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1.
Biochim Biophys Acta Gene Regul Mech ; 1863(3): 194506, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32068131

RESUMO

Discovered in the 1980s, small regulatory RNAs (sRNAs) are now considered key actors in virtually all aspects of bacterial physiology and virulence. Together with transcriptional and translational regulatory proteins, they integrate and often are hubs of complex regulatory networks, responsible for bacterial response/adaptation to various perceived stimuli. The recent development of powerful RNA sequencing technologies has facilitated the identification and characterization of sRNAs (length, structure and expression conditions) and their RNA targets in several bacteria. Nevertheless, it could be very difficult for non-experts to understand the advantages and drawbacks related to each offered option and, consequently, to make an informed choice. Therefore, the main goal of this review is to provide a guide to navigate through the twists and turns of high-throughput RNA sequencing technologies, with a specific focus on those applied to the study of sRNAs. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.


Assuntos
RNA Bacteriano/química , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Análise de Sequência de RNA , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico
2.
BMC Bioinformatics ; 21(1): 15, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931703

RESUMO

BACKGROUND: Seed and accessibility constraints are core features to enable highly accurate sRNA target screens based on RNA-RNA interaction prediction. Currently, available tools provide different (sets of) constraints and default parameter sets. Thus, it is hard to impossible for users to estimate the influence of individual restrictions on the prediction results. RESULTS: Here, we present a systematic assessment of the impact of established and new constraints on sRNA target prediction both on a qualitative as well as computational level. This is done exemplarily based on the performance of IntaRNA, one of the most exact sRNA target prediction tools. IntaRNA provides various ways to constrain considered seed interactions, e.g. based on seed length, its accessibility, minimal unpaired probabilities, or energy thresholds, beside analogous constraints for the overall interaction. Thus, our results reveal the impact of individual constraints and their combinations. CONCLUSIONS: This provides both a guide for users what is important and recommendations for existing and upcoming sRNA target prediction approaches.We show on a large sRNA target screen benchmark data set that only by altering the parameter set, IntaRNA recovers 30% more verified interactions while becoming 5-times faster. This exemplifies the potential of seed, accessibility and interaction constraints for sRNA target prediction.


Assuntos
Bactérias/genética , Biologia Computacional/métodos , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Bactérias/química , Bactérias/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo
3.
Biochim Biophys Acta Gene Regul Mech ; 1863(1): 194477, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31884117

RESUMO

Cyanobacteria are photosynthetic bacteria that populate widely different habitats. Accordingly, cyanobacteria exhibit a wide spectrum of lifestyles, physiologies, and morphologies and possess genome sizes and gene numbers which may vary by up to a factor of ten within the phylum. Consequently, large differences exist between individual species in the size and complexity of their regulatory networks. Several non-coding RNAs have been identified that play crucial roles in the acclimation responses of cyanobacteria to changes in the environment. Some of these regulatory RNAs are conserved throughout the cyanobacterial phylum, while others exist only in a few taxa. Here we give an overview on characterized regulatory RNAs in cyanobacteria, with a focus on regulators of photosynthesis, carbon and nitrogen metabolism. However, chances are high that these regulators represent just the tip of the iceberg.


Assuntos
Carbono/metabolismo , Cianobactérias/genética , Nitrogênio/metabolismo , Fotossíntese/genética , RNA não Traduzido/fisiologia , Cianobactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Fixação de Nitrogênio/genética , RNA Antissenso/biossíntese , Pequeno RNA não Traduzido/biossíntese , Pequeno RNA não Traduzido/química , RNA não Traduzido/biossíntese , Riboswitch
4.
Microbiol Res ; 229: 126319, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31479952

