◽

Single Nucleotide ◽

Rna Integrity ◽

C Elegans ◽

Micro Rnas ◽

Functional Rnas

ABSTRACTChanges in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that northern blotting of small RNAs is biased against short sequences, and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm C. elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq. When using northern blotting during these studies, we discovered that miRNA-length probes can have a ~360-fold bias against detecting even synthetic sequences that are 8 nt shorter. By using shorter probes and by performing hybridization and washes at low temperatures, we greatly reduced this bias to enable equivalent detection of 24 nt to 14 nt RNAs. Our protocol can better discriminate RNAs that differ by a single nucleotide and can detect specific miRNAs present in total RNA from C. elegans. This improved northern blotting is particularly useful to obtain a measure of small RNA integrity, analyze products of RNA processing or turnover, and analyze functional RNAs that are shorter than typical miRNAs.

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Using pan RNA-seq analysis to reveal the ubiquitous existence of 5’ end and 3’ end small RNAs

10.1101/444117 ◽

2018 ◽

Author(s):

Xiaofeng Xu ◽

Haishuo Ji ◽

Zhi Cheng ◽

Xiufeng Jin ◽

Xue Yao ◽

...

Keyword(s):

Gene Expression ◽

Steady State ◽

Small Rnas ◽

Gene Expression Regulation ◽

Regulation Of Gene Expression ◽

Cost Effective ◽

Rna Degradation ◽

Point Of View ◽

Rna Seq ◽

Dsrna Cleavage

AbstractIn this study, we used pan RNA-seq analysis to reveal the ubiquitous existence of 5’ end and 3’ end small RNAs. 5’ and 3’ sRNAs alone can be used to annotate mitochondrial with 1-bp resolution and nuclear non-coding genes and identify new steady-state RNAs, which are usually from functional genes. Using 5’, 3’ and intronic sRNAs, we revealed that the enzymatic dsRNA cleavage and RNAi could involve in the RNA degradation and gene expression regulation of U1 snRNA in human. The further study of 5’, 3’ and intronic sRNAs help rediscover double-stranded RNA (dsRNA) cleavage, RNA interference (RNAi) and the regulation of gene expression, which challenges the classical theories. In this study, we provided a simple and cost effective way for the annotation of mitochondrial and nuclear non-coding genes and the identification of new steady-state RNAs, particularly long non-coding RNAs (lncRNAs). We also provided a different point of view for cancer and virus, based on the new discoveries of dsRNA cleavage, RNAi and the regulation of gene expression.

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Refining the pneumococcal competence regulon by RNA-sequencing

10.1101/497099 ◽

2018 ◽

Author(s):

Jelle Slager ◽

Rieza Aprianto ◽

Jan-Willem Veening

Keyword(s):

Small Rnas ◽

Antigenic Variation ◽

Stress Factors ◽

Rna Seq ◽

Exogenous Dna ◽

Genomic Plasticity ◽

Resistance Markers ◽

Opportunistic Human Pathogen ◽

Competence System ◽

Generation Sequencing

ABSTRACTCompetence for genetic transformation allows the opportunistic human pathogenStreptococcus pneumoniaeto take up exogenous DNA for incorporation into its own genome. This ability may account for the extraordinary genomic plasticity of this bacterium, leading to antigenic variation, vaccine escape, and the spread of antibiotic resistance markers. The competence system has been thoroughly studied and its regulation is well-understood. Additionally, over the last decade, several stress factors have been shown to trigger the competent state, leading to the activation of several stress response regulons. The arrival of next-generation sequencing techniques allowed us to update the competence regulon, the latest report of which still depended on DNA microarray technology. Enabled by the availability of an up-to-date genome annotation, including transcript boundaries, we assayed time-dependent expression of all annotated features in response to competence induction, were able to identify the affected promoters and produced a more complete overview of the various regulons activated during competence. We show that 4% of all annotated genes are under direct control of competence regulators ComE and ComX, while the expression of a total of up to 17% of all genes is, either directly or indirectly, affected. Among the affected genes are various small RNAs with an as-of-yet unknown function. Besides the ComE and ComX regulons, we were also able to refine the CiaR, VraR (LiaR) and BlpR regulons, underlining the strength of combining RNA-seq with a well-annotated genome.

