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1.
Cell ; 185(8): 1308-1324.e23, 2022 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-35325593

RESUMO

Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of client proteins. Genetic modification of scaffold granule proteins or tethering the intrinsically disordered region of human fused in sarcoma (FUS) to oskar mRNA allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate the localization and expression of a maternal RNA that instructs germline formation.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Animais , Grânulos de Ribonucleoproteínas Citoplasmáticas , Drosophila/embriologia , Proteínas de Drosophila/genética , Desenvolvimento Embrionário , Oócitos/metabolismo , RNA/metabolismo
2.
Cell ; 181(2): 346-361.e17, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302572

RESUMO

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.


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 , Ribonucleoproteínas/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Células HeLa , Humanos , Conformação de Ácido Nucleico , Organelas/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Estresse Fisiológico/genética
3.
Mol Cell ; 84(19): 3692-3705, 2024 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-39366355

RESUMO

RNAs and RNA-binding proteins can undergo spontaneous or active condensation into phase-separated liquid-like droplets. These condensates are cellular hubs for various physiological processes, and their dysregulation leads to diseases. Although RNAs are core components of many cellular condensates, the underlying molecular determinants for the formation, regulation, and function of ribonucleoprotein condensates have largely been studied from a protein-centric perspective. Here, we highlight recent developments in ribonucleoprotein condensate biology with a particular emphasis on RNA-driven phase transitions. We also present emerging future directions that might shed light on the role of RNA condensates in spatiotemporal regulation of cellular processes and inspire bioengineering of RNA-based therapeutics.


Assuntos
Condensados Biomoleculares , Transição de Fase , Proteínas de Ligação a RNA , RNA , Ribonucleoproteínas , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Humanos , RNA/metabolismo , RNA/química , RNA/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Animais
4.
Mol Cell ; 78(4): 670-682.e8, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32343944

RESUMO

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.


Assuntos
Caulobacter crescentus/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Organelas/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/crescimento & desenvolvimento , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Humanos , Complexos Multienzimáticos/genética , Organelas/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Helicases/genética , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
5.
Mol Cell ; 74(3): 521-533.e6, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30952514

RESUMO

Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA-processing enzymes, termed processing bodies (PBs). Here we track the dynamic localization of individual miRNAs, mRNAs, and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs, and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and 3' versus 5' placement of miRNA target sites significantly affect the PB localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches, we show that partitioning in the PB phase attenuates mRNA silencing, suggesting that physiological mRNA turnover occurs predominantly outside of PBs. Instead, our data support a PB role in sequestering unused miRNAs for surveillance and provide a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution.


Assuntos
MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Grânulos Citoplasmáticos/genética , Inativação Gênica , Células HeLa , Humanos , Processamento Pós-Transcricional do RNA/genética , RNA não Traduzido/genética , Imagem Individual de Molécula
6.
Mol Cell ; 74(3): 598-608.e6, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31051140

RESUMO

RNA flow between organisms has been documented within and among different kingdoms of life. Recently, we demonstrated horizontal RNA transfer between honeybees involving secretion and ingestion of worker and royal jellies. However, how the jelly facilitates transfer of RNA is still unknown. Here, we show that worker and royal jellies harbor robust RNA-binding activity. We report that a highly abundant jelly component, major royal jelly protein 3 (MRJP-3), acts as an extracellular non-sequence-specific RNA-aggregating factor. Multivalent RNA binding stimulates higher-order assembly of MRJP-3 into extracellular ribonucleoprotein granules that protect RNA from degradation and enhance RNA bioavailability. These findings reveal that honeybees have evolved a secreted dietary RNA-binding factor to concentrate, stabilize, and share RNA among individuals. Our work identifies high-order ribonucleoprotein assemblies with functions outside cells and organisms.