RESUMO

Methionine is critical for variety of metabolic processes in biological organisms, acting as a precursor or intermediate for many final products. The last step for the synthesis of methionine is the methylation of homocysteine, which is catalyzed by MetE. Here, we use Salmonella enterica serovar Typhimurium LT2 to study the regulation of the metE+ gene by an anaerobically induced small non-coding RNA-FnrS, the expression of which is strictly dependent on the anaerobic regulator-FNR. The MetE-HA protein was expressed at an increased level in the fnrS- and hfq- deficient strains under anaerobic conditions. The Hfq protein is predicted to stabilize the binding between small RNA(s) and their target mRNA(s). A transcriptional (op) and translational (pr) metE::lacZ fusion gene were separately constructed, with the metE+-promoter fused to a lacZ reporter gene. In an anaerobic environment, the metE::lacZ (pr) fusion gene and reverse transcription-PCR identified that FnrS and/or FNR negatively regulate metE+ mRNA levels in the rich media. Analysis of FnrS revealed a sequence complementary to the 5' mRNA translational initiation region (TIR) of the metE+ gene. Mutation(s) predicted to disrupt base pairing between FnrS and metE+ TIR were constructed in fnrS, and most of those resulted in the loss of repressive activity. When compensatory mutation(s) were made in metE+ 5' TIR to restore base pairing with FnrS, the repressive regulation was completely restored. Therefore, in this study, we identified that in anaerobic phase, there is a repression of metE+ gene expression by FnrS and that base-paring, between both expressive transcripts, plays an important role for this negative regulation.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Metiltransferases/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Salmonella typhimurium/enzimologia , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Regulação Enzimológica da Expressão Gênica , Metiltransferases/química , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
5.
EMBO J ; 38(16): e101650, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31313835

RESUMO

Small regulatory RNAs (sRNAs) are crucial components of many stress response systems. The envelope stress response (ESR) of Gram-negative bacteria is a paradigm for sRNA-mediated stress management and involves, among other factors, the alternative sigma factor E (σE ) and one or more sRNAs. In this study, we identified the MicV sRNA as a new member of the σE regulon in Vibrio cholerae. We show that MicV acts redundantly with another sRNA, VrrA, and that both sRNAs share a conserved seed-pairing domain allowing them to regulate multiple target mRNAs. V. cholerae lacking σE displayed increased sensitivity toward antimicrobials, and over-expression of either of the sRNAs suppressed this phenotype. Laboratory selection experiments using a library of synthetic sRNA regulators revealed that the seed-pairing domain of σE -dependent sRNAs is strongly enriched among sRNAs identified under membrane-damaging conditions and that repression of OmpA is crucial for sRNA-mediated stress relief. Together, our work shows that MicV and VrrA act as global regulators in the ESR of V. cholerae and provides evidence that bacterial sRNAs can be functionally annotated by their seed-pairing sequences.


Assuntos
Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Vibrio cholerae/genética , Proteínas da Membrana Bacteriana Externa/genética , Sequência Conservada , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , Estresse Fisiológico
6.
Nucleic Acids Res ; 47(12): 6396-6410, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-30993322

RESUMO

Ribosome-mediated transcription attenuation is a basic posttranscriptional regulation mechanism in bacteria. Liberated attenuator RNAs arising in this process are generally considered nonfunctional. In Sinorhizobium meliloti, the tryptophan (Trp) biosynthesis genes are organized into three operons, trpE(G), ppiD-trpDC-moaC-moeA, and trpFBA-accD-folC, of which only the first one, trpE(G), contains a short ORF (trpL) in the 5'-UTR and is regulated by transcription attenuation. Under conditions of Trp sufficiency, transcription is terminated between trpL and trpE(G), and a small attenuator RNA, rnTrpL, is produced. Here, we show that rnTrpL base-pairs with trpD and destabilizes the polycistronic trpDC mRNA, indicating rnTrpL-mediated downregulation of the trpDC operon in trans. Although all three trp operons are regulated in response to Trp availability, only in the two operons trpE(G) and trpDC the Trp-mediated regulation is controlled by rnTrpL. Together, our data show that the trp attenuator coordinates trpE(G) and trpDC expression posttranscriptionally by two fundamentally different mechanisms: ribosome-mediated transcription attenuation in cis and base-pairing in trans. Also, we present evidence that rnTrpL-mediated regulation of trpDC genes expression in trans is conserved in Agrobacterium and Bradyrhizobium, suggesting that the small attenuator RNAs may have additional conserved functions in the control of bacterial gene expression.