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Dual RNA-seq in Streptococcus pneumoniae Infection Reveals Compartmentalized Neutrophil Responses in Lung and Pleural Space

mSystems ◽

10.1128/msystems.00216-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 6

Author(s):

Neil D. Ritchie ◽

Tom J. Evans

Keyword(s):

Streptococcus Pneumoniae ◽

Small Rnas ◽

Pneumococcal Infection ◽

Pleural Space ◽

Type I Interferons ◽

Type I ◽

Bacterial Cells ◽

Rna Seq ◽

Bacterial Killing ◽

Content Type

ABSTRACT Streptococcus pneumoniae is the dominant cause of community-acquired pneumonia worldwide. Invasion of the pleural space is common and results in increased mortality. We set out to determine the bacterial and host factors that influence invasion of the pleural space. In a murine model of pneumococcal infection, we isolated neutrophil-dominated samples of bronchoalveolar and pleural fluid containing bacteria 48 hours after infection. Using dual RNA sequencing (RNA-seq), we characterized bacterial and host transcripts that were differentially regulated between these compartments and bacteria in broth and resting neutrophils, respectively. Pleural and lung samples showed upregulation of genes involved in the positive regulation of neutrophil extravasation but downregulation of genes mediating bacterial killing. Compared to the lung samples, cells within the pleural space showed marked upregulation of many genes induced by type I interferons, which are cytokines implicated in preventing bacterial transmigration across epithelial barriers. Differences in the bacterial transcripts between the infected samples and bacteria grown in broth showed the upregulation of genes in the bacteriocin locus, the pneumococcal surface adhesin PsaA, and the glycopeptide resistance gene vanZ; the gene encoding the ClpP protease was downregulated in infection. One hundred sixty-nine intergenic putative small bacterial RNAs were also identified, of which 43 (25.4%) small RNAs had been previously described. Forty-two of the small RNAs were upregulated in pleura compared to broth, including many previously identified as being important in virulence. Our results have identified key host and bacterial responses to invasion of the pleural space that can be potentially exploited to develop alternative antimicrobial strategies for the prevention and treatment of pneumococcal pleural disease. IMPORTANCE The factors that regulate the passage of bacteria between different anatomical compartments are unclear. We have used an experimental model of infection with Streptococcus pneumoniae to examine the host and bacterial factors involved in the passage of bacteria from the lung to the pleural space. The transcriptional profile of host and bacterial cells within the pleural space and lung was analyzed using deep sequencing of the entire transcriptome using the technique of dual RNA-seq. We found significant differences in the host and bacterial RNA profiles in infection, which shed light on the key factors that allow passage of this bacterium into the pleural space.

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MiR&moRe2: A Bioinformatics Tool to Characterize microRNAs and microRNA-Offset RNAs from Small RNA-Seq Data

International Journal of Molecular Sciences ◽

10.3390/ijms21051754 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1754 ◽

Cited By ~ 3

Author(s):

Enrico Gaffo ◽

Michele Bortolomeazzi ◽

Andrea Bisognin ◽

Piero Di Battista ◽

Federica Lovisa ◽

...

