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1.
Biochemistry (Mosc) ; 84(11): 1221-1232, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760913

RESUMO

The review covers three independent blocks of research. The first one is discovery, isolation, and investigation of snake venom RNases and their use in studying RNA macrostructure. It has been established that snake venom RNases are not specific to the primary RNA structure but rather to the RNA helical conformation (double, single, or hybrid helix). Snake venom RNases hydrolyze RNA to short oligomers with the 5'-terminal phosphate. Analysis of the kinetics and products of tRNA hydrolysis exemplifies the use of snake venom RNases for deciphering RNA macrostructure. The second block is devoted to the principle formulated by the author for analyzing the primary structure of nucleic acids and describes the method of direct RNA sequencing that has been developed with author's participation. The third block describes the results of genotyping and etiologic control of epidemic influenza A viruses circulating in the Soviet Union in 1968 to 1992. The method for comparative analysis of genome sequences of viral isolates has made it possible to detect and characterize epidemic influenza virus strains that had emerged in the circulation as a result of reactivation of inactivated vaccines.


Assuntos
RNA/química , Humanos , Hidrólise , Vírus da Influenza A/genética , Cinética , Conformação de Ácido Nucleico , RNA/metabolismo , RNA/ultraestrutura , RNA Viral/química , RNA Viral/metabolismo , Ribonucleases/metabolismo , Venenos de Serpentes/metabolismo
2.
J Chem Theory Comput ; 15(11): 5829-5844, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31593627

RESUMO

A powerful computational strategy to determine the equilibrium association constant of two macromolecules with explicit-solvent molecular dynamics (MD) simulations is the "geometric route", which considers the reversible physical separation of the bound complex in solution. Nonetheless, multiple challenges remain to render this type of methodology reliable and computationally efficient in practice. In particular, in one, formulation of the geometric route relies on the potential of mean force (PMF) for physically separating the two binding partners restrained along a straight axis, which must be selected prior to the calculation. However, practical applications indicate that the calculation of the separation PMF along the predefined rectilinear pathway may be suboptimal and slowly convergent. Recognizing that a rectilinear straight separation pathway is generally not representative of how the protein complex physically separates in solution, we put forth a novel theoretical framework for binding free-energy calculations, leaning on the optimal curvilinear minimum free-energy path (MFEP) determined from the string method. The proposed formalism is validated by comparing the results obtained using both rectilinear and curvilinear pathways for a prototypical host-guest complex formed by cucurbit[7]uril (CB[7]) binding benzene, and for the barnase-barstar protein complex. On the basis of multi-microsecond MD calculations, we find that the calculations following the traditional rectilinear pathway and the string-based curvilinear pathway agree quantitatively, but convergence is faster with the latter.


Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Benzeno/química , Benzeno/metabolismo , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Ligação Proteica , Proteínas/metabolismo , Ribonucleases/química , Ribonucleases/metabolismo , Termodinâmica
3.
Nat Commun ; 10(1): 4302, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541109

RESUMO

Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA4 binding site and a ssRNA catalytic pocket. cOA4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5'-C-C-3'. The degradation of cOA4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.


Assuntos
Nucleotídeos de Adenina/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Endorribonucleases/química , Oligorribonucleotídeos/química , Estabilidade de RNA , Proteínas de Ligação a RNA/química , Sulfolobus/química , Sítio Alostérico , Proteínas de Bactérias/química , Sítios de Ligação , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cristalografia por Raios X , Proteínas de Membrana/química , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA/genética , Ribonucleases/metabolismo , Sistemas do Segundo Mensageiro , Sulfolobus/genética
4.
Nat Protoc ; 14(10): 2986-3012, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548639

RESUMO

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/genética , Ácidos Nucleicos/análise , Primers do DNA , Endonucleases/isolamento & purificação , Endonucleases/metabolismo , Humanos , Leptotrichia/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Engenharia de Proteínas/métodos , RNA Guia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Ribonucleases/isolamento & purificação , Ribonucleases/metabolismo , Fluxo de Trabalho , Zika virus/genética , Infecção por Zika virus/sangue , Infecção por Zika virus/urina
5.
Mol Cell ; 75(5): 933-943.e6, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31326272

RESUMO

Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cAn) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cAn second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppAn), and products (cAn), to decipher mechanistic aspects of cAn formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppAn intermediates and subsequent cAn formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cAn signaling pathway.