Assuntos
Abelhas/genética , Ácidos Graxos/genética , Transferência Genética Horizontal/genética , Glicoproteínas/genética , Proteínas de Insetos/genética , Animais , Ácidos Graxos/biossíntese , Transição de Fase , RNA/genética , Transporte de RNA/genética , Proteínas de Ligação a RNA/genética
7.
EMBO J ; 41(9): e110137, 2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35355287

RESUMO

Numerous membrane-less organelles, composed of a combination of RNA and proteins, are observed in the nucleus and cytoplasm of eukaryotic cells. These RNP granules include stress granules (SGs), processing bodies (PBs), Cajal bodies, and nuclear speckles. An unresolved question is how frequently RNA molecules are required for the integrity of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. We find the majority of RNP granules, including SGs, Cajal bodies, nuclear speckles, and the nucleolus, are altered by the degradation of their RNA components. In contrast, PBs and super-enhancer complexes were largely not affected by RNA degradation in their respective compartments. RNA degradation overall led to the apparent dissolution of some membrane-less organelles, whereas others reorganized into structures with altered morphology. These findings highlight a critical and widespread role of RNA in the organization of several RNP granules.


Assuntos
Grânulos Citoplasmáticos , RNA , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo
8.
EMBO J ; 40(21): e107711, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34524703

RESUMO

RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches.


Assuntos
Grânulos de Ribonucleoproteínas Citoplasmáticas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Rotavirus/genética , Proteínas não Estruturais Virais/metabolismo , Animais , Bovinos , Linhagem Celular , Grânulos de Ribonucleoproteínas Citoplasmáticas/efeitos dos fármacos , Grânulos de Ribonucleoproteínas Citoplasmáticas/ultraestrutura , Grânulos de Ribonucleoproteínas Citoplasmáticas/virologia , Regulação Viral da Expressão Gênica , Genes Reporter , Glicóis/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Haplorrinos , Interações Hospedeiro-Patógeno/genética , Humanos , Concentração Osmolar , Fosforilação , Propilenoglicol/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Rotavirus/efeitos dos fármacos , Rotavirus/crescimento & desenvolvimento , Rotavirus/ultraestrutura , Transdução de Sinais , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
9.
Development ; 149(21)2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36196602

RESUMO

In animals with germ plasm, specification of the germline involves 'germ granules', cytoplasmic condensates that enrich maternal transcripts in the germline founder cells. In Caenorhabditis elegans embryos, P granules enrich maternal transcripts, but surprisingly P granules are not essential for germ cell fate specification. Here, we describe a second condensate in the C. elegans germ plasm. Like canonical P-bodies found in somatic cells, 'germline P-bodies' contain regulators of mRNA decapping and deadenylation and, in addition, the intrinsically-disordered proteins MEG-1 and MEG-2 and the TIS11-family RNA-binding protein POS-1. Embryos lacking meg-1 and meg-2 do not stabilize P-body components, misregulate POS-1 targets, mis-specify the germline founder cell and do not develop a germline. Our findings suggest that specification of the germ line involves at least two distinct condensates that independently enrich and regulate maternal mRNAs in the germline founder cells. This article has an associated 'The people behind the papers' interview.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Corpos de Processamento , Células Germinativas/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciação Celular/genética , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
10.
Mol Cell ; 68(1): 144-157.e5, 2017 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-28965817

RESUMO

Within cells, soluble RNPs can switch states to coassemble and condense into liquid or solid bodies. Although these phase transitions have been reconstituted in vitro, for endogenous bodies the diversity of the components, the specificity of the interaction networks, and the function of the coassemblies remain to be characterized. Here, by developing a fluorescence-activated particle sorting (FAPS) method to purify cytosolic processing bodies (P-bodies) from human epithelial cells, we identified hundreds of proteins and thousands of mRNAs that structure a dense network of interactions, separating P-body from non-P-body RNPs. mRNAs segregating into P-bodies are translationally repressed, but not decayed, and this repression explains part of the poor genome-wide correlation between RNA and protein abundance. P-bodies condense thousands of mRNAs that strikingly encode regulatory processes. Thus, we uncovered how P-bodies, by condensing and segregating repressed mRNAs, provide a physical substrate for the coordinated regulation of posttranscriptional mRNA regulons.