Assuntos
Regulação Bacteriana da Expressão Gênica , Pequeno RNA não Traduzido/metabolismo , Sinorhizobium meliloti/genética , Triptofano/biossíntese , Pareamento de Bases , Óperon , Estabilidade de RNA , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Sinorhizobium meliloti/metabolismo , Transcrição Genética
7.
RNA Biol ; 16(6): 719-726, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30870072

RESUMO

Small RNAs are important for post-transcriptional regulation of gene expression, affecting stability and activity of their target mRNAs. The bacterial Sm-like protein Hfq is required to promote pairing between both RNAs when their sequence complementarity is limited. To provide a first global view on the post-transcriptional landscape of the α-proteobacterium Caulobacter crescentus, we have identified the Hfq-binding RNAs employing High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). A total of 261 RNAs, including 3 unannotated RNAs, were successfully identified and classified according to putative function. Moreover, possible interactions between the identified sRNAs with mRNA targets were postulated through computational target predictions.


Assuntos
Caulobacter crescentus/genética , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Pequeno RNA não Traduzido/química , Análise de Sequência de RNA
8.
RNA Biol ; 16(6): 830-837, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30870076

RESUMO

In this study, we used a small RNA sequencing (sRNA-seq) based method to annotate the mitochondrial genome of the insect Erthesina fullo Thunberg at 1 bp resolution. The high-resolution annotations cover both entire strands of the mitochondrial genome without any gaps or overlaps. Most of the new annotations were consistent with the previous annotations which had been obtained using PacBio full-length transcripts. Two important findings were that animals transcribe both entire strands of mitochondrial genomes and the tandem repeats in the control region of the E. fullo mitochondrial genome contains the repeated Transcription Initiation Sites (TISs) of the heavy strand. In addition, we found that the copy numbers of tandem repeats showed a great diversity within an individual, suggesting that mitochondrial DNA recombination occurs in an individual. In conclusion, the sRNA-seq based method uses 5' and 3' end small RNAs to annotate nuclear non-coding and mitochondrial genes at 1 bp resolution, and can be used to identify new steady RNAs, particularly long non-coding RNAs (lncRNAs). The high-resolution annotations of mitochondrial genomes can also be used to study the molecular phylogenetics and evolution of animals or to investigate mitochondrial gene transcription, RNA processing, RNA maturation and several other related topics. The complete mitochondrial genome sequence of E. fullo with the new annotations using the sRNA-seq based method is available at the NCBI GenBank database under the accession number MK374364. We publish our theories, methods, the high quality sRNA-seq and RNA-seq data (SRA: SRP174926) for extensive use.


Assuntos
DNA Mitocondrial/química , Genoma Mitocondrial , Heterópteros/genética , Anotação de Sequência Molecular , Sequências de Repetição em Tandem , Animais , Genes de Insetos , RNA Mitocondrial/química , Pequeno RNA não Traduzido/química , Análise de Sequência de RNA , Sítio de Iniciação de Transcrição
9.
RNA Biol ; 16(3): 270-281, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30706751

RESUMO

The bacterial pathogen Listeria monocytogenes encodes seven homologous small regulatory RNAs, named the LhrC family of sRNAs. The LhrCs are highly induced under infection-relevant conditions and are known to inhibit the expression of multiple target mRNAs encoding virulence-associated surface proteins. In all cases studied so far, the LhrCs use their CU-rich regions for base pairing to complementary AG-rich sequences of the ribosomal binding site (RBS) of specific target mRNAs. Consequently, LhrC-mRNA interaction results in inhibition of translation followed by mRNA degradation, corresponding to the canonical model for sRNA-mediated gene regulation in bacteria. Here, we demonstrate that the LhrC sRNAs employ a different regulatory mechanism when acting to down-regulate the expression of tcsA, encoding a T cell-stimulating antigen. In this case, LhrC base pairs to an AG-rich site located well upstream of the RBS in tcsA mRNA. Using an in vitro translation assay, we found that LhrC could not prevent the ribosome from translating the tcsA messenger. Rather, the LhrC sRNAs act to decrease the half-life of tcsA mRNA in vivo. Importantly, LhrC-mediated destabilization of tcsA mRNA relies on an intact LhrC binding site near the 5´-end of the tcsA mRNA and occurs independently of translation. Based on these findings, we propose an alternative mechanism for LhrC-mediated control in L. monocytogenes that relies solely on sRNA-induced degradation of a target mRNA.


