BR-bodies provide selectively permeable condensates that stimulate mRNA decay and prevent release of decay intermediates

AbstractBiomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA-seq. We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved ncRNAs are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the build-up of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both, enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

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Using Pan RNA-Seq Analysis to Reveal the Ubiquitous Existence of 5′ and 3′ End Small RNAs

Frontiers in Genetics ◽

10.3389/fgene.2019.00105 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 3

Author(s):

Xiaofeng Xu ◽

Haishuo Ji ◽

Xiufeng Jin ◽

Zhi Cheng ◽

Xue Yao ◽

...

Keyword(s):

Small Rnas ◽

Rna Seq

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Nonsense-Mediated mRNA Decay Is Essential for the Hematopietic Compartement.

Blood ◽

10.1182/blood.v110.11.506.506 ◽

2007 ◽

Vol 110 (11) ◽

pp. 506-506

Author(s):

Joachim Weischenfeldt ◽

Inge Damgaard ◽

David Bryder ◽

Claus Nerlov ◽

Bo Porse

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

T Cells ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Mrna Decay ◽

Double Positive ◽

Mrna Pool ◽

Nonsense Mediated Mrna Decay

Abstract Nonsense-mediated mRNA decay (NMD) is a conserved cellular surveillance system that degrades mRNAs with premature termination codons (PTCs). PTC-containing transcripts can arise from faulty events such as erroneous mRNA processing events as well as mutations, and their translation may lead to the synthesis of deleterious proteins. In addition to serving as a genomic protection system, experiments in tissue culture cells have demonstrated that NMD regulates 5% of the normal mRNA pool suggesting that the NMD pathway may have a broader role in gene regulation. Finally, NMD has also been proposed to be important during lymphocyte development as a tool of riding the cells of transcripts resulting from unproductive re-arrangements events of T cell receptor and immunoglobulin genes. Although NMD has been studied extensively at the biochemical level, the actual role and importance of NMD in the mammalian organism has not been investigated. We therefore generated a conditional Upf2 knock-out mouse line (UPF2 being an essential NMD factor) which we crossed to different hematopoietic relevant Cre expressing lines. Full ablation of UPF2 (using the inducible Mx1-Cre deleter) led to complete loss of all nucleated cells in the bone marrow and death of the animals within 10 days. A similar phenotype was observed when Upf2fl/fl; Mx1Cre BM cells were transplanted into lethally irradiated WT recipients and induced with poly-IC, demonstrating the cell autonomous nature of the phenotype. Deletion of UPF2 in the myeloid lineage using the LysM-Cre deleter resulted in efficient ablation of UPF2 and the absence of NMD in reporter transfected bone marrow derived macrophages (BMDMs). However, the steady state levels of myeloid cells appeared unaltered. Finally, deletion of UPF2 in T cells using a Lck-Cre deleter led to a marked reduction of both CD4/CD8 double-positive and single-positive T cells and accumulation of PTC containing transcripts. Gene expression profiling experiments of BMDM and thymocytes from WT and UPF2-ablated animals identified a common core set of 27 up-regulated genes consistent with the role of NMD as a mRNA degrading system. The gene expression profiling data suggest that ablation of NMD leads to accumulation of unfolded proteins. In summary, these studies demonstrate the vital and cell-autonomous role of NMD in the hematopoietic system.

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Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2269 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2269-2284 ◽

Cited By ~ 229

Author(s):

D Herrick ◽

R Parker ◽

A Jacobson

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase Ii ◽

Mrna Decay ◽

Thermal Inactivation ◽

Decay Rates ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Mrna Decay Rate ◽

Approach To Steady State

We developed a procedure to measure mRNA decay rates in the yeast Saccharomyces cerevisiae and applied it to the determination of half-lives for 20 mRNAs encoded by well-characterized genes. The procedure utilizes Northern (RNA) or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant. We compared the results of this procedure with results obtained by two other procedures (approach to steady-state labeling and inhibition of transcription with Thiolutin) and also evaluated whether heat shock alter mRNA decay rates. We found that there are no significant differences in the mRNA decay rates measured in heat-shocked and non-heat-shocked cells and that, for most mRNAs, different procedures yield comparable relative decay rates. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MAT alpha 1, were unstable (t1/2 less than 7 min) and 4, including those encoded by ACT1 and PGK1, were stable (t1/2 greater than 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. Our results indicate that (i) stable and unstable mRNAs do not differ significantly in their poly(A) metabolism; (ii) deadenylation does not destabilize stable mRNAs; (iii) there is no correlation between mRNA decay rate and mRNA size; (iv) the degradation of both stable and unstable mRNAs depends on concomitant translational elongation; and (v) the percentage of rare codons present in most unstable mRNAs is significantly higher than in stable mRNAs.

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SIKs control osteocyte responses to parathyroid hormone

Nature Communications ◽

10.1038/ncomms13176 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 57

Author(s):

Marc N. Wein ◽

Yanke Liang ◽

Olga Goransson ◽

Thomas B. Sundberg ◽

Jinhua Wang ◽

...

Keyword(s):

Parathyroid Hormone ◽

Bone Resorption ◽

Bone Formation ◽

Small Molecule ◽

Nuclear Translocation ◽

Rna Seq ◽

Once Daily ◽

Skeletal Effects ◽

Stimulation Of

Abstract Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.

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

PLoS ONE ◽

10.1371/journal.pone.0107519 ◽

2014 ◽

Vol 9 (9) ◽

pp. e107519 ◽

Cited By ~ 15

Author(s):

Baoyan Bai ◽

Srinivasan Yegnasubramanian ◽

Sarah J. Wheelan ◽

Marikki Laiho

Keyword(s):

Small Rnas ◽

Rna Seq

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Analysis of RDR1/RDR2/RDR6-independent small RNAs in Arabidopsis thaliana improves MIRNA annotations and reveals novel siRNA loci

10.1101/238691 ◽

2017 ◽

Cited By ~ 1

Author(s):

Seth Polydore ◽

Michael J. Axtell

Keyword(s):

Gene Expression ◽

Arabidopsis Thaliana ◽

Small Rna ◽

Small Rnas ◽

Rna Seq ◽

Triple Mutant ◽

Physiological Mechanisms ◽

Protein Coding ◽

Regulate Gene Expression ◽

Rna Biogenesis

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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