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1.
Nat Commun ; 11(1): 1113, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111843

RESUMO

Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.


Assuntos
Sistemas CRISPR-Cas , Vesículas Extracelulares/metabolismo , Pequeno RNA não Traduzido/metabolismo , Transporte Biológico , Comunicação Celular , Linhagem Celular , Endocitose/genética , Vesículas Extracelulares/genética , Fluorescência , Genes Reporter/genética , Células HEK293 , Humanos , RNA Guia/genética , RNA Guia/metabolismo , Pequeno RNA não Traduzido/genética
2.
Mol Genet Genomics ; 295(3): 775-785, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32170429

RESUMO

The regulatory networks involved in the uptake and metabolism of different nitrogen sources in response to their availability are crucial in all organisms. Nitrogen metabolism pathways have been studied in detail in archaea such as the extreme halophilic archaeon Haloferax mediterranei. However, knowledge about nitrogen metabolism regulation in haloarchaea is very scarce, and no transcriptional regulators involved in nitrogen metabolism have been identified to date. Advances in the molecular biology field have revealed that many small RNAs (sRNAs) are involved in the regulation of a diverse metabolic pathways. Surprisingly, no studies on regulation mediated by sRNAs have focused on the response to environmental fluctuations in nitrogen in haloarchaea. To identify sRNAs involved in the transcriptional regulation of nitrogen assimilation genes in Haloferax mediterranei and, thus, propose a novel regulatory mechanism, RNA-Seq was performed using cells grown in the presence of two different nitrogen sources. The differential transcriptional expression analysis of the RNA-Seq data revealed differences in the transcription patterns of 102 sRNAs according to the nitrogen source, and the molecular functions, cellular locations and biological processes with which the target genes were associated were predicted. These results enabled the identification of four sRNAs that could be directly related to the regulation of genes involved in nitrogen metabolism. This work provides the first proposed regulatory mechanism of nitrogen assimilation-related gene expression by sRNAs in haloarchaea as an alternative to transcriptional regulation mediated by proteins.


Assuntos
Proteínas Arqueais/genética , Regulação da Expressão Gênica em Archaea , Haloferax mediterranei/genética , Haloferax mediterranei/metabolismo , Nitrogênio/metabolismo , RNA Arqueal/genética , Pequeno RNA não Traduzido/genética , Perfilação da Expressão Gênica , Haloferax mediterranei/crescimento & desenvolvimento
3.
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
4.
Nat Commun ; 11(1): 168, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924754

RESUMO

Variations in transcription start site (TSS) selection reflect diversity of preinitiation complexes and can impact on post-transcriptional RNA fates. Most metazoan polymerase II-transcribed genes carry canonical initiation with pyrimidine/purine (YR) dinucleotide, while translation machinery-associated genes carry polypyrimidine initiator (5'-TOP or TCT). By addressing the developmental regulation of TSS selection in zebrafish we uncovered a class of dual-initiation promoters in thousands of genes, including snoRNA host genes. 5'-TOP/TCT initiation is intertwined with canonical initiation and used divergently in hundreds of dual-initiation promoters during maternal to zygotic transition. Dual-initiation in snoRNA host genes selectively generates host and snoRNA with often different spatio-temporal expression. Dual-initiation promoters are pervasive in human and fruit fly, reflecting evolutionary conservation. We propose that dual-initiation on shared promoters represents a composite promoter architecture, which can function both coordinately and divergently to diversify RNAs.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Regiões Promotoras Genéticas/genética , Sítio de Iniciação de Transcrição , Transcrição Genética , Animais , Sequência de Bases , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Humanos , RNA/genética , RNA/fisiologia , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/fisiologia , RNA não Traduzido/genética , RNA não Traduzido/fisiologia , Elementos Reguladores de Transcrição , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Zigoto
5.
Nat Cell Biol ; 22(2): 187-199, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932738

RESUMO

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.