Assuntos
Nucleotídeos de Adenina/química , Proteínas Arqueais/química , Sistemas CRISPR-Cas , Oligorribonucleotídeos/química , Ribonucleases/química , Sistemas do Segundo Mensageiro , Thermococcus/química , Nucleotídeos de Adenina/metabolismo , Proteínas Arqueais/metabolismo , Microscopia Crioeletrônica , Oligorribonucleotídeos/metabolismo , Ribonucleases/metabolismo , Thermococcus/metabolismo , Thermococcus/ultraestrutura
6.
Chem Commun (Camb) ; 55(62): 9112-9115, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31298670

RESUMO

We designed novel 4'-C-guanidinocarbohydrazidomethyl-5-methyl uridine (GMU) modified small interfering RNA (siRNA) and evaluated its biophysical and biochemical properties. Incorporation of GMU units significantly increased the thermodynamic stability as well as the enzymatic stability against nucleases in human serum. A gene silencing experiment indicated that GMU modfied siRNA (siRNA6) resulted in ≈4.9-fold more efficient knockdown than unmodified siRNA.


Assuntos
Guanidina/química , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Guanidina/análogos & derivados , Modelos Moleculares , RNA Interferente Pequeno/genética , Ribonucleases/sangue , Ribonucleases/metabolismo , Termodinâmica
7.
Adv Exp Med Biol ; 1157: 85-98, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31342438

RESUMO

RNA degradation is considered a critical posttranscriptional regulatory checkpoint, maintaining the correct functioning of organisms. When a specific RNA transcript is no longer required in the cell, it is signaled for degradation through a number of highly regulated steps. Ribonucleases (or simply RNases) are key enzymes involved in the control of RNA stability. These enzymes can perform the RNA degradation alone or cooperate with other proteins in RNA degradation complexes. Important findings over the last years have shed light into eukaryotic RNA degradation by members of the RNase II/RNB family of enzymes. DIS3 enzyme belongs to this family and represents one of the catalytic subunits of the multiprotein complex exosome. This RNase has a diverse range of functions, mainly within nuclear RNA metabolism. Humans encode two other DIS3-like enzymes: DIS3L (DIS3L1) and DIS3L2. DIS3L1 also acts in association with the exosome but is strictly cytoplasmic. In contrast, DIS3L2 acts independently of the exosome and shows a distinctive preference for uridylated RNAs. These enzymes have been shown to be involved in important cellular processes, such as mitotic control, and associated with human disorders like cancer. This review shows how the impairment of function of each of these enzymes is implicated in human disease.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo , Neoplasias , RNA , Ribonucleases , Endorribonucleases , Exorribonucleases , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos , Humanos , Neoplasias/fisiopatologia , RNA/metabolismo , Estabilidade de RNA , Ribonucleases/metabolismo
8.
Nat Commun ; 10(1): 3173, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320642

RESUMO

CCR4-NOT is a conserved multiprotein complex which regulates eukaryotic gene expression principally via shortening of poly(A) tails of messenger RNA or deadenylation. Here, we reconstitute a complete, recombinant human CCR4-NOT complex. Our reconstitution strategy permits strict compositional control to test mechanistic hypotheses with purified component variants. CCR4-NOT is more active and selective for poly(A) than the isolated exonucleases, CCR4a and CAF1, which have distinct deadenylation profiles in vitro. The exonucleases require at least two out of three conserved non-enzymatic modules (CAF40, NOT10:NOT11 or NOT) for full activity in CCR4-NOT. CAF40 and the NOT10:NOT11 module both bind RNA directly and stimulate deadenylation in a partially redundant manner. Linear motifs from different RNA-binding factors that recruit CCR4-NOT to specific mRNAs via protein-protein interactions with CAF40 can inhibit bulk deadenylation. We reveal an additional layer of regulatory complexity to the human deadenylation machinery, which may prime it either for general or target-specific degradation.