Assuntos
Regulação da Expressão Gênica , Proteoma/genética , RNA Mensageiro/genética , Regulon , Ribonucleoproteínas/genética , Fracionamento Celular , Citoplasma/metabolismo , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Ontologia Genética , Células HEK293 , Células HeLa , Humanos , Anotação de Sequência Molecular , Transição de Fase , Biossíntese de Proteínas , Proteoma/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo
11.
Mol Cell ; 68(4): 808-820.e5, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29129640

RESUMO

Stress granules are mRNA-protein assemblies formed from nontranslating mRNAs. Stress granules are important in the stress response and may contribute to some degenerative diseases. Here, we describe the stress granule transcriptome of yeast and mammalian cells through RNA-sequencing (RNA-seq) analysis of purified stress granule cores and single-molecule fluorescence in situ hybridization (smFISH) validation. While essentially every mRNA, and some noncoding RNAs (ncRNAs), can be targeted to stress granules, the targeting efficiency varies from <1% to >95%. mRNA accumulation in stress granules correlates with longer coding and UTR regions and poor translatability. Quantifying the RNA-seq analysis by smFISH reveals that only 10% of bulk mRNA molecules accumulate in mammalian stress granules and that only 185 genes have more than 50% of their mRNA molecules in stress granules. These results suggest that stress granules may not represent a specific biological program of messenger ribonucleoprotein (mRNP) assembly, but instead form by condensation of nontranslating mRNPs in proportion to their length and lack of association with ribosomes.


Assuntos
Grânulos Citoplasmáticos/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcriptoma/fisiologia , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/genética , Humanos , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética
12.
Biochem Soc Trans ; 2024 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-39324618

RESUMO

Understanding of the physicochemical properties and functions of biomolecular condensates has rapidly advanced over the past decade. More recently, many RNA viruses have been shown to form cytoplasmic replication factories, or viroplasms, via phase separation of their components, akin to numerous cellular membraneless organelles. Notably, diverse viruses from the Reoviridae family containing 10-12 segmented double-stranded RNA genomes induce the formation of viroplasms in infected cells. Little is known about the inner workings of these membraneless cytoplasmic inclusions and how they may support stoichiometric RNA assembly in viruses with segmented RNA genomes, raising questions about the roles of phase separation in coordinating viral genome packaging. Here, we discuss how the molecular composition of viroplasms determines their properties, highlighting the interplay between RNA structure, RNA remodelling, and condensate self-organisation. Advancements in RNA structural probing and theoretical modelling of condensates can reveal the mechanisms through which these ribonucleoprotein complexes support the selective enrichment and stoichiometric assembly of distinct viral RNAs.

13.
Can J Microbiol ; 70(1): 32-39, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-37826860

RESUMO

Ribonucleoprotein granules are bio-condensates that form a diverse group of dynamic membrane-less organelles implicated in several cellular functions, including stress response and cellular survival. In Toxoplasma gondii, a type of bio-condensates referred to as stress granules (SGs) are formed prior to the parasites' egress from the host cell and are implicated in the survival and invasion competency of extracellular tachyzoites. We used paraformaldehyde to fix and cross-link SG proteins to allow purification by centrifugation and analysis by mass spectrometry. We profiled protein components of SGs at 10 and 30 min post-egress when parasite's invasion ability is significantly diminished. Thirty-three proteins were identified from 10 min SGs, and additional 43 proteins were identified from 30 min SGs. Notably, common SG components such as proteins with intrinsically disordered domains were not identified. Gene ontology analysis of both 10 and 30 min SGs shows that overall molecular functions of SGs' proteins are ATP-binding, GTP-binding, and GTPase activity. Discernable differences between 10 and 30 min SGs are in the proportions of translation and microtubule-related proteins. Ten-minute SGs have a higher proportion of microtubule-related proteins and a lower proportion of ribosome-related proteins, while a reverse correlation was identified for those of 30 min. It remains to be investigated whether this reverse correlation contributes to the ability of extracellular tachyzoites to reinvade host cells.


