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1.
Life Sci ; 242: 117207, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31863777

RESUMO

Abdominal Aortic Aneurysm (AAA) is a severe cardiovascular disease, with high mortality rate after acute rupture of blood vessels. However, the underlying pathogenesis of different morbidity between men and women remains unclear. In the present study, we first selected four datasets including 68 AAA and 32 control samples from published data on GEO database, and analyzed them by data mining. The integrative analysis found a total of 368 differentially expressed genes in E2-related AAA. Next, regulatory mechanism networks among these target genes were predicted, and four genes were identified as key nodes in the network, which play a major role in the immune system. We focused on the role of monocytes/macrophages in the development of cardiovascular diseases to further explore the role of estrogen in the polarization of monocytes/macrophage, the mRNA level of the four genes was validated by RT-PCR in RAW264.7 cells treated with ß-estradiol (E2), diarylpropionitrile (DPN), 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), fulvestrant or vehicle. The results showed that the mRNA level and protein level of TROVE2 was significantly increased in estrogen or estrogen receptor agonist-treated groups. Moreover, estrogen affected the transformation of macrophages to M2 phenotype by detecting M1- and M2-related indicator genes at the mRNA level. Flow cytometry demonstrated that the TROVE2 deficiency led to a notable decrease in the level of M2 phenotype marker protein CD206. In conclusion, our results suggest that E2 can promote the expression of TROVE2, which is closely related to the M2-phenotype transformation of macrophages.


Assuntos
Aneurisma da Aorta Abdominal/fisiopatologia , Autoantígenos/fisiologia , Estradiol/metabolismo , Ativação de Macrófagos , RNA Citoplasmático Pequeno/fisiologia , Ribonucleoproteínas/fisiologia , Animais , Aneurisma da Aorta Abdominal/metabolismo , Autoantígenos/metabolismo , Western Blotting , Estradiol/fisiologia , Citometria de Fluxo , Humanos , Camundongos , Células RAW 264.7 , RNA Citoplasmático Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Ribonucleoproteínas/metabolismo , Transcriptoma
2.
Adv Exp Med Biol ; 1203: 83-112, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31811631

RESUMO

Serine- and arginine-rich proteins (SR proteins) are a family of multitasking RNA-binding proteins (RBPs) that are key determinants of messenger ribonucleoprotein (mRNP) formation, identity and fate. Apart from their essential functions in pre-mRNA splicing, SR proteins display additional pre- and post-splicing activities and connect nuclear and cytoplasmic gene expression machineries. Through changes in their post-translational modifications (PTMs) and their subcellular localization, they provide functional specificity and adjustability to mRNPs. Transcriptome-wide UV crosslinking and immunoprecipitation (CLIP-Seq) studies revealed that individual SR proteins are present in distinct mRNPs and act in specific pairs to regulate different gene expression programmes. Adopting an mRNP-centric viewpoint, we discuss the roles of SR proteins in the assembly, maturation, quality control and turnover of mRNPs and describe the mechanisms by which they integrate external signals, coordinate their multiple tasks and couple subsequent mRNA processing steps.


Assuntos
Processamento de RNA , Proteínas de Ligação a RNA , Ribonucleoproteínas , Regulação da Expressão Gênica , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo
3.
Adv Exp Med Biol ; 1203: 285-312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31811638

RESUMO

RNA-protein interactions are essential to a variety of biological processes. The realization that mammalian genomes are pervasively transcribed brought a tidal wave of tens of thousands of newly identified long noncoding RNAs (lncRNAs) and raised questions about their purpose in cells. The vast majority of lncRNAs have yet to be studied, and it remains to be determined to how many of these transcripts a function can be ascribed. However, results gleaned from studying a handful of these macromolecules have started to reveal common themes of biological function and mechanism of action involving intricate RNA-protein interactions. Some lncRNAs were shown to regulate the chromatin and transcription of distant and neighboring genes in the nucleus, while others regulate the translation or localization of proteins in the cytoplasm. Some lncRNAs were found to be crucial during development, while mutations and aberrant expression of others have been associated with several types of cancer and a plethora of diseases. Over the last few years, the establishment of new technologies has been key in providing the tools to decode the rules governing lncRNA-protein interactions and functions. This chapter will highlight the general characteristics of lncRNAs, their function, and their mode of action, with a special focus on protein interactions. It will also describe the methods at the disposition of scientists to help them cross this next frontier in our understanding of lncRNA biology.


