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1.
Biol Chem ; 401(10): 1123-1141, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32229649

RESUMO

PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that associate with members of the PIWI clade of the Argonaute superfamily of proteins. piRNAs are predominantly found in animal gonads. There they silence transposable elements (TEs), regulate gene expression and participate in DNA methylation, thus orchestrating proper germline development. Furthermore, PIWI proteins are also indispensable for the maintenance and differentiation capabilities of pluripotent stem cells in free-living invertebrate species with regenerative potential. Thus, PIWI proteins and piRNAs seem to constitute an essential molecular feature of somatic pluripotent stem cells and the germline. In keeping with this hypothesis, both PIWI proteins and piRNAs are enriched in neoblasts, the adult stem cells of planarian flatworms, and their presence is a prerequisite for the proper regeneration and perpetual tissue homeostasis of these animals. The piRNA pathway is required to maintain the unique biology of planarians because, in analogy to the animal germline, planarian piRNAs silence TEs and ensure stable genome inheritance. Moreover, planarian piRNAs also contribute to the degradation of numerous protein-coding transcripts, a function that may be critical for neoblast differentiation. This review gives an overview of the planarian piRNA pathway and of its crucial function in neoblast biology.


Assuntos
Planárias/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , RNA Interferente Pequeno/genética
2.
Bioorg Chem ; 97: 103703, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32143017

RESUMO

Three N-metallocenoylsphingosines with variance in the central metal (Fe, Co, Ru), the charge (neutral or cationic), and the arene ligands (Cp2, Cp*Ph) were synthesized from serine and metallocene carboxylic acids as substrate-analogous inhibitors of human acid ceramidase (AC). Their inhibitory potential was examined using the recombinant full length ASAH1 enzyme, expressed and secreted from High Five insect cells, and the fluorescent substrate Rbm14-12. All complexes inhibited AC, most strongly so ruthenium(II) complex 13a. Some antitumoral effects of the complexes, such as the interference with the microtubular and F-actin cytoskeleton of cancer cells, were correlated to their AC-inhibition, whereas others, e.g. their cytotoxicity and their induction of caspase-3/-7 activity in cancer cells, were not. All complexes accumulated preferentially in the lysosomes of cancer cells like their target AC, arrested the cells in G1 phase of the cell cycle, and displayed cytotoxicity with mostly single-digit micromolar IC50 values while inducing cancer cell apoptosis.


Assuntos
Ceramidase Ácida/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Ceramidase Ácida/metabolismo , Animais , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismo , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Esfingosina/síntese química
3.
Proc Natl Acad Sci U S A ; 117(6): 2894-2905, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31988137

RESUMO

The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an "activation helix" close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.


Assuntos
Quinase 8 Dependente de Ciclina/metabolismo , Complexo Mediador/metabolismo , Domínio Catalítico , Ciclina C/genética , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/química , Quinase 8 Dependente de Ciclina/genética , Ativação Enzimática , Humanos , Complexo Mediador/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos
4.
BMC Genomics ; 20(1): 909, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783730

RESUMO

BACKGROUND: The astounding regenerative abilities of planarian flatworms prompt steadily growing interest in examining their molecular foundation. Planarian regeneration was found to require hundreds of genes and is hence a complex process. Thus, RNA interference followed by transcriptome-wide gene expression analysis by RNA-seq is a popular technique to study the impact of any particular planarian gene on regeneration. Typically, the removal of ribosomal RNA (rRNA) is the first step of all RNA-seq library preparation protocols. To date, rRNA removal in planarians was primarily achieved by the enrichment of polyadenylated (poly(A)) transcripts. However, to better reflect transcriptome dynamics and to cover also non-poly(A) transcripts, a procedure for the targeted removal of rRNA in planarians is needed. RESULTS: In this study, we describe a workflow for the efficient depletion of rRNA in the planarian model species S. mediterranea. Our protocol is based on subtractive hybridization using organism-specific probes. Importantly, the designed probes also deplete rRNA of other freshwater triclad families, a fact that considerably broadens the applicability of our protocol. We tested our approach on total RNA isolated from stem cells (termed neoblasts) of S. mediterranea and compared ribodepleted libraries with publicly available poly(A)-enriched ones. Overall, mRNA levels after ribodepletion were consistent with poly(A) libraries. However, ribodepleted libraries revealed higher transcript levels for transposable elements and histone mRNAs that remained underrepresented in poly(A) libraries. As neoblasts experience high transposon activity this suggests that ribodepleted libraries better reflect the transcriptional dynamics of planarian stem cells. Furthermore, the presented ribodepletion procedure was successfully expanded to the removal of ribosomal RNA from the gram-negative bacterium Salmonella typhimurium. CONCLUSIONS: The ribodepletion protocol presented here ensures the efficient rRNA removal from low input total planarian RNA, which can be further processed for RNA-seq applications. Resulting libraries contain less than 2% rRNA. Moreover, for a cost-effective and efficient removal of rRNA prior to sequencing applications our procedure might be adapted to any prokaryotic or eukaryotic species of choice.