Assuntos
Toxoplasma , Toxoplasma/fisiologia , Grânulos de Estresse , Proteômica , Espectrometria de Massas , Estresse Fisiológico
14.
EMBO Rep ; 22(12): e53632, 2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34787357

RESUMO

TDP-43 is an RNA-binding protein that forms ribonucleoprotein condensates via liquid-liquid phase separation (LLPS) and regulates gene expression through specific RNA interactions. Loss of TDP-43 protein homeostasis and dysfunction are tied to neurodegenerative disorders, mainly amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Alterations of TDP-43 LLPS properties may be linked to protein aggregation. However, the mechanisms regulating TDP-43 LLPS are ill-defined, particularly how TDP-43 association with specific RNA targets regulates TDP-43 condensation remains unclear. We show that RNA binding strongly promotes TDP-43 LLPS through sequence-specific interactions. RNA-driven condensation increases with the number of adjacent TDP-43-binding sites and is also mediated by multivalent interactions involving the amino and carboxy-terminal TDP-43 domains. The physiological relevance of RNA-driven TDP-43 condensation is supported by similar observations in mammalian cellular lysate. Importantly, we find that TDP-43-RNA association maintains liquid-like properties of the condensates, which are disrupted in the presence of ALS-linked TDP-43 mutations. Altogether, RNA binding plays a central role in modulating TDP-43 condensation while maintaining protein solubility, and defects in this RNA-mediated activity may underpin TDP-43-associated pathogenesis.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/genética , RNA/genética , Proteínas de Ligação a RNA/genética
15.
J Mol Cell Cardiol ; 165: 115-129, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35041844

RESUMO

Dilated cardiomyopathy (DCM) is a heritable and genetically heterogenous disease often idiopathic and a leading cause of heart failure with high morbidity and mortality. DCM caused by RNA binding motif protein 20 (RBM20) mutations is diverse and needs a more complete mechanistic understanding. RBM20 mutation S637G (S639G in mice) is linked to severe DCM and early death in human patients. In this study, we generated a RBM20 S639G mutation knock-in (KI) mouse model to validate the function of S639G mutation and examine the underlying mechanisms. KI mice exhibited severe DCM and premature death with a ~ 50% mortality in two months old homozygous (HM) mice. KI mice had enlarged atria and increased ANP and BNP biomarkers. The S639G mutation promoted RBM20 trafficking and ribonucleoprotein (RNP) granules in the sarcoplasm. RNA Seq data revealed differentially expressed and spliced genes were associated with arrhythmia, cardiomyopathy, and sudden death. KI mice also showed a reduction of diastolic stiffness and impaired contractility at both the left ventricular (LV) chamber and cardiomyocyte levels. Our results indicate that the RBM20 S639G mutation leads to RNP granules causing severe heart failure and early death and this finding strengthens the novel concept that RBM20 cardiomyopathy is a RNP granule disease.


Assuntos
Cardiomiopatia Dilatada , Insuficiência Cardíaca , Animais , Cardiomiopatia Dilatada/metabolismo , Insuficiência Cardíaca/genética , Humanos , Camundongos , Mortalidade Prematura , Mutação , RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Risco
16.
Trends Genet ; 34(8): 612-626, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29908710

RESUMO

P-bodies (PBs) are cytosolic RNP granules that are conserved among eukaryotic organisms. In the past few years, major progress has been made in understanding the biochemical and biophysical mechanisms that lead to their formation. However, whether they play a role in mRNA storage or decay remains actively debated. P-bodies were recently isolated from human cells by a novel fluorescence-activated particle sorting (FAPS) approach that enabled the characterization of their protein and RNA content, providing new insights into their function. Together with recent innovative imaging studies, these new data show that mammalian PBs are primarily involved not in RNA decay but rather in the coordinated storage of mRNAs encoding regulatory functions. These small cytoplasmic droplets could thus be important for cell adaptation to the environment.