Assuntos
Neoplasias , RNA Longo não Codificante , Ribonucleoproteínas , Animais , Biotecnologia/tendências , Cromatina , Regulação da Expressão Gênica , Genoma , RNA Longo não Codificante/metabolismo , Ribonucleoproteínas/metabolismo
4.
PLoS Pathog ; 15(9): e1007921, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31568537

RESUMO

Humans are frequently exposed to bacterial genotoxins involved in digestive cancers, colibactin and Cytolethal Distending Toxin (CDT), the latter being secreted by many pathogenic bacteria. Our aim was to evaluate the effects induced by these genotoxins on nuclear remodeling in the context of cell survival. Helicobacter infected mice, coculture experiments with CDT- and colibactin-secreting bacteria and hepatic, intestinal and gastric cells, and xenograft mouse-derived models were used to assess the nuclear remodeling in vitro and in vivo. Our results showed that CDT and colibactin induced-nuclear remodeling can be associated with the formation of deep cytoplasmic invaginations in the nucleus of giant cells. These structures, observed both in vivo and in vitro, correspond to nucleoplasmic reticulum (NR). The core of the NR was found to concentrate ribosomes, proteins involved in mRNA translation, polyadenylated RNA and the main components of the complex mCRD involved in mRNA turnover. These structures are active sites of mRNA translation, correlated with a high degree of ploidy, and involve MAPK and calcium signaling. Additional data showed that insulation and concentration of these adaptive ribonucleoprotein particles within the nucleus are dynamic, transient and protect the cell until the genotoxic stress is relieved. Bacterial genotoxins-induced NR would be a privileged gateway for selected mRNA to be preferably transported therein for local translation. These findings offer new insights into the context of NR formation, a common feature of many cancers, which not only appears in response to therapies-induced DNA damage but also earlier in response to genotoxic bacteria.


Assuntos
Toxinas Bacterianas/toxicidade , Helicobacter/patogenicidade , Ribonucleoproteínas/metabolismo , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Sobrevivência Celular , Dano ao DNA , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mutagênicos/toxicidade , Peptídeos/toxicidade , Policetídeos/toxicidade , RNA Mensageiro/metabolismo
5.
Nat Commun ; 10(1): 4502, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31582740

RESUMO

The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s.


Assuntos
Lisina/metabolismo , Ribonucleoproteínas/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Motivos de Aminoácidos/genética , Biocatálise , Cristalografia por Raios X , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismo
6.
Mol Cell Biol ; 39(24)2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31591142

RESUMO

The eukaryotic cytosol contains multiple RNP granules, including P-bodies and stress granules. Three different methods have been used to describe the transcriptome of stress granules or P-bodies, but how these methods compare and how RNA partitioning occurs between P-bodies and stress granules have not been addressed. Here, we compare the analysis of the stress granule transcriptome based on differential centrifugation with and without subsequent stress granule immunopurification. We find that while differential centrifugation alone gives a first approximation of the stress granule transcriptome, this methodology contains nonspecific transcripts that play a confounding role in the interpretation of results. We also immunopurify and compare the RNAs in stress granules and P-bodies under arsenite stress and compare those results to those for the P-body transcriptome described under nonstress conditions. We find that the P-body transcriptome is dominated by poorly translated mRNAs under nonstress conditions, but during arsenite stress, when translation is globally repressed, the P-body transcriptome is very similar to the stress granule transcriptome. This suggests that translation is a dominant factor in targeting mRNAs into both P-bodies and stress granules, and during stress, when most mRNAs are untranslated, the composition of P-bodies reflects this broader translation repression.