Assuntos
Autofagossomos/metabolismo , Autofagia/genética , Vesículas Extracelulares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Autofagossomos/química , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Transporte Biológico , Biotinilação , Vesículas Extracelulares/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteômica/métodos , Células RAW 264.7 , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo
6.
Nucleic Acids Res ; 48(4): 2126-2143, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31863581

RESUMO

Small noncoding RNAs (sRNAs) from mRNA 3' UTRs seem to present a previously unrecognized layer of bacterial post-transcriptional control whereby mRNAs influence each other's expression, independently of transcriptional control. Studies in Escherichia coli and Salmonella enterica showed that such sRNAs are natural products of RNase E-mediated mRNA decay and associate with major RNA-binding proteins (RBPs) such as Hfq and ProQ. If so, there must be additional sRNAs from mRNAs that accumulate only under specific physiological conditions. We test this prediction by characterizing candidate NarS that represents the 3' UTR of nitrate transporter NarK whose gene is silent during standard aerobic growth. We find that NarS acts by Hfq-dependent base pairing to repress the synthesis of the nitrite transporter, NirC, resulting in mRNA cross-regulation of nitrate and nitrite transporter genes. Interestingly, the NarS-mediated repression selectively targets the nirC cistron of the long nirBDC-cysG operon, an observation that we rationalize as a mechanism to protect the bacterial cytoplasm from excessive nitrite toxicity during anaerobic respiration with abundant nitrate. Our successful functional assignment of a 3' UTR sRNA from a non-standard growth condition supports the notion that mRNA crossregulation is more pervasive than currently appreciated.


Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Metiltransferases/genética , Pequeno RNA não Traduzido/genética , Regiões 3' não Traduzidas/genética , Endorribonucleases/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Nitratos/metabolismo , Óperon/genética , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Respiração/genética , Salmonella enterica/genética
7.
Nucleic Acids Res ; 48(4): e21, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31879784

RESUMO

Many organisms exchange small RNAs (sRNAs) during their interactions, that can target or bolster defense strategies in host-pathogen systems. Current sRNA-Seq technology can determine the sRNAs present in any symbiotic system, but there are very few bioinformatic tools available to interpret the results. We show that one of the biggest challenges comes from sequences that map equally well to the genomes of both interacting organisms. This arises due to the small size of the sRNAs compared to large genomes, and because a large portion of sequenced sRNAs come from genomic regions that encode highly conserved miRNAs, rRNAs or tRNAs. Here, we present strategies to disentangle sRNA-Seq data from samples of communicating organisms, developed using diverse plant and animal species that are known to receive or exchange RNA with their symbionts. We show that sequence assembly, both de novo and genome-guided, can be used for these sRNA-Seq data, greatly reducing the ambiguity of mapping reads. Even confidently mapped sequences can be misleading, so we further demonstrate the use of differential expression strategies to determine true parasite-derived sRNAs within host cells. We validate our methods on new experiments designed to probe the nature of the extracellular vesicle sRNAs from the parasitic nematode Heligmosomoides bakeri that get into mouse intestinal epithelial cells.


Assuntos
Interações Hospedeiro-Patógeno/genética , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Simbiose/genética , Animais , Arabidopsis/genética , Arabidopsis/microbiologia , Botrytis/genética , Biologia Computacional , Genoma Bacteriano/genética , Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , MicroRNAs/genética , RNA Ribossômico/genética , RNA de Transferência/genética , Análise de Sequência de RNA
8.
Elife ; 82019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31845648

RESUMO

Trans-species small regulatory RNAs (sRNAs) are delivered to host plants from diverse pathogens and parasites and can target host mRNAs. How trans-species sRNAs can be effective on diverse hosts has been unclear. Multiple species of the parasitic plant Cuscuta produce trans-species sRNAs that collectively target many host mRNAs. Confirmed target sites are nearly always in highly conserved, protein-coding regions of host mRNAs. Cuscuta trans-species sRNAs can be grouped into superfamilies that have variation in a three-nucleotide period. These variants compensate for synonymous-site variation in host mRNAs. By targeting host mRNAs at highly conserved protein-coding sites, and simultaneously expressing multiple variants to cover synonymous-site variation, Cuscuta trans-species sRNAs may be able to successfully target multiple homologous mRNAs from diverse hosts.