Assuntos
Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Interferência de RNA , RNA Bacteriano/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Regiões 5' não Traduzidas , Sequência de Bases , Sítios de Ligação , Listeriose/imunologia , Modelos Biológicos , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Bacteriano/química , Pequeno RNA não Traduzido/química
10.
Parasit Vectors ; 12(1): 36, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30646930

RESUMO

BACKGROUND: Transfer RNA (tRNA)-derived fragments (tRFs) have been widely identified in nature, functioning in diverse biological and pathological situations. Yet, the presence of these small RNAs in Plasmodium spp. remains unknown. Systematic identification and characterization of tRFs is therefore highly needed to understand further their roles in Plasmodium parasites, particularly in the virulent Plasmodium falciparum parasite. RESULTS: Genome-wide small RNAs with sizes ranging from 18-30 nucleotides from P. falciparum were deep-sequenced via Illumina HiSeq 2000 technology. In-depth analysis revealed the presence of a vast number of small RNAs originating from tRNA-coding genes, responsible for 22.4% of the total reads as the second predominant group. Three P. falciparum-derived tRF types (ptRFs) were identified as 5'ptRFs, mid-ptRFs and 3'ptRFs. The majority (90%) of ptRFs were derived from tRNAs that coded eight amino acids: Pro, Phe, Asn, Gly, Cys, Gln, His and Ala. Stem-loop reverse transcription polymerase chain reaction further confirmed the presence of tRFs in the blood stages of P. falciparum. Four new motifs with an enriched G/C feature were determined at cleavage sites that might guide the generation of ptRFs. CONCLUSIONS: To our knowledge, this is the first report of a genome-wide investigation of ptRFs from Plasmodium species. The identification of ptRFs reveals a complex small RNA system manipulated by the malaria parasite, and might promote research on the function of tRFs in the pathogenesis of Plasmodium infections.


Assuntos
Genoma de Protozoário , Plasmodium falciparum/genética , Pequeno RNA não Traduzido/genética , RNA de Transferência/genética , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Reação em Cadeia da Polimerase , Pequeno RNA não Traduzido/química , RNA de Transferência/química , Análise de Sequência de DNA
11.
Nucleic Acids Res ; 47(3): 1482-1492, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30462307

RESUMO

Increasing 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 better understand the sequence and structure determinants for the precise processing of a 3' UTR-derived sRNA, 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 probably other 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.


Assuntos
Endorribonucleases/química , Escherichia coli/genética , Pequeno RNA não Traduzido/genética , Regiões 3' não Traduzidas/genética , Endorribonucleases/genética , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , Pequeno RNA não Traduzido/química
12.
RNA ; 24(12): 1761-1784, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30217864

RESUMO

MgrR is an Hfq-dependent sRNA, whose transcription is controlled by the level of Mg2+ ions in Escherichia coli MgrR belongs to Class II sRNAs because its stability in the cell is affected by mutations in Hfq differently than canonical, Class I sRNAs. Here, we examined the effect of mutations in RNA binding sites of Hfq on the kinetics of the annealing of MgrR to two different target mRNAs, eptB and ygdQ, by global data fitting of the reaction kinetics monitored by gel electrophoresis of intermediates and products. The data showed that the mutation on the rim of the Hfq ring trapped MgrR on Hfq preventing the annealing of MgrR to either mRNA. The mutation in the distal face slowed the ternary complex formation and affected the release of MgrR-mRNA complexes from Hfq, while the mutation in the proximal face weakened the MgrR binding to Hfq and in this way affected the annealing. Moreover, competition assays established that MgrR bound to both faces of Hfq and competed against other sRNAs. Further studies showed that uridine-rich sequences located in less structurally stable regions served as Hfq binding sites in each mRNA. Overall, the data show that the binding of MgrR sRNA to both faces of the Hfq ring enables it to efficiently anneal to target mRNAs. It also confers on MgrR a competitive advantage over other sRNAs, which could contribute to efficient cellular response to changes in magnesium homeostasis.