Assuntos
Planárias/genética , RNA Ribossômico , Análise de Sequência de RNA/métodos , Animais , Sondas de DNA , Salmonella typhimurium/genética
5.
Genes Dev ; 33(21-22): 1575-1590, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31537626

RESUMO

PIWI proteins utilize small RNAs called piRNAs to silence transposable elements, thereby protecting germline integrity. In planarian flatworms, PIWI proteins are essential for regeneration, which requires adult stem cells termed neoblasts. Here, we characterize planarian piRNAs and examine the roles of PIWI proteins in neoblast biology. We find that the planarian PIWI proteins SMEDWI-2 and SMEDWI-3 cooperate to degrade active transposons via the ping-pong cycle. Unexpectedly, we discover that SMEDWI-3 plays an additional role in planarian mRNA surveillance. While SMEDWI-3 degrades numerous neoblast mRNAs in a homotypic ping-pong cycle, it is also guided to another subset of neoblast mRNAs by antisense piRNAs and binds these without degrading them. Mechanistically, the distinct activities of SMEDWI-3 are primarily dictated by the degree of complementarity between target mRNAs and antisense piRNAs. Thus, PIWI proteins enable planarians to repurpose piRNAs for potentially critical roles in neoblast mRNA turnover.


Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Helminto/metabolismo , Planárias/citologia , Planárias/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Pareamento de Bases , Elementos de DNA Transponíveis , Imunoprecipitação , Ligação Proteica , Estabilidade de RNA
6.
Bioessays ; 38(5): 465-73, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26990636

RESUMO

tRNAs undergo multiple conformational changes during the translation cycle that are required for tRNA translocation and proper communication between the ribosome and translation factors. Recent structural data on how destabilized tRNAs utilize the CCA-adding enzyme to proofread themselves put a spotlight on tRNA flexibility beyond the translation cycle. In analogy to tRNA surveillance, this review finds that other processes also exploit versatile tRNA folding to achieve, amongst others, specific aminoacylation, translational regulation by riboswitches or a block of bacterial translation. tRNA flexibility is thereby not restricted to the hinges utilized during translation. In contrast, the flexibility of tRNA is distributed all over its L-shape and is actively exploited by the tRNA-interacting partners to discriminate one tRNA from another. Since the majority of tRNA modifications also modulate tRNA flexibility it seems that cells devote enormous resources to tightly sense and regulate tRNA structure. This is likely required for error-free protein synthesis.


Assuntos
Bactérias/genética , Biossíntese de Proteínas , RNA de Transferência/genética , Ribossomos/metabolismo , Aminoacilação , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Cinamatos/farmacologia , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Transporte de RNA , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/efeitos dos fármacos , Riboswitch
7.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 9): 1850-5, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26327374

RESUMO

RNA polymerase I (Pol I) is the central, 14-subunit enzyme that synthesizes the ribosomal RNA (rRNA) precursor in eukaryotic cells. The recent crystal structure of Pol I at 2.8 Šresolution revealed two novel elements: the `expander' in the active-centre cleft and the `connector' that mediates Pol I dimerization [Engel et al. (2013), Nature (London), 502, 650-655]. Here, a Pol I structure in an alternative crystal form that was solved by molecular replacement using the original atomic Pol I structure is reported. The resulting alternative structure lacks the expander but still shows an expanded active-centre cleft. The neighbouring Pol I monomers form a homodimer with a relative orientation distinct from that observed previously, establishing the connector as a hinge between Pol I monomers.


Assuntos
RNA Polimerase I/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Conformação Proteica
8.
Cell ; 160(4): 644-658, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25640237

RESUMO

Transcription in eukaryotes produces a number of long noncoding RNAs (lncRNAs). Two of these, MALAT1 and Menß, generate a tRNA-like small RNA in addition to the mature lncRNA. The stability of these tRNA-like small RNAs and bona fide tRNAs is monitored by the CCA-adding enzyme. Whereas CCA is added to stable tRNAs and tRNA-like transcripts, a second CCA repeat is added to certain unstable transcripts to initiate their degradation. Here, we characterize how these two scenarios are distinguished. Following the first CCA addition cycle, nucleotide binding to the active site triggers a clockwise screw motion, producing torque on the RNA. This ejects stable RNAs, whereas unstable RNAs are refolded while bound to the enzyme and subjected to a second CCA catalytic cycle. Intriguingly, with the CCA-adding enzyme acting as a molecular vise, the RNAs proofread themselves through differential responses to its interrogation between stable and unstable substrates.