Assuntos
Arabidopsis/parasitologia , Cuscuta/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Sequência de Bases , Códon , Biologia Computacional , Sequência Conservada , Cuscuta/crescimento & desenvolvimento , Cuscuta/metabolismo , Variação Genética , Interações Hospedeiro-Parasita , Fases de Leitura Aberta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/classificação , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Pequeno RNA não Traduzido/classificação , Pequeno RNA não Traduzido/metabolismo , Alinhamento de Sequência , Tabaco/genética , Tabaco/crescimento & desenvolvimento , Tabaco/parasitologia
9.
Nat Commun ; 10(1): 5118, 2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31712554

RESUMO

KRAS receives and relays signals at the plasma membrane (PM) where it transmits extracellular growth factor signals to downstream effectors. SNORD50A/B were recently found to bind KRAS and inhibit its tumorigenic action by unknown mechanisms. KRAS proximity protein labeling was therefore undertaken in SNORD50A/B wild-type and knockout cells, revealing that SNORD50A/B RNAs shape the composition of proteins proximal to KRAS, notably by inhibiting KRAS proximity to the SNARE vesicular transport proteins SNAP23, SNAP29, and VAMP3. To remain enriched on the PM, KRAS undergoes cycles of endocytosis, solubilization, and vesicular transport to the PM. Here we report that SNAREs are essential for the final step of this process, with KRAS localization to the PM facilitated by SNAREs but antagonized by SNORD50A/B. Antagonism between SNORD50A/B RNAs and specific SNARE proteins thus controls KRAS localization, signaling, and tumorigenesis, and disrupting SNARE-enabled KRAS function represents a potential therapeutic opportunity in KRAS-driven cancer.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas p21(ras)/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas SNARE/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Humanos , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pequeno RNA não Traduzido/genética , Transdução de Sinais
10.
Int J Mol Sci ; 20(20)2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31623090

RESUMO

The floral development in an important legume crop yellow lupine (Lupinus luteus L., Taper cv.) is often affected by the abscission of flowers leading to significant economic losses. Small non-coding RNAs (sncRNAs), which have a proven effect on almost all developmental processes in other plants, might be of key players in a complex net of molecular interactions regulating flower development and abscission. This study represents the first comprehensive sncRNA identification and analysis of small RNA, transcriptome and degradome sequencing data in lupine flowers to elucidate their role in the regulation of lupine generative development. As shedding in lupine primarily concerns flowers formed at the upper part of the inflorescence, we analyzed samples from extreme parts of raceme separately and conducted an additional analysis of pedicels from abscising and non-abscising flowers where abscission zone forms. A total of 394 known and 28 novel miRNAs and 316 phased siRNAs were identified. In flowers at different stages of development 59 miRNAs displayed differential expression (DE) and 46 DE miRNAs were found while comparing the upper and lower flowers. Identified tasiR-ARFs were DE in developing flowers and were strongly expressed in flower pedicels. The DEmiR-targeted genes were preferentially enriched in the functional categories related to carbohydrate metabolism and plant hormone transduction pathways. This study not only contributes to the current understanding of how lupine flowers develop or undergo abscission but also holds potential for research aimed at crop improvement.


Assuntos
Flores/genética , Regulação da Expressão Gênica de Plantas , Lupinus/genética , Desenvolvimento Vegetal/genética , RNA de Plantas/genética , Pequeno RNA não Traduzido/genética , Transcriptoma , Biologia Computacional/métodos , Evolução Molecular , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Redes e Vias Metabólicas , Família Multigênica , Fenótipo , Interferência de RNA , Estabilidade de RNA , Reprodutibilidade dos Testes
11.
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
12.
Mol Genet Genomics ; 294(5): 1359-1371, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31363904