Assuntos
Exorribonucleases/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Receptores CCR4/genética , Humanos , Complexos Multiproteicos/síntese química , Complexos Multiproteicos/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Poli A/metabolismo , RNA Mensageiro/genética , Receptores CCR4/metabolismo , Proteínas Recombinantes/genética , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo
9.
Int J Mol Sci ; 20(13)2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31261992

RESUMO

Focal cerebral ischemia can cause blood-brain barrier (BBB) breakdown, which is implicated in neuroinflammation and progression of brain damage. Monocyte chemotactic protein 1-induced protein 1 (MCPIP1) is a newly identified zinc-finger protein that negatively regulates inflammatory signaling pathways. We aimed to evaluate the impact of genetic MCPIP1 deletion on BBB breakdown and expression of BBB-related matrix metalloproteinases (MMPs) and tight junction proteins after cerebral ischemia/reperfusion (I/R) using MCPIP1-deficient (MCPIP1-/-) mice. Transient middle cerebral artery occlusion was induced in the MCPIP1-/- mice and their wild-type littermates for 2 h followed by reperfusion for 24 h. The degree of BBB breakdown was evaluated by injection of fluorescein isothiocyanate (FITC)-dextran. Quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry were performed to compare the expression of MMPs and claudin-5 and zonula occludens-1 (ZO-1). MCPIP1 deficiency in mice resulted in enhanced leakage of FITC-dextran, increased expression of MMP-9/3, and reduced expression of claudin-5 and ZO-1 in the brain compared to that seen in their wild-type littermates subjected to cerebral I/R. These results demonstrate that absence of MCPIP1 exacerbates cerebral I/R-induced BBB disruption by enhancing the expression of MMP-9/3 and the degradation of claudin-5 and ZO-1, providing novel insights into the mechanisms underlying BBB breakdown after cerebral ischemia/reperfusion.


Assuntos
Barreira Hematoencefálica/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Ribonucleases/metabolismo , Animais , Permeabilidade Capilar , Claudina-5/genética , Claudina-5/metabolismo , Infarto da Artéria Cerebral Média/genética , Masculino , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ribonucleases/genética , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
10.
Prep Biochem Biotechnol ; 49(9): 916-926, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31322478

RESUMO

A Gram-positive, rod-shaped, endospore-forming, and RNA-degrading bacterium RB-5 was isolated from a soil sample. Based on 16-rDNA gene sequence, the bacterium RB-5 was identified as Bacillus safensis (Accession number KX443714.1). The bacterium appeared to be related to Bacillus safensis KL-052, an other-member of genus Bacillus. One-factor-at-a-time (OFAT) and Response Surface Methodology (RSM) statistical approaches were used to optimize the fermentation broth to obtain an improved extracellular RNase production from B. safensis RB-5. These approaches improved RNase activity of B. safensis KL-052 from 4.26 to 7.85 U/mL. The OFAT approach was used to study the effects of supplementation of carbon, nitrogen and physical conditions, which included temperature, pH and agitation rate on extracellular RNase production by B. safensis KL-052. Five variables screened by Central Composite Design (CCD) were employed to evaluate their interactive effects on RNase production by the organism. CCD selected 25 factorial values obtained by the statistical approach were peptone 1.13% (w/v), sodium nitrate 1.13% (w/v), MgSO4 0.06% (w/v), pH 8.5, and temperature 35 °C for RNase production by B. safensis. The highest predicted value of RNase was 7.05 U/ml while actual obtained value was 7.85 U/ml that was ∼84% and 1.84-fold higher than OFAT approach.