Assuntos
Grânulos Citoplasmáticos/genética , Perfilação da Expressão Gênica/métodos , Estabilidade de RNA/genética , Linhagem Celular Tumoral , Citosol/metabolismo , Células Eucarióticas , Humanos , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Estresse Fisiológico/genética , Transcriptoma/genética
7.
Genome Biol ; 20(1): 216, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640799

RESUMO

BACKGROUND: Cells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via ribosome-associated quality control. RESULTS: Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during ribosome-associated quality control. We show that MKRN1 directly binds to the cytoplasmic poly(A)-binding protein (PABPC1) and associates with polysomes. MKRN1 is positioned upstream of poly(A) tails in mRNAs in a PABPC1-dependent manner. Ubiquitin remnant profiling and in vitro ubiquitylation assays uncover PABPC1 and ribosomal protein RPS10 as direct ubiquitylation substrates of MKRN1. CONCLUSIONS: We propose that MKRN1 mediates the recognition of poly(A) tails to prevent the production of erroneous proteins from prematurely polyadenylated transcripts, thereby maintaining proteome integrity.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Biossíntese de Proteínas , Ribonucleoproteínas/metabolismo , Regiões 3' não Traduzidas , Células HEK293 , Humanos , Proteína I de Ligação a Poli(A)/metabolismo , RNA Mensageiro/metabolismo , Ubiquitinação
8.
Nat Commun ; 10(1): 4906, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31659165

RESUMO

The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Endonucleases/metabolismo , Células Epiteliais/metabolismo , Pneumopatias/terapia , Pulmão/metabolismo , Peptídeos/genética , Animais , Proteínas de Bactérias/genética , Brônquios/citologia , Brônquios/metabolismo , Endonucleases/genética , Terapia Genética , Humanos , Pneumopatias/genética , Pneumopatias/metabolismo , Camundongos , Peptídeos/administração & dosagem , Peptídeos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Suínos
9.
Mol Cell Biol ; 39(22)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31481451

RESUMO

Stress granules (SGs) are ribonucleoprotein aggregates that form in response to stress conditions. The regulation of SG dynamics is not fully understood. Permanent pathological SG-like structures were reported in neurodegenerative diseases such as amyotrophic lateral sclerosis. The Ras GTPase-activating protein-binding protein G3BP1 is a central regulator of SG dynamics. We found that the lysine 376 residue (K376) of G3BP1, which is in the RRM RNA binding domain, was acetylated. Consequently, G3BP1 RNA binding was impaired by K376 acetylation. In addition, the acetylation-mimicking mutation K376Q impaired the RNA-dependent interaction of G3BP1 with poly(A)-binding protein 1 (PABP1), but its RNA-independent interactions with caprin-1 and USP10 were little affected. The formation of G3BP1 SGs depended on G3BP1 RNA binding; thus, replacement of endogenous G3BP1 with the K376Q mutant or the RNA binding-deficient F380L/F382L mutant interfered with SG formation. Significant G3BP1 K376 acetylation was detected during SG resolution, and K376-acetylated G3BP1 was seen outside SGs. G3BP1 acetylation is regulated by histone deacetylase 6 (HDAC6) and CBP/p300. Our data suggest that the acetylation of G3BP1 facilitates the disassembly of SGs, offering a potential avenue to mitigate hyperactive stress responses under pathological conditions.


Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , Células HEK293 , Desacetilase 6 de Histona/metabolismo , Humanos , Lisina/metabolismo , Camundongos , Camundongos Knockout , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA/genética , RNA/metabolismo , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/genética , Ribonucleoproteínas/metabolismo , Estresse Fisiológico/fisiologia , Fatores de Transcrição de p300-CBP/metabolismo
10.
Exp Appl Acarol ; 78(4): 505-520, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31375950