Assuntos
Organelas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Humanos , Organelas/ultraestrutura , Estabilidade de RNA , RNA Mensageiro Estocado/genética , RNA Mensageiro Estocado/metabolismo , Ribonucleoproteínas/metabolismo
17.
Proc Natl Acad Sci U S A ; 115(11): 2734-2739, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29483269

RESUMO

Stress granules are higher order assemblies of nontranslating mRNAs and proteins that form when translation initiation is inhibited. Stress granules are thought to form by protein-protein interactions of RNA-binding proteins. We demonstrate RNA homopolymers or purified cellular RNA forms assemblies in vitro analogous to stress granules. Remarkably, under conditions representative of an intracellular stress response, the mRNAs enriched in assemblies from total yeast RNA largely recapitulate the stress granule transcriptome. We suggest stress granules are formed by a summation of protein-protein and RNA-RNA interactions, with RNA self-assembly likely to contribute to other RNP assemblies wherever there is a high local concentration of RNA. RNA assembly in vitro is also increased by GR and PR dipeptide repeats, which are known to increase stress granule formation in cells. Since GR and PR dipeptides are involved in neurodegenerative diseases, this suggests that perturbations increasing RNA-RNA assembly in cells could lead to disease.


Assuntos
Grânulos Citoplasmáticos/genética , RNA/genética , Saccharomyces cerevisiae/genética , Transcriptoma , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , RNA/química , RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
EMBO J ; 35(15): 1603-12, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27357569

RESUMO

Cells contain numerous, molecularly distinct cellular compartments that are not enclosed by lipid bilayers. These compartments are implicated in a wide range of cellular activities, and they have been variously described as bodies, granules, or organelles. Recent evidence suggests that a liquid-liquid phase separation (LLPS) process may drive their formation, possibly justifying the unifying term "droplet organelle". A veritable deluge of recent publications points to the importance of low-complexity proteins and RNA in determining the physical properties of phase-separated structures. Many of the proteins linked to such structures are implicated in human diseases, such as amyotrophic lateral sclerosis (ALS). We provide an overview of the organizational principles that characterize putative "droplet organelles" in healthy and diseased cells, connecting protein biochemistry with cell physiology.


Assuntos
Fenômenos Fisiológicos Celulares , Citosol/química , Substâncias Macromoleculares/metabolismo , Complexos Multienzimáticos/metabolismo
19.
Proc Natl Acad Sci U S A ; 114(45): E9569-E9578, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29078371

RESUMO

Eukaryotic cells contain multiple RNA-protein assemblies referred to as RNP granules, which are thought to form through multiple protein-protein interactions analogous to a liquid-liquid phase separation. One class of RNP granules consists of P bodies, which consist of nontranslating mRNAs and the general translation repression and mRNA degradation machinery. P bodies have been suggested to form predominantly through interactions of Edc3 and a prion-like domain on Lsm4. In this work, we provide evidence that P-body assembly can be driven by multiple different protein-protein and/or protein-RNA interactions, including interactions involving Dhh1, Psp2, and Pby1. Moreover, the relative importance of specific interactions can vary with different growth conditions. Based on these observations, we develop a summative model wherein the P-body assembly phenotype of a given mutant can be predicted from the number of currently known protein-protein interactions between P-body components.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Células Eucarióticas/metabolismo , Biossíntese de Proteínas/fisiologia , Mapas de Interação de Proteínas/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo
20.
Int J Mol Sci ; 19(8)2018 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-30081499

RESUMO

The timing, dosage and location of gene expression are fundamental determinants of brain architectural complexity. In neurons, this is, primarily, achieved by specific sets of trans-acting RNA-binding proteins (RBPs) and their associated factors that bind to specific cis elements throughout the RNA sequence to regulate splicing, polyadenylation, stability, transport and localized translation at both axons and dendrites. Not surprisingly, misregulation of RBP expression or disruption of its function due to mutations or sequestration into nuclear or cytoplasmic inclusions have been linked to the pathogenesis of several neuropsychiatric and neurodegenerative disorders such as fragile-X syndrome, autism spectrum disorders, spinal muscular atrophy, amyotrophic lateral sclerosis and frontotemporal dementia. This review discusses the roles of Pumilio, Staufen, IGF2BP, FMRP, Sam68, CPEB, NOVA, ELAVL, SMN, TDP43, FUS, TAF15, and TIA1/TIAR in RNA metabolism by analyzing their specific molecular and cellular function, the neurological symptoms associated with their perturbation, and their axodendritic transport/localization along with their target mRNAs as part of larger macromolecular complexes termed ribonucleoprotein (RNP) granules.


Assuntos
Doenças Neurodegenerativas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Humanos , Modelos Biológicos , Doenças Neurodegenerativas/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética
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