Assuntos
Bacillus/enzimologia , Ribonucleases/metabolismo , Bacillus/genética , Bacillus/isolamento & purificação , Bacillus/metabolismo , Carbono/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Nitrogênio/metabolismo , Peptonas/metabolismo , Filogenia , Ribonucleases/genética , Microbiologia do Solo , Temperatura Ambiente
11.
Immunohorizons ; 3(5): 172-185, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31356171

RESUMO

Cytokine IL-17A (IL-17) acts on various cell types, including epidermal keratinocytes, and induces antimicrobial peptide and chemokine production to elicit antibacterial and antifungal defense responses. Excess IL-17 leads to inflammatory skin diseases such as psoriasis. The IκB family protein IκB-ζ mediates IL-17-induced responses. However, the mechanism controlling IκB-ζ expression in IL-17-stimulated cells remains elusive. In this study, we showed that JAK kinase TYK2 positively regulates IL-17-induced IκB-ζ expression. TYK2-deficient mice showed reduced inflammation and concomitant reduction of IκB-ζ mRNA compared with wild-type mice in imiquimod-induced skin inflammation. The analysis of the IκB-ζ promoter activity using human cell lines (HaCaT and HeLa) revealed that catalytic activity of TYK2 and its substrate transcription factor STAT3, but not IL-17, is required for IκB-ζ promoter activity. In contrast, IL-17-induced signaling, which did not activate STAT3, posttranscriptionally stabilized IκB-ζ mRNA via its 3'-untranslated region. IL-17 signaling protein ACT1 was required to counteract constitutive IκB-ζ mRNA degradation by RNase Regnase-1. These results suggested that transcriptional activation by TYK2-STAT3 pathway and mRNA stabilization by IL-17-mediated signals act separately from each other but complementarily to achieve IκB-ζ induction. Therefore, JAK/TYK2 inhibition might be of significance in regulation of IL-17-induced inflammatory reactions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Interleucina-17/metabolismo , Estabilidade de RNA , Fator de Transcrição STAT3/metabolismo , TYK2 Quinase/metabolismo , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Células HeLa , Humanos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Psoríase/induzido quimicamente , Psoríase/metabolismo , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , TYK2 Quinase/genética , Fatores de Transcrição/metabolismo
12.
Microb Pathog ; 135: 103648, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31356928

RESUMO

Norovirus is a highly infectious human pathogen that causes acute foodborne diseases worldwide. As global diet patterns have begun to incorporate a higher consumption of fresh agricultural products, the internalization of norovirus into plants has emerged as a potential threat to human health. Here, we demonstrated that murine norovirus (MNV1) was internalized into Arabidopsis in multiple phases, and this internalization was correlated with Arabidopsis innate immunity responses. Under hydroponic conditions, continuous treatment of MNV1 retarded root growth and facilitated flower development of Arabidopsis without causing necrotic lesions. Examination of viral titers and RNA levels revealed that MNV1 was internalized into Arabidopsis in at least three different phases. In response to MNV1 treatment, the Arabidopsis defensive marker PR1 (a salicylic acid signaling marker) was transiently up-regulated at the early stage. PDF1.2, a jasmonic acid signaling marker, exhibited a gradual induction over time. Noticeably, Arabidopsis RNS1 (T2 ribonuclease) was rapidly induced by MNV1 and exhibited anti-correlation with the internalization of MNV1. Exposure to recombinant Arabidopsis RNS1 protein reduced the viral titers and degraded MNV1 RNA in vitro. In conclusion, the internalization of MNV1 into Arabidopsis was fluctuated by mutual interactions that were potentially regulated by Arabidopsis immune systems containing RNS1.