RESUMO

Phytoseiulus persimilis is one of the most important biological control agents of spider mites. Multiple studies have been conducted on factors affecting its reproduction, but limited research on related molecular mechanisms has been carried out. In this study, RNA interference of three genes, ribosomal protein L11 (RpL11), ribosomal protein S2 (RpS2), and transformer-2 (tra-2), to newly emerged females were performed through oral delivery of double-stranded RNA, and knockdown of target genes was verified using qRT-PCR analysis. When RpL11 or RpS2 was interfered, 42 and 30% P. persimilis individuals either laid no egg or had no egg hatched, whereas the remaining females had their oviposition duration reduced by 31.8 and 49.9%, fecundity reduced by 48.1 and 67.8%, and egg hatching rate reduced by 20.4 and 22.4%, respectively. In addition, offspring sex ratios were significantly male biased especially at low fecundities. When tra-2 was interfered, no significant difference in fecundity was detected, but egg hatching rate reduced by 30.6%. This study verified the possibility of RNA interference in Phytoseiidae through oral delivery, and indicated that RpL11 and RpS2 are involved in egg formation, whereas tra-2 is involved in embryo development in P. persimilis. Phytoseiid mites have different sex determination pathways compared to insects. The present study provides data and evidence at molecular biological level for future research on reproduction and sex determination of phytoseiid mites.


Assuntos
Proteínas de Artrópodes/genética , Ácaros/fisiologia , Interferência de RNA , Animais , Proteínas de Artrópodes/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Ácaros/genética , Reprodução/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
11.
EMBO J ; 38(21): e101365, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31468569

RESUMO

Inflammasomes are potent innate immune signalling complexes that couple cytokine release with pro-inflammatory cell death. However, pathogens have evolved strategies to evade this cell autonomous system. Here, we show how antibodies combine with innate sensors in primary human macrophages to detect viral infection and activate the inflammasome. Our data demonstrate that antibody opsonisation of virions can activate macrophages in multiple ways. In the first, antibody binding of adenovirus causes lysosomal damage, activating NLRP3 to drive inflammasome formation and IL-1ß release. Importantly, this mechanism enhances virion capture but not infection and is accompanied by cell death, denying the opportunity for viral replication. Unexpectedly, we also find that antibody-coated viruses, which successfully escape into the cytosol, trigger a second system of inflammasome activation. These viruses are intercepted by the cytosolic antibody receptor TRIM21 and the DNA sensor cGAS. Together, these sensors stimulate both NLRP3 inflammasome formation and NFκB activation, driving dose-dependent IL-1ß and TNF secretion, without inducing cell death. Our data highlight the importance of cooperativity between multiple sensing networks to expose viruses to the inflammasome pathway, which is particularly important for how our innate immune system responds to infection in the presence of pre-existing immunity.


Assuntos
Infecções por Adenoviridae/imunologia , Anticorpos Antivirais/imunologia , Inflamassomos/imunologia , Macrófagos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleotidiltransferases/metabolismo , Ribonucleoproteínas/metabolismo , Replicação Viral/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Infecções por Adenoviridae/metabolismo , Infecções por Adenoviridae/virologia , Animais , Células Cultivadas , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nucleotidiltransferases/genética , Ribonucleoproteínas/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
12.
Elife ; 82019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31436530

RESUMO

Extracellular vesicles (EVs) encompass a variety of vesicles secreted into the extracellular space. EVs have been implicated in promoting tumor metastasis, but the molecular composition of tumor-derived EV sub-types and the mechanisms by which molecules are sorted into EVs remain mostly unknown. We report the separation of two small EV sub-populations from a metastatic breast cancer cell line, with biochemical features consistent with different sub-cellular origins. These EV sub-types use different mechanisms of miRNA sorting (selective and non-selective), suggesting that sorting occurs via fundamentally distinct processes, possibly dependent on EV origin. Using biochemical and genetic tools, we identified the Lupus La protein as mediating sorting of selectively packaged miRNAs. We found that two motifs embedded in miR-122 are responsible for high-affinity binding to Lupus La and sorting into vesicles formed in a cell-free reaction. Thus, tumor cells can simultaneously deploy multiple EV species using distinct sorting mechanisms that may enable diverse functions in normal and cancer biology.