Assuntos
Archaeoglobus fulgidus/enzimologia , Mitocôndrias/enzimologia , RNA Nucleotidiltransferases/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Archaeoglobus fulgidus/metabolismo , Sequência de Bases , Domínio Catalítico , Humanos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/genética , Estabilidade de RNA , Pequeno RNA não Traduzido/metabolismo
9.
Trends Biochem Sci ; 38(5): 263-71, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23541793

RESUMO

Despite the fact that different classes of small RNAs are generated by largely different biogenesis pathways, all mature small RNAs associate with an Argonaute family member to form the RNA-induced silencing complex (RISC). Gene silencing by RISC could not be studied in molecular detail because structural information on eukaryotic Argonautes was lacking. Recently, however, the structure of human Argonaute-2 (hAgo2), a model for RISC function, was determined in complexes with heterogeneous guide RNA and in complexes with a specific miRNA. We review the exciting advances that these two structures, together with the structure of a budding yeast Argonaute, brought to the field of eukaryotic RNA interference (RNAi), and how they will enable a more detailed mechanistic understanding of eukaryotic RISC.


Assuntos
Proteínas Argonauta/metabolismo , Animais , Proteínas Argonauta/química , Proteínas Argonauta/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Moleculares , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
10.
Genes Dev ; 26(21): 2392-407, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23073843

RESUMO

The MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is misregulated in many human cancers and produces an abundant long nuclear-retained noncoding RNA. Despite being transcribed by RNA polymerase II, the 3' end of MALAT1 is produced not by canonical cleavage/polyadenylation but instead by recognition and cleavage of a tRNA-like structure by RNase P. Mature MALAT1 thus lacks a poly(A) tail yet is expressed at a level higher than many protein-coding genes in vivo. Here we show that the 3' ends of MALAT1 and the MEN ß long noncoding RNAs are protected from 3'-5' exonucleases by highly conserved triple helical structures. Surprisingly, when these structures are placed downstream from an ORF, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail. The triple helix therefore also functions as a translational enhancer, and mutations in this region separate this translation activity from simple effects on RNA stability or transport. We further found that a transcript ending in a triple helix is efficiently repressed by microRNAs in vivo, arguing against a major role for the poly(A) tail in microRNA-mediated silencing. These results provide new insights into how transcripts that lack poly(A) tails are stabilized and regulated and suggest that RNA triple-helical structures likely have key regulatory functions in vivo.


Assuntos
RNA Longo não Codificante/genética , RNA Mensageiro/genética , Motivos de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Regulação da Expressão Gênica , Células HeLa , Humanos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Desnaturação Proteica , Estrutura Secundária de Proteína , Processamento de Terminações 3' de RNA/genética , Estabilidade de RNA , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , Alinhamento de Sequência
11.
Cell ; 150(1): 100-10, 2012 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-22682761

RESUMO

Argonaute proteins lie at the heart of the RNA-induced silencing complex (RISC), wherein they use small RNA guides to recognize targets. Initial insight into the architecture of Argonautes came from studies of prokaryotic proteins, revealing a crescent-shaped base made up of the amino-terminal, PAZ, middle, and PIWI domains. The recently reported crystal structure of human Argonaute-2 (hAgo2), the "slicer" in RNA interference, in complex with a mixed population of RNAs derived from insect cells provides insight into the architecture of a eukaryotic Argonaute protein with defined biochemical and biological functions. Here, we report the structure of human Ago2 bound to a physiologically relevant microRNA, microRNA-20a, at 2.2 Å resolution. The miRNA is anchored at both ends by the Mid and PAZ domains and makes several kinks and turns along the binding groove. Interestingly, miRNA binding confers remarkable stability on hAgo2, locking this otherwise flexible enzyme into a stable conformation.


Assuntos
Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , MicroRNAs/química , MicroRNAs/metabolismo , Proteínas Argonauta/isolamento & purificação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-18453714

RESUMO

The removal of flexible protein regions is generally used to promote crystallization, but advanced strategies to quickly remove multiple flexible regions from proteins or protein complexes are lacking. Here, it is shown how a protein heterodimer with multiple flexibilities, the RNA polymerase I subcomplex A14/A43, could be crystallized with the use of an iterative procedure of predicting flexible regions, experimentally testing and improving these predictions and combining deletions of flexible regions in a stepwise manner. This strategy should enable the crystallization of other proteins and subcomplexes with multiple flexibilities, as required for hybrid structure solution of large macromolecular assemblies.


Assuntos
Cristalização/métodos , Engenharia de Proteínas , RNA Polimerase I/química , Sequência de Aminoácidos , Clonagem Molecular , Biologia Computacional , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Polimerase I/metabolismo , RNA Polimerase I/fisiologia , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
13.
Cell ; 131(7): 1260-72, 2007 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-18160037

RESUMO

Synthesis of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. Here we report the 12 A cryo-electron microscopic structure for the complete 14-subunit yeast Pol I, a homology model for the core enzyme, and the crystal structure of the subcomplex A14/43. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3'-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2 and, apparently, enables ribosomal RNA proofreading and 3'-end trimming.


Assuntos
DNA Polimerase I/química , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Transcrição Genética , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Modelos Moleculares , Mutação , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Regiões Promotoras Genéticas , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Subunidades Proteicas , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo
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