Assuntos
Proteínas de Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Pequeno RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Sítios de Ligação , Escherichia coli/genética , Proteínas de Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/química , Magnésio/química , Magnésio/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/química , Proteínas de Ligação a RNA/química
13.
Nucleic Acids Res ; 46(20): 10969-10982, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30165530

RESUMO

Bacterial small RNAs (sRNAs) are a heterogeneous group of post-transcriptional regulators that often act at the heart of large networks. Hundreds of sRNAs have been discovered by genome-wide screens and most of these sRNAs exert their functions by base-pairing with target mRNAs. However, studies addressing the molecular roles of sRNAs have been largely confined to gamma-proteobacteria, such as Escherichia coli. Here we identify and characterize a novel sRNA, ChvR, from the alpha-proteobacterium Caulobacter crescentus. Transcription of chvR is controlled by the conserved two-component system ChvI-ChvG and it is expressed in response to DNA damage, low pH, and growth in minimal medium. Transient over-expression of ChvR in combination with genome-wide transcriptome profiling identified the mRNA of the TonB-dependent receptor ChvT as the sole target of ChvR. Genetic and biochemical analyses showed that ChvR represses ChvT at the post-transcriptional level through direct base-pairing. Fine-mapping of the ChvR-chvT interaction revealed the requirement of two distinct base-pairing sites for full target regulation. Finally, we show that ChvR-controlled repression of chvT is independent of the ubiquitous RNA-chaperone Hfq, and therefore distinct from previously reported mechanisms employed by prototypical bacterial sRNAs. These findings have implications for the mechanism and evolution of sRNA function across bacterial species.


Assuntos
Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Caulobacter crescentus/metabolismo , Dano ao DNA , Perfilação da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo
14.
Biomolecules ; 8(3)2018 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-30012983

RESUMO

tRNA-derived fragments (tRFs) are a new class of non-coding RNA that play an important role in regulating cellular RNA processing and protein translation. However, there is currently no study reporting the influence of tRFs on myocardial hypertrophy. In this study, we used an isoproterenol (ISO)-induced myocardial hypertrophy rat model. Small RNA (<40 nts) transcriptome sequencing was used to select differentially expressed tRFs. We also compared the tRFs expression pattern in F0 sperm and the hearts of F1 offspring between the myocardial hypertrophy group (Hyp) and the control group (Con). Isoproterenol successfully induced a typical cardiac hypertrophy model in our study. Small RNA-seq revealed that tRFs were extremely enriched (84%) in the Hyp heart. Overexpression of tRFs1 and tRFs2 both enlarged the surface area of cardiac cells and increased expression of hypertrophic markers (ANF, BNP, and ß-MHC). Luciferase reporter assay identified that tRFs1 directly target 3'UTR of Timp3. tRFs1, tRFs2, tRFs3, and tRFs4 were also highly expressed in Hyp F0 sperm and in Hyp F1 offspring hearts, but there was no differential expression of tRFs7, tRFs9, and tRFs10. Compared to Con F1 offspring, Hyp F1 offspring had elevated expression levels of ß-MHC and ANP genes, and they had increased fibrosis and apoptosis in their hearts. These results demonstrated that tRFs are involved in regulating the response of myocardial hypertrophy. Besides, tRFs might serve as novel epigenetic factors that contribute to the intergenerational inheritance of cardiac hypertrophy.


Assuntos
Cardiomegalia/patologia , Perfilação da Expressão Gênica/métodos , Isoproterenol/efeitos adversos , Pequeno RNA não Traduzido/genética , RNA de Transferência/química , Regulação para Cima , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Modelos Animais de Doenças , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Herança Materna , Herança Paterna , Pequeno RNA não Traduzido/química , Ratos , Análise de Sequência de RNA , Espermatozoides/química
15.
FEBS Lett ; 592(17): 2874-2883, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29749606