Assuntos
Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Autoantígenos/metabolismo , Transporte Biológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Humanos , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo
13.
Genetics ; 213(1): 251-265, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31285256

RESUMO

The eukaryotic cell is highly compartmentalized, and contains a variety of both membrane-bound and membraneless organelles. The latter include the cytoplasmic ribonucleoprotein (RNP) granules, known as the processing body (P-body) and the stress granule. These RNP structures are thought to be involved in the storage of particular mRNAs during periods of stress. Here, we find that a mutant lacking both P-bodies and stress granules exhibits phenotypes suggesting that these structures also have a role in the maintenance of protein homeostasis. In particular, there was an increased occurrence of specific protein quality control (PQC) compartments in this mutant, an observation that is consistent with there being an elevated level of protein misfolding. These compartments normally house soluble misfolded proteins and allow the cell to sequester these polypeptides away from the remaining cellular milieu. Moreover, specific proteins that are normally targeted to both P-bodies and stress granules were found to instead associate with these PQC compartments in this granuleless mutant. This observation is interesting as our data indicate that this association occurs specifically in cells that have been subjected to an elevated level of proteotoxic stress. Altogether, the results here are consistent with P-bodies and stress granules having a role in normal protein homeostasis in eukaryotic cells.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteostase , Proteínas de Saccharomyces cerevisiae/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae , Resposta a Proteínas não Dobradas
14.
Int J Mol Sci ; 20(13)2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31323950

RESUMO

Short tandem repeat (STR) or microsatellite, expansions underlie more than 50 hereditary neurological, neuromuscular and other diseases, including myotonic dystrophy types 1 (DM1) and 2 (DM2). Current disease models for DM1 and DM2 propose a common pathomechanism, whereby the transcription of mutant DMPK (DM1) and CNBP (DM2) genes results in the synthesis of CUG and CCUG repeat expansion (CUGexp, CCUGexp) RNAs, respectively. These CUGexp and CCUGexp RNAs are toxic since they promote the assembly of ribonucleoprotein (RNP) complexes or RNA foci, leading to sequestration of Muscleblind-like (MBNL) proteins in the nucleus and global dysregulation of the processing, localization and stability of MBNL target RNAs. STR expansion RNAs also form phase-separated gel-like droplets both in vitro and in transiently transfected cells, implicating RNA-RNA multivalent interactions as drivers of RNA foci formation. Importantly, the nucleation and growth of these nuclear foci and transcript misprocessing are reversible processes and thus amenable to therapeutic intervention. In this review, we provide an overview of potential DM1 and DM2 pathomechanisms, followed by a discussion of MBNL functions in RNA processing and how multivalent interactions between expanded STR RNAs and RNA-binding proteins (RBPs) promote RNA foci assembly.


Assuntos
Processamento Alternativo/genética , Repetições de Microssatélites/genética , Distrofia Miotônica/genética , RNA/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Humanos , Músculo Esquelético/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo
15.
Nat Commun ; 10(1): 3230, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324804

RESUMO

Liquid-liquid phase separation is thought to be a key organizing principle in eukaryotic cells to generate highly concentrated dynamic assemblies, such as the RNP granules. Numerous in vitro approaches have validated this model, yet a missing aspect is to take into consideration the complex molecular mixture and promiscuous interactions found in vivo. Here we report the versatile scaffold ArtiG to generate concentration-dependent RNA-protein condensates within living cells, as a bottom-up approach to study the impact of co-segregated endogenous components on phase separation. We demonstrate that intracellular RNA seeds the nucleation of the condensates, as it provides molecular cues to locally coordinate the formation of endogenous high-order RNP assemblies. Interestingly, the co-segregation of intracellular components ultimately impacts the size of the phase-separated condensates. Thus, RNA arises as an architectural element that can influence the composition and the morphological outcome of the condensate phases in an intracellular context.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Grânulos Citoplasmáticos/química , Células HeLa , Humanos , Cinética , Microscopia Eletrônica de Transmissão , Ligação Proteica , Mapas de Interação de Proteínas , RNA/química , Proteínas de Ligação a RNA/química , Ribonucleoproteínas/química , Ribonucleoproteínas/ultraestrutura
16.
J Biol Chem ; 294(28): 10758-10759, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300590

RESUMO

Despite the advances in understanding the assembly of yeast preribosomes using affinity purification and structural analysis, studies on mammalian ribosome biogenesis have lagged behind. Using an unbiased method to purify native mammalian preribosomal complexes from the nucleus, Abetov et al. now uncover two types of premature ribonucleoprotein complexes that are nutrient- and mTOR-dependent. This purification scheme, combined with genome-editing techniques, could be exploited to untangle the complexities underlying human ribosome biogenesis and ribosomopathies.