RESUMO

Previous studies revealed important roles of small RNAs (sRNAs) in regulation of bacterial metabolism, stress responses and virulence. However, only a minor fraction of sRNAs is well characterized with respect to the spectra of their targets, conditional expression profiles and actual mechanisms they use to regulate gene expression to control particular biological pathways. To learn more about the specific contribution of sRNAs to the global regulatory network controlling the Escherichia coli central carbon metabolism (CCM), we employed microarray analysis and compared transcriptome profiles of E. coli cells grown on two alternative minimal media supplemented with either pyruvate or glucose, respectively. Microarray analysis revealed that utilization of these alternative carbon sources led to profound differences in gene expression affecting all major gene clusters associated with CCM as well as expression of several known (CyaR, RyhB, GcvB and RyeA) and putative (C0652) sRNAs. To assess the impact of transcriptional reprogramming of gene expression on E. coli protein abundance, we also employed two-dimensional protein gel electrophoresis. Our experimental data made it possible to determine the major pathways for pyruvate assimilation when it is used as a sole carbon source and reveal the impact of other key processes (i.e., energy production, molecular transport and cell resistance to stress) associated with the CCM in E. coli. Moreover, some of these processes were apparently controlled by GcvB, RyhB and CyaR at the post-transcriptional level, thus indicating the complexity and interconnection of the regulatory networks that control CCM in bacteria.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Glucose/metabolismo , Ácido Pirúvico/metabolismo , Proteínas de Escherichia coli/genética , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Transcrição Genética/genética , Transcriptoma/genética
13.
Nat Commun ; 10(1): 3728, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427601

RESUMO

Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Escherichia coli/genética , Pseudomonas aeruginosa/genética , RNA Bacteriano/biossíntese , Pequeno RNA não Traduzido/genética , Fator Rho/antagonistas & inibidores , Terminação da Transcrição Genética/fisiologia , Bacteriófagos/genética , Sistemas CRISPR-Cas/genética , Ensaios de Triagem em Larga Escala , RNA Bacteriano/genética
14.
Microbiol Res ; 229: 126295, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31450184

RESUMO

Vibrio alginolyticus is an opportunistic pathogen that is a threat to the aquaculture industry. Evidence has revealed critical roles for small RNAs (sRNAs) in bacterial physiology and pathology by modulating gene expression post transcription. However, little information about sRNA-mediated regulation in V. alginolyticus is available. We experimentally verified the existence and characterized the function of sRNA srvg17985 in V. alginolyticus ZJ-T. We identified a 179 nt and growth-phase-dependent transcript with a σ70 promoter and a ρ-independent terminator. The transcript consisted of five stem-loops and was conserved in Vibrio spp. Phenotype microarray assays showed that deletion of srvg17985 led to less use of Gly-Glu as a carbon source but a gain in ability to use l-phenylalanine as a nitrogen source. Srvg17985 regulated the osmotic stress response with stronger tolerance to NaCl but weaker tolerance to urea. In addition, srvg17985 inhibited the deamination of l-serine at pH 9.5 and promoted the hydrolysis of X-beta-d-glucuronide, thus affecting the pH stress response. Bioinformatics by IntaRNA and TargetRNA2 identified 45 common target mRNAs, some of which probably contributed to the observed phenotypes. These results indicated that srvg17985 regulated environmental adaptation. The results provide valuable information for in-depth studies of sRNA-mediated regulation mechanisms of the complex physiological processes of V alginolyticus and provide new targets for antibacterial therapeutics or attenuated vaccines for Vibrio spp.