Keyword(s):

Small Rna ◽

Small Rnas ◽

Cell Types ◽

Small Rna Sequencing ◽

Rna Seq ◽

Sequencing Data ◽

Research Findings ◽

Human Blood Cell ◽

And Function ◽

Comprehensive Study

MicroRNA-offset RNAs (moRNAs) are microRNA-like small RNAs generated by microRNA precursors. To date, little is known about moRNAs and bioinformatics tools to inspect their expression are still missing. We developed miR&moRe2, the first bioinformatics method to consistently characterize microRNAs, moRNAs, and their isoforms from small RNA sequencing data. To illustrate miR&moRe2 discovery power, we applied it to several published datasets. MoRNAs identified by miR&moRe2 were in agreement with previous research findings. Moreover, we observed that moRNAs and new microRNAs predicted by miR&moRe2 were downregulated upon the silencing of the microRNA-biogenesis pathway. Further, in a sizeable dataset of human blood cell populations, tens of novel miRNAs and moRNAs were discovered, some of them with significantly varied expression levels among the cell types. Results demonstrate that miR&moRe2 is a valid tool for a comprehensive study of small RNAs generated from microRNA precursors and could help to investigate their biogenesis and function.

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Bacterial Small RNAs in the Genus Herbaspirillum spp.

International Journal of Molecular Sciences ◽

10.3390/ijms20010046 ◽

2018 ◽

Vol 20 (1) ◽

pp. 46 ◽

Cited By ~ 1

Author(s):

Amanda Carvalho Garcia ◽

Vera dos Santos ◽

Teresa Santos Cavalcanti ◽

Luiz Collaço ◽

Hans Graf

Keyword(s):

Small Rnas ◽

Physiological Responses ◽

Rna Seq ◽

Herbaspirillum Seropedicae ◽

Experimental Conditions ◽

Environmental Signals ◽

Base Sequences ◽

Non Coding Rnas ◽

Rna Interaction ◽

Spacer Dna

The genus Herbaspirillum includes several strains isolated from different grasses. The identification of non-coding RNAs (ncRNAs) in the genus Herbaspirillum is an important stage studying the interaction of these molecules and the way they modulate physiological responses of different mechanisms, through RNA–RNA interaction or RNA–protein interaction. This interaction with their target occurs through the perfect pairing of short sequences (cis-encoded ncRNAs) or by the partial pairing of short sequences (trans-encoded ncRNAs). However, the companion Hfq can stabilize interactions in the trans-acting class. In addition, there are Riboswitches, located at the 5′ end of mRNA and less often at the 3′ end, which respond to environmental signals, high temperatures, or small binder molecules. Recently, CRISPR (clustered regularly interspaced palindromic repeats), in prokaryotes, have been described that consist of serial repeats of base sequences (spacer DNA) resulting from a previous exposure to exogenous plasmids or bacteriophages. We identified 285 ncRNAs in Herbaspirillum seropedicae (H. seropedicae) SmR1, expressed in different experimental conditions of RNA-seq material, classified as cis-encoded ncRNAs or trans-encoded ncRNAs and detected RNA riboswitch domains and CRISPR sequences. The results provide a better understanding of the participation of this type of RNA in the regulation of the metabolism of bacteria of the genus Herbaspirillum spp.

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Genome Sequence of Southern tomato virus in Asymptomatic Tomato ‘Sweet Hearts’

Genome Announcements ◽

10.1128/genomea.01374-16 ◽

2017 ◽

Vol 5 (7) ◽

Cited By ~ 6

Author(s):

Ricardo I. Alcalá-Briseño ◽

Sevgi Coşkan ◽

Maria A. Londoño ◽

Jane E. Polston

Keyword(s):

United States ◽

North Carolina ◽

Solanum Lycopersicum ◽

Genome Sequence ◽

Small Rnas ◽

The United States ◽

Full Genome ◽

Rna Seq ◽

Genome Sequences

ABSTRACT The genome sequence of Southern tomato virus in asymptomatic Solanum lycopersicum ‘Sweet Hearts’ (STV-Florida) in Florida was assembled from small RNAs sequenced by Illumina RNA-seq. The STV-Florida genome shared 99.0 to 99.9% similarity with full genome sequences from Bangladesh, China, Mexico, and the United States (Mississippi and North Carolina).

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