RESUMO

It is now evident that noncoding RNAs play key roles in regulatory networks determining cell fate and behavior, in a myriad of different conditions, and across all species. Among these noncoding RNAs are short RNAs, such as MicroRNAs, snoRNAs, and Piwi-interacting RNAs, and the functions of those are relatively well understood. Other noncoding RNAs are longer, and their modes of action and functions are also increasingly explored and deciphered. Short RNAs and long noncoding RNAs (lncRNAs) interact with each other with reciprocal consequences for their fates and functions. LncRNAs serve as precursors for many types of small RNAs and, therefore, the pathways for small RNA biogenesis can impinge upon the fate of lncRNAs. In addition, lncRNA expression can be repressed by small RNAs, and lncRNAs can affect small RNA activity and abundance through competition for binding or by triggering small RNA degradation. Here, I review the known types of interactions between small and long RNAs, discuss their outcomes, and bring representative examples from studies in mammals.


Assuntos
RNA Longo não Codificante/metabolismo , Pequeno RNA não Traduzido/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética
16.
Genomics Proteomics Bioinformatics ; 16(2): 144-151, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29730207

RESUMO

High-throughput RNA-seq has revolutionized the process of small RNA (sRNA) discovery, leading to a rapid expansion of sRNA categories. In addition to the previously well-characterized sRNAs such as microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and small nucleolar RNA (snoRNAs), recent emerging studies have spotlighted on tRNA-derived sRNAs (tsRNAs) and rRNA-derived sRNAs (rsRNAs) as new categories of sRNAs that bear versatile functions. Since existing software and pipelines for sRNA annotation are mostly focused on analyzing miRNAs or piRNAs, here we developed the sRNA annotation pipelineoptimized for rRNA- and tRNA-derived sRNAs (SPORTS1.0). SPORTS1.0 is optimized for analyzing tsRNAs and rsRNAs from sRNA-seq data, in addition to its capacity to annotate canonical sRNAs such as miRNAs and piRNAs. Moreover, SPORTS1.0 can predict potential RNA modification sites based on nucleotide mismatches within sRNAs. SPORTS1.0 is precompiled to annotate sRNAs for a wide range of 68 species across bacteria, yeast, plant, and animal kingdoms, while additional species for analyses could be readily expanded upon end users' input. For demonstration, by analyzing sRNA datasets using SPORTS1.0, we reveal that distinct signatures are present in tsRNAs and rsRNAs from different mouse cell types. We also find that compared to other sRNA species, tsRNAs bear the highest mismatch rate, which is consistent with their highly modified nature. SPORTS1.0 is an open-source software and can be publically accessed at https://github.com/junchaoshi/sports1.0.


Assuntos
RNA Ribossômico/química , Pequeno RNA não Traduzido/química , RNA de Transferência/química , Análise de Sequência de RNA/métodos , Software , Animais , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , MicroRNAs/química , MicroRNAs/metabolismo , Anotação de Sequência Molecular , RNA Ribossômico/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/metabolismo
17.
Cell Mol Life Sci ; 75(20): 3857-3875, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29808415

RESUMO

The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs.


Assuntos
Células Dendríticas/metabolismo , Vesículas Extracelulares/genética , Transcriptoma , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Colecalciferol/farmacologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Corantes Fluorescentes/química , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Microscopia Eletrônica , Nanopartículas/química , RNA Nucleolar Pequeno/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/isolamento & purificação , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/metabolismo , Análise de Sequência de RNA
18.
Nat Cell Biol ; 20(5): 535-540, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29695786