Assuntos
Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/genética , Vibrio alginolyticus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sequências Repetidas Invertidas , Conformação de Ácido Nucleico , Filogenia , Regiões Promotoras Genéticas , RNA Bacteriano/química , RNA Bacteriano/genética , Pequeno RNA não Traduzido/metabolismo , Estresse Fisiológico , Regiões Terminadoras Genéticas , Vibrio alginolyticus/química , Vibrio alginolyticus/classificação , Vibrio alginolyticus/fisiologia
15.
Microbiology ; 165(10): 1135-1150, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31464662

RESUMO

Small non-coding sRNAs have versatile roles in regulating bacterial metabolism. Four short homologous Burkholderia cenocepacia sRNAs strongly expressed under conditions of growth arrest were recently identified. Here we report the detailed investigation of one of these, NcS27. sRNA NcS27 contains a short putative target recognition sequence, which is conserved throughout the order Burkholderiales. This sequence is the reverse complement of the Shine-Dalgarno sequence of a large number of genes involved in transport and metabolism of amino acids and carbohydrates. Overexpression of NcS27 sRNA had a distinct impact on growth, attenuating growth on a variety of substrates such as phenylalanine, tyrosine, glycerol and galactose, while having no effect on growth on other substrates. Transcriptomics and proteomics of NcS27 overexpression and silencing mutants revealed numerous predicted targets changing expression, notably of genes involved in degradation of aromatic amino acids phenylalanine and tyrosine, and in transport of carbohydrates. The conserved target recognition sequence was essential for growth phenotypes and gene expression changes. Cumulatively, our data point to a role of NcS27 in regulating the shutdown of metabolism upon nutrient deprivation in B. cenocepacia. We propose Burkholderiadouble-hairpin sRNA regulator bdhR1 as designation for ncS27.


Assuntos
Burkholderia cenocepacia/metabolismo , Carbono/metabolismo , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mutação , Proteômica , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética
16.
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
17.
Nucleic Acids Res ; 47(16): 8821-8837, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31329973

RESUMO

In many Gram-negative and some Gram-positive bacteria, small regulatory RNAs (sRNAs) that bind the RNA chaperone Hfq have a pivotal role in modulating virulence, stress responses, metabolism and biofilm formation. These sRNAs recognize transcripts through base-pairing, and sRNA-mRNA annealing consequently alters the translation and/or stability of transcripts leading to changes in gene expression. We have previously found that the highly conserved 3'-to-5' exoribonuclease polynucleotide phosphorylase (PNPase) has an indispensable role in paradoxically stabilizing Hfq-bound sRNAs and promoting their function in gene regulation in Escherichia coli. Here, we report that PNPase contributes to the degradation of specific short mRNA fragments, the majority of which bind Hfq and are derived from targets of sRNAs. Specifically, we found that these mRNA-derived fragments accumulate in the absence of PNPase or its exoribonuclease activity and interact with PNPase. Additionally, we show that mutations in hfq or in the seed pairing region of some sRNAs eliminated the requirement of PNPase for their stability. Altogether, our results are consistent with a model that PNPase degrades mRNA-derived fragments that could otherwise deplete cells of Hfq-binding sRNAs through pairing-mediated decay.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Pareamento de Bases , Sequência de Bases , Domínio Catalítico , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Cinética , Mutação , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Clivagem do RNA , Estabilidade de RNA , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo
18.
Biochim Biophys Acta Gene Regul Mech ; 1862(11-12): 194406, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31323432

RESUMO

Splicing and alternative splicing (AS), which occur in the endogenous spliceosome, play major roles in regulating gene expression, and defects in them are involved in numerous human diseases including cancer. Although the mechanism of the splicing reaction is well understood, the regulation of AS remains to be elucidated. A group of essential regulatory factors in gene expression are small non-coding RNAs (sncRNA): e.g. microRNA, mainly known for their inhibitory role in translation in the cytoplasm; and small nucleolar RNA, known for their role in methylating non-coding RNA in the nucleolus. Here I highlight a new aspect of sncRNAs found within the endogenous spliceosome. Assembled in non-canonical complexes and through different base pairing than their canonical ones, spliceosomal sncRNAs can potentially target different RNAs. Examples of spliceosomal sncRNAs regulating AS, regulating gene expression, and acting in a quality control of AS are reviewed, suggesting novel functions for spliceosomal sncRNAs. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.