Assuntos
Arabidopsis/imunologia , Arabidopsis/virologia , Norovirus/fisiologia , Plântula/imunologia , Plântula/virologia , Internalização do Vírus , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclopentanos , Defensinas/metabolismo , Doenças Transmitidas por Alimentos/virologia , Imunidade Inata , Camundongos , Oxilipinas , Desenvolvimento Vegetal , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/virologia , Proteínas Recombinantes , Ribonucleases/genética , Ribonucleases/metabolismo , Plântula/genética , Plântula/metabolismo , Regulação para Cima , Carga Viral
13.
Cell Host Microbe ; 25(6): 815-826.e4, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31155345

RESUMO

CRISPR-Cas12a (Cpf1), a type V CRISPR-associated nuclease, provides bacterial immunity against bacteriophages and plasmids but also serves as a tool for genome editing. Foreign nucleic acids are integrated into the CRISPR locus, prompting transcription of CRISPR RNAs (crRNAs) that guide Cas12a cleavage of foreign complementary DNA. However, mobile genetic elements counteract Cas12a with inhibitors, notably type V-A anti-CRISPRs (AcrVAs). We present cryoelectron microscopy structures of Cas12a-crRNA bound to AcrVA1 and AcrVA4 at 3.5 and 3.3 Å resolutions, respectively. AcrVA1 is sandwiched between the recognition (REC) and nuclease (NUC) lobes of Cas12a and inserts into the binding pocket for the protospacer-adjacent motif (PAM), a short DNA sequence guiding Cas12a targeting. AcrVA1 cleaves crRNA in a Cas12a-dependent manner, inactivating Cas12a-crRNA complexes. The AcrVA4 dimer is anchored around the crRNA pseudoknot of Cas12a-crRNA, preventing required conformational changes for crRNA-DNA heteroduplex formation. These results uncover molecular mechanisms for CRISPR-Cas12a inhibition, providing insights into bacteria-phage dynamics.


Assuntos
Sistemas CRISPR-Cas , Endodesoxirribonucleases/antagonistas & inibidores , Endodesoxirribonucleases/metabolismo , Inibidores Enzimáticos/metabolismo , RNA Guia/metabolismo , Ribonucleases/metabolismo , Microscopia Crioeletrônica , Endodesoxirribonucleases/ultraestrutura , Ligação Proteica , Conformação Proteica , RNA Guia/ultraestrutura , Ribonucleases/ultraestrutura
14.
Nat Commun ; 10(1): 2544, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186424

RESUMO

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3'-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ribonucleases/metabolismo , Ruminococcus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Conformação de Ácido Nucleico , Domínios Proteicos , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Guia/genética , Ribonucleases/química , Ribonucleases/genética , Ruminococcus/enzimologia
15.
Int J Biol Macromol ; 136: 625-631, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31220501

RESUMO

Ribotoxins are fungal proteins that serve as weapons against parasites and insects. They are strongly toxic due to their ability to enter host cells and inactivate ribosomes. Ageritin is the prototype of a new ribotoxin-like protein family present in basidiomycetes. We demonstrate that this enzyme has peculiar binding and enzymatic features. Different from other ribotoxins, its ribonucleolytic activity requires the presence of divalent cations, with a maximum activation in the presence of zinc ions, for which Ageritin exhibits the strongest affinity of binding. We modeled the catalytic metal binding site of Ageritin, made of the putative triad Asp68, Asp70 and His77. This report highlights that Ageritin has the structure and function of an RNase but a Mg2+/Zn2+-dependent mechanism of action, a new finding for ribotoxins. As a zinc-dependent toxin, Ageritin can be classified among the arsenal of zinc-binding proteins involved in fungal virulence.