RESUMO

The discovery of RNAs (for example, messenger RNAs, non-coding RNAs) in sperm has opened the possibility that sperm may function by delivering additional paternal information aside from solely providing the DNA 1 . Increasing evidence now suggests that sperm small non-coding RNAs (sncRNAs) can mediate intergenerational transmission of paternally acquired phenotypes, including mental stress2,3 and metabolic disorders4-6. How sperm sncRNAs encode paternal information remains unclear, but the mechanism may involve RNA modifications. Here we show that deletion of a mouse tRNA methyltransferase, DNMT2, abolished sperm sncRNA-mediated transmission of high-fat-diet-induced metabolic disorders to offspring. Dnmt2 deletion prevented the elevation of RNA modifications (m5C, m2G) in sperm 30-40 nt RNA fractions that are induced by a high-fat diet. Also, Dnmt2 deletion altered the sperm small RNA expression profile, including levels of tRNA-derived small RNAs and rRNA-derived small RNAs, which might be essential in composing a sperm RNA 'coding signature' that is needed for paternal epigenetic memory. Finally, we show that Dnmt2-mediated m5C contributes to the secondary structure and biological properties of sncRNAs, implicating sperm RNA modifications as an additional layer of paternal hereditary information.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Transtornos do Metabolismo de Glucose/enzimologia , Transtornos do Metabolismo de Glucose/genética , Herança Paterna , Pequeno RNA não Traduzido/genética , Espermatozoides/enzimologia , Animais , Biomarcadores/sangue , Glicemia/metabolismo , DNA (Citosina-5-)-Metiltransferases/deficiência , DNA (Citosina-5-)-Metiltransferases/genética , Dieta Hiperlipídica , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Interação Gene-Ambiente , Predisposição Genética para Doença , Transtornos do Metabolismo de Glucose/sangue , Transtornos do Metabolismo de Glucose/diagnóstico , Hereditariedade , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Conformação de Ácido Nucleico , Fenótipo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Relação Estrutura-Atividade , Transcriptoma
19.
BMC Genomics ; 19(1): 247, 2018 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-29642859

RESUMO

BACKGROUND: Coxiella burnetii is a Gram-negative gammaproteobacterium and zoonotic agent of Q fever. C. burnetii's genome contains an abundance of pseudogenes and numerous selfish genetic elements. MITEs (miniature inverted-repeat transposable elements) are non-autonomous transposons that occur in all domains of life and are thought to be insertion sequences (ISs) that have lost their transposase function. Like most transposable elements (TEs), MITEs are thought to play an active role in evolution by altering gene function and expression through insertion and deletion activities. However, information regarding bacterial MITEs is limited. RESULTS: We describe two MITE families discovered during research on small non-coding RNAs (sRNAs) of C. burnetii. Two sRNAs, Cbsr3 and Cbsr13, were found to originate from a novel MITE family, termed QMITE1. Another sRNA, CbsR16, was found to originate from a separate and novel MITE family, termed QMITE2. Members of each family occur ~ 50 times within the strains evaluated. QMITE1 is a typical MITE of 300-400 bp with short (2-3 nt) direct repeats (DRs) of variable sequence and is often found overlapping annotated open reading frames (ORFs). Additionally, QMITE1 elements possess sigma-70 promoters and are transcriptionally active at several loci, potentially influencing expression of nearby genes. QMITE2 is smaller (150-190 bps), but has longer (7-11 nt) DRs of variable sequences and is mainly found in the 3' untranslated region of annotated ORFs and intergenic regions. QMITE2 contains a GTAG repetitive extragenic palindrome (REP) that serves as a target for IS1111 TE insertion. Both QMITE1 and QMITE2 display inter-strain linkage and sequence conservation, suggesting that they are adaptive and existed before divergence of C. burnetii strains. CONCLUSIONS: We have discovered two novel MITE families of C. burnetii. Our finding that MITEs serve as a source for sRNAs is novel. QMITE2 has a unique structure and occurs in large or small versions with unique DRs that display linkage and sequence conservation between strains, allowing for tracking of genomic rearrangements. QMITE1 and QMITE2 copies are hypothesized to influence expression of neighboring genes involved in DNA repair and virulence through transcriptional interference and ribonuclease processing.


Assuntos
Coxiella burnetii/genética , Elementos de DNA Transponíveis , Sequências Repetidas Invertidas , Pequeno RNA não Traduzido/genética , Proteínas de Bactérias/genética , Sequência de Bases , Sequência Conservada , Evolução Molecular , Ligação Genética , Loci Gênicos , Genoma Bacteriano , Conformação de Ácido Nucleico , Peptídeos/genética , Pequeno RNA não Traduzido/química , Alinhamento de Sequência
20.
Methods Mol Biol ; 1737: 231-248, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29484597

RESUMO

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. The information described in this chapter provides a guideline to help develop a protocol in order to assess these important parameters and to identify RNA-processing enzymes involved in sRNA degradation processes.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Exorribonucleases/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética
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