Assuntos
Processamento Alternativo , Pequeno RNA não Traduzido/genética , Spliceossomos/genética , Pareamento de Bases , Regulação da Expressão Gênica , Humanos , Pequeno RNA não Traduzido/metabolismo , Spliceossomos/metabolismo
19.
Nucleic Acids Res ; 47(14): e84, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31165880

RESUMO

In small RNA (smRNA) sequencing studies, highly abundant molecules such as adapter dimer products and tissue-specific microRNAs (miRNAs) inhibit accurate quantification of lowly expressed species. We previously developed a method to selectively deplete highly abundant miRNAs. However, this method does not deplete adapter dimer ligation products that, unless removed by gel-separation, comprise most of the library. Here, we have adapted and modified recently described methods for CRISPR/Cas9-based Depletion of Abundant Species by Hybridization ('DASH') to smRNA-seq, which we have termed miRNA and Adapter Dimer-DASH (MAD-DASH). In MAD-DASH, Cas9 is complexed with single guide RNAs (sgRNAs) targeting adapter dimer ligation products, alongside highly expressed tissue-specific smRNAs, for cleavage in vitro. This process dramatically reduces adapter dimer and targeted smRNA sequences, can be multiplexed, shows minimal off-target effects, improves the quantification of lowly expressed miRNAs from human plasma and tissue derived RNA, and obviates the need for gel-separation, greatly increasing sample throughput. Additionally, the method is fully customizable to other smRNA-seq preparation methods. Like depletion of ribosomal RNA for mRNA-seq and mitochondrial DNA for ATAC-seq, our method allows for greater proportional read-depth of non-targeted sequences.


Assuntos
Sistemas CRISPR-Cas , Biblioteca Gênica , Hibridização de Ácido Nucleico/métodos , Pequeno RNA não Traduzido/genética , Sequência de Bases , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , MicroRNAs/genética , Modelos Genéticos , RNA Ribossômico/genética , Análise de Sequência de RNA/métodos
20.
Int J Mol Sci ; 20(12)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31207900

RESUMO

Small noncoding RNAs (sncRNAs) are key regulators of the majority of human reproduction events. Understanding their function in the context of gametogenesis and embryogenesis will allow insight into the possible causes of in vitro fertilization (IVF) implantation failure. The aim of this study was to analyze the sncRNA expression profile of the spent culture media on day 4 after fertilization and to reveal a relationship with the morphofunctional characteristics of gametes and resultant embryos, in particular, with the embryo development and implantation potential. Thereto, cell-free, embryo-specific sncRNAs were identified by next generation sequencing (NGS) and quantified by reverse transcription coupled with polymerase chain reaction (RT-PCR) in real-time. Significant differences in the expression level of let-7b-5p, let-7i-5p, piR020401, piR16735, piR19675, piR20326, and piR17716 were revealed between embryo groups of various morphological gradings. Statistically significant correlations were found between the expression profiles of piR16735 and piR020401 with the oocyte-cumulus complex number, let-7b-5p and piR020401 with metaphase II oocyte and two pronuclei embryo numbers, let-7i-5p and piR20497 with the spermatozoid count per milliliter of ejaculate, piR19675 with the percentage of linearly motile spermatozoids, let-7b-5p with the embryo development grade, and let-7i-5p with embryo implantation. According to partial least squares discriminant analysis (PLS-DA), the expression levels of let-7i-5p (Variable Importance in Projection score (VIP) = 1.6262), piR020401 (VIP = 1.45281), and piR20497 (VIP = 1.42765) have the strongest influences on the implantation outcome.


Assuntos
Blastocisto/metabolismo , Fertilização In Vitro/estatística & dados numéricos , Infertilidade/genética , Pequeno RNA não Traduzido/genética , Adulto , Biomarcadores/análise , Biomarcadores/metabolismo , Meios de Cultura/química , Feminino , Humanos , Infertilidade/metabolismo , Infertilidade/terapia , Masculino , Oócitos/metabolismo , Pequeno RNA não Traduzido/análise , Pequeno RNA não Traduzido/metabolismo
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