Assuntos
Agrocybe/enzimologia , Ribonucleases/química , Ribonucleases/metabolismo , Zinco/metabolismo , Domínio Catalítico , Colicinas/metabolismo , Modelos Moleculares , Ligação Proteica
16.
Molecules ; 24(12)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31207974

RESUMO

A novel RNase R, psrnr, was cloned from the Antarctic bacterium Psychrobacter sp. ANT206 and expressed in Escherichia coli (E. coli). A bioinformatics analysis of the psrnr gene revealed that it contained an open reading frame of 2313 bp and encoded a protein (PsRNR) of 770 amino acids. Homology modeling indicated that PsRNR had reduced hydrogen bonds and salt bridges, which might be the main reason for the catalytic efficiency at low temperatures. A site directed mutation exhibited that His 667 in the active site was absolutely crucial for the enzyme catalysis. The recombinant PsRNR (rPsRNR) showed maximum activity at 30 °C and had thermal instability, suggesting that rPsRNR was a cold-adapted enzyme. Interestingly, rPsRNR displayed remarkable salt tolerance, remaining stable at 0.5-3.0 M NaCl. Furthermore, rPsRNR had a higher kcat value, contributing to its efficient catalytic activity at a low temperature. Overall, cold-adapted RNase R in this study was an excellent candidate for antimicrobial treatment.


Assuntos
Adaptação Biológica , Temperatura Baixa , Microbiologia Ambiental , Camada de Gelo/microbiologia , Psychrobacter/fisiologia , Ribonucleases/metabolismo , Tolerância ao Sal , Sequência de Aminoácidos , Regiões Antárticas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ativação Enzimática , Cinética , Modelos Biológicos , Conformação Molecular , Estrutura Molecular , Psychrobacter/isolamento & purificação , Ribonucleases/genética
17.
RNA Biol ; 16(7): 972-987, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31043113

RESUMO

CsrA is a widely conserved, abundant small RNA binding protein that has been found in E. coli and other Gram-negative bacteria where it is involved in the regulation of carbon metabolism, biofilm formation and virulence. CsrA binds to single-stranded GGA motifs around the SD sequence of target mRNAs where it inhibits or activates translation or influences RNA processing. Small RNAs like CsrB or CsrC containing 13-22 GGA motifs can sequester CsrA, thereby abrogating the effect of CsrA on its target mRNAs. In B. subtilis, CsrA has so far only been found to regulate one target, hag mRNA and to be sequestered by a protein (FliW) and not by an sRNA. Here, we employ a combination of in vitro and in vivo methods to investigate the effect of CsrA on the small regulatory RNA SR1 from B. subtilis, its primary target ahrC mRNA and its downstream targets, the rocABC and rocDEF operons. We demonstrate that CsrA can promote the base-pairing interactions between SR1 and ahrC mRNA, a function that has so far only been found for Hfq or ProQ. Abbreviations: aa, amino acid; bp, basepair; nt, nucleotide; PAA, polyacrylamide; SD, Shine Dalgarno.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , RNA Bacteriano/metabolismo , Arginina/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Pareamento de Bases/genética , Sequência de Bases , Carbono/farmacologia , Regulação Bacteriana da Expressão Gênica , Mutação/genética , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Ligação Proteica , Biossíntese de Proteínas , Estabilidade de RNA/genética , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Transcrição Genética
18.
Nucleic Acids Res ; 47(11): 5892-5905, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30957850

RESUMO

The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets.


Assuntos
Mycobacterium tuberculosis/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA/análise , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complexos Multienzimáticos , Mycobacterium smegmatis/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/genética , Proteoma , Proteômica , RNA/química , RNA Helicases/metabolismo , Estabilidade de RNA , RNA Bacteriano/metabolismo , Ribonuclease III/metabolismo , Ribonucleases/metabolismo , Tiouridina/química , Transcriptoma
19.
Plant Mol Biol ; 100(4-5): 367-378, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30937702