Assuntos
Ribossomos/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Humanos , RNA Ribossômico/metabolismo , Ribonucleoproteínas/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
17.
Pediatr Hematol Oncol ; 36(4): 236-243, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31361176

RESUMO

Here we report a case of refractory macrocytic anemia with a spliceosomal point mutation involving the ZRSR2 gene in a child with Down syndrome (DS). Such mutations have been shown to cause refractory macrocytic anemia and myelodysplastic syndrome (MDS) in elderly individuals. We report the hematological indices of a child with DS and a ZRSR2 spliceosomal mutation. DS is known to produce macrocytic anemia but does not lead to transfusion dependence. In this case, the ZRSR2 mutation was the likely implicating factor for severe transfusion-dependent anemia in a child with DS. The clinical implication of a ZRSR2 mutation in a child with DS has not been previously described and warrants close surveillance to detect potential insidious transformation to MDS.


Assuntos
Anemia Macrocítica/genética , Síndrome de Down/genética , Mutação Puntual , Ribonucleoproteínas/genética , Anemia Macrocítica/sangue , Anemia Macrocítica/terapia , Criança , Síndrome de Down/sangue , Síndrome de Down/terapia , Humanos , Masculino , Ribonucleoproteínas/metabolismo
18.
Nat Commun ; 10(1): 2806, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243272

RESUMO

CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.


Assuntos
Bacteriófagos/metabolismo , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Sistemas CRISPR-Cas , Ribonucleoproteínas/metabolismo , Proteínas Virais/farmacologia , Escherichia coli/metabolismo , Edição de Genes , Regulação Bacteriana da Expressão Gênica , Neisseria/virologia , Ribonucleoproteínas/genética , Proteínas Virais/metabolismo
19.
PLoS Comput Biol ; 15(6): e1007150, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194731

RESUMO

A coarse-grain computational method integrates biophysical and structural data to generate models of HIV-1 genomic RNA, nucleocapsid and integrase condensed into a mature ribonucleoprotein complex. Several hypotheses for the initial structure of the genomic RNA and oligomeric state of integrase are tested. In these models, integrase interaction captures features of the relative distribution of gRNA in the immature virion and increases the size of the RNP globule, and exclusion of nucleocapsid from regions with RNA secondary structure drives an asymmetric placement of the dimerized 5'UTR at the surface of the RNP globule.


Assuntos
HIV-1 , RNA Viral , Ribonucleoproteínas , Proteínas Virais , Biologia Computacional , HIV-1/química , HIV-1/metabolismo , Simulação de Dinâmica Molecular , RNA Viral/química , RNA Viral/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Vírion , Montagem de Vírus
20.
Nat Commun ; 10(1): 2593, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197139

RESUMO

Prion-like domains (PLDs), defined by their low sequence complexity and intrinsic disorder, are present in hundreds of human proteins. Although gain-of-function mutations in the PLDs of neuronal RNA-binding proteins have been linked to neurodegenerative disease progression, the physiological role of PLDs and their range of molecular functions are still largely unknown. Here, we show that the PLD of Drosophila Imp, a conserved component of neuronal ribonucleoprotein (RNP) granules, is essential for the developmentally-controlled localization of Imp RNP granules to axons and regulates in vivo axonal remodeling. Furthermore, we demonstrate that Imp PLD restricts, rather than promotes, granule assembly, revealing a novel modulatory function for PLDs in RNP granule homeostasis. Swapping the position of Imp PLD compromises RNP granule dynamic assembly but not transport, suggesting that these two functions are uncoupled. Together, our study uncovers a physiological function for PLDs in the spatio-temporal control of neuronal RNP assemblies.


Assuntos
Transporte Axonal/fisiologia , Grânulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/metabolismo , Domínios Proteicos/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Microscopia de Fluorescência , Modelos Animais , Príons/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
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