RESUMO

KEY MESSAGE: S-RNase was demonstrated to be predominantly recognized by an S locus F-box-like protein and an S haplotype-specific F-box-like protein in compatible pollen tubes of sweet cherry. Self-incompatibility (SI) is a reproductive barrier that rejects self-pollen and inhibits self-fertilization to promote outcrossing. In Solanaceae and Rosaceae, S-RNase-based gametophytic SI (GSI) comprises S-RNase and F-box protein(s) as the pistil and pollen S determinants, respectively. Compatible pollen tubes are assumed to detoxify the internalized cytotoxic S-RNases to maintain growth. S-RNase detoxification is conducted by the Skp1-cullin1-F-box protein complex (SCF) formed by pollen S determinants, S locus F-box proteins (SLFs), in Solanaceae. In Prunus, the general inhibitor (GI), but not pollen S determinant S haplotype-specific F-box protein (SFB), is hypothesized to detoxify S-RNases. Recently, SLF-like proteins 1-3 (SLFL1-3) were suggested as GI candidates, although it is still possible that other proteins function predominantly in GI. To identify the other GI candidates, we isolated four other pollen-expressed SLFL and SFB-like (SFBL) proteins PavSLFL6, PavSLFL7A, PavSFBL1, and PavSFBL2 in sweet cherry. Binding assays with four PavS-RNases indicated that PavSFBL2 bound to PavS1, 6-RNase while the others bound to nothing. PavSFBL2 was confirmed to form an SCF complex in vitro. A co-immunoprecipitation assay using the recombinant PavS6-RNase as bait against pollen extracts and a mass spectrometry analysis identified the SCF complex components of PavSLFLs and PavSFBL2, M-locus-encoded glutathione S-transferase (MGST), DnaJ-like protein, and other minor proteins. These results suggest that SLFLs and SFBLs could act as predominant GIs in Prunus-specific S-RNase-based GSI.


Assuntos
Proteínas F-Box/fisiologia , Proteínas de Plantas/fisiologia , Prunus/metabolismo , Autoincompatibilidade em Angiospermas , Clonagem Molecular , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Espectrometria de Massas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubo Polínico/metabolismo , Tubo Polínico/fisiologia , Prunus/enzimologia , Reprodução , Ribonucleases/química , Ribonucleases/metabolismo
20.
RNA ; 25(7): 840-856, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30988100

RESUMO

ZC3H12B is the most enigmatic member of the ZC3H12 protein family. The founding member of this family, Regnase-1/MCPIP1/ZC3H12A, is a well-known modulator of inflammation and is involved in the degradation of inflammatory mRNAs. In this study, for the first time, we characterized the properties of the ZC3H12B protein. We show that the biological role of ZC3H12B depends on an intact NYN/PIN RNase domain. Using RNA immunoprecipitation, experiments utilizing actinomycin D and ELISA, we show that ZC3H12B binds interleukin-6 (IL-6) mRNA in vivo, regulates its turnover, and results in reduced production of IL-6 protein upon stimulation with IL-1ß. We verified that regulation of IL-6 mRNA stability occurs via interaction of ZC3H12B with the stem-loop structure present in the IL-6 3'UTR. The IL-6 transcript is not the only target of ZC3H12B. ZC3H12B also interacts with other known substrates of Regnase-1 and ZC3H12D, such as the 3'UTRs of IER3 and Regnase-1, and binds IER3 mRNA in vivo. Using immunofluorescence, we examined the localization of ZC3H12B within the cell. ZC3H12B forms small, granule-like structures in the cytoplasm that are characteristic of proteins involved in mRNA turnover. The overexpression of ZC3H12B inhibits proliferation by stalling the cell cycle in the G2 phase. This effect of ZC3H12B is also NYN/PIN dependent. The analysis of the ZC3H12B mRNA level reveals its highest expression in the human brain and the neuroblastoma cell line SH-SY5Y, although the factors regulating its expression remain elusive. Down-regulation of ZC3H12B in SH-SY5Y cells by specific shRNAs results in up-regulation of ZC3H12B-target mRNAs.


Assuntos
Regiões 3' não Traduzidas/genética , Regulação da Expressão Gênica , Interleucina-6/genética , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Encéfalo/metabolismo , Células HeLa , Humanos , Interleucina-1beta/farmacologia , Interleucina-6/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Domínios Proteicos , Estabilidade de RNA , RNA Mensageiro/genética , Ribonucleases/genética , Homologia de Sequência , Fatores de Transcrição/genética
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