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1.
RNA ; 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493599

RESUMO

RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two color imaging directly revealed single mRNAs transported by single KIF1C motors, with the 3'UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal a widespread RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role in transporting RNAs and clustering them within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA suggesting a possible feedback loop acting at the level of mRNA transport.

2.
Nat Plants ; 7(8): 1050-1064, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373603

RESUMO

Plants are constantly adapting to ambient fluctuations through spatial and temporal transcriptional responses. Here, we implemented the latest-generation RNA imaging system and combined it with microfluidics to visualize transcriptional regulation in living Arabidopsis plants. This enabled quantitative measurements of the transcriptional activity of single loci in single cells, in real time and under changing environmental conditions. Using phosphate-responsive genes as a model, we found that active genes displayed high transcription initiation rates (one initiation event every ~3 s) and frequently clustered together in endoreplicated cells. We observed gene bursting and large allelic differences in single cells, revealing that at steady state, intrinsic noise dominated extrinsic variations. Moreover, we established that transcriptional repression triggered in roots by phosphate, a crucial macronutrient limiting plant development, occurred with unexpectedly fast kinetics (on the order of minutes) and striking heterogeneity between neighbouring cells. Access to single-cell RNA polymerase II dynamics in live plants will benefit future studies of signalling processes.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Fosfatos/metabolismo , Células Vegetais/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Transcrição Genética , Regulação da Expressão Gênica de Plantas , Cinética , RNA Polimerase II/genética
3.
Nat Commun ; 12(1): 4810, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376666

RESUMO

The R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium and death within 10 days. Levels of R2TP substrates decreased, with strong effects on mTOR, ATM and ATR. Proliferative stem cells and progenitors deficient for Rpap3 failed to import RNA polymerase II into the nucleus and they induced p53, cell cycle arrest and apoptosis. Post-mitotic, differentiated cells did not display these alterations, suggesting that R2TP clients are preferentially built in actively proliferating cells. In addition, high RPAP3 levels in colorectal tumors from patients correlate with bad prognosis. Here, we show that, in the intestine, the R2TP chaperone plays essential roles in normal and tumoral proliferation.


Assuntos
Proliferação de Células , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mucosa Intestinal/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Células Cultivadas , Células Epiteliais/citologia , Humanos , Mucosa Intestinal/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Ligação Proteica , Células-Tronco/citologia , Células-Tronco/metabolismo
4.
Nat Commun ; 12(1): 4503, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301927

RESUMO

Promoter-proximal pausing of RNA polymerase II is a key process regulating gene expression. In latent HIV-1 cells, it prevents viral transcription and is essential for latency maintenance, while in acutely infected cells the viral factor Tat releases paused polymerase to induce viral expression. Pausing is fundamental for HIV-1, but how it contributes to bursting and stochastic viral reactivation is unclear. Here, we performed single molecule imaging of HIV-1 transcription. We developed a quantitative analysis method that manages multiple time scales from seconds to days and that rapidly fits many models of promoter dynamics. We found that RNA polymerases enter a long-lived pause at latent HIV-1 promoters (>20 minutes), thereby effectively limiting viral transcription. Surprisingly and in contrast to current models, pausing appears stochastic and not obligatory, with only a small fraction of the polymerases undergoing long-lived pausing in absence of Tat. One consequence of stochastic pausing is that HIV-1 transcription occurs in bursts in latent cells, thereby facilitating latency exit and providing a rationale for the stochasticity of viral rebounds.


Assuntos
Regulação Viral da Expressão Gênica , Infecções por HIV/genética , HIV-1/genética , Regiões Promotoras Genéticas/genética , Latência Viral/genética , Algoritmos , RNA Polimerases Dirigidas por DNA/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Células HeLa , Humanos , Modelos Genéticos , Processos Estocásticos , Fatores de Tempo , Ativação Viral/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
5.
Nat Commun ; 12(1): 4504, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301936

RESUMO

Genes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. We investigate two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, we demonstrate that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, our results provide a rationale for promoter switching during zygotic genome activation.


Assuntos
Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Regiões Promotoras Genéticas/genética , TATA Box/genética , Imagem com Lapso de Tempo/métodos , Transcrição Genética/genética , Algoritmos , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/embriologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Modelos Teóricos
6.
Nat Commun ; 12(1): 3646, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131137

RESUMO

U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before the next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.


Assuntos
Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Regulação para Baixo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Fatores de Alongamento de Peptídeos , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Splicing de RNA , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão , Ribonucleoproteínas Nucleares Pequenas/química , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética
7.
Nat Commun ; 12(1): 3158, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039974

RESUMO

The carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.


Assuntos
Microscopia Intravital/métodos , RNA Polimerase II/metabolismo , Imagem Individual de Molécula/métodos , Transcrição Genética , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Microscopia de Fluorescência , Fosforilação , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Serina/metabolismo , Análise Espaço-Temporal , Imagem com Lapso de Tempo
8.
J Cell Sci ; 134(9)2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33785534

RESUMO

MicroRNAs (miRNAs), the tiny regulators of gene expression, can be transferred between neighbouring cells via extracellular vesicles (EVs) to control the expression of genes in both donor and recipient cells. How the EV-derived miRNAs are internalized and become functional in target cells is an unresolved question. We have expressed a liver-specific miRNA, miR-122, in non-hepatic cells for packaging in released EVs. With these EVs, we have followed the trafficking of miR-122 to recipient HeLa cells that otherwise do not express this miRNA. We found that EV-associated miR-122 is primarily single-stranded and, to become functional, is loaded onto the recipient cell argonaute proteins without requiring host Dicer1. Following endocytosis, EV-associated miR-122 is loaded onto the host cell argonaute proteins on the endosomal membrane, where the release of internalized miRNAs occurs in a pH-dependent manner, facilitating the formation of the exogenous miRNP pool in the recipient cells. Endosome maturation defects affect EV-mediated entry of exogeneous miRNAs in mammalian cells. This article has an associated First Person interview with the first author of the paper.

9.
Nat Commun ; 12(1): 1859, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767140

RESUMO

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ativação Transcricional/fisiologia , Proliferação de Células/fisiologia , Cromatina/genética , Cristalografia por Raios X , Histonas/metabolismo , Ressonância Magnética Nuclear Biomolecular , RNA Polimerase II/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Genética/genética
10.
Nat Commun ; 12(1): 1352, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649340

RESUMO

Local translation allows for a spatial control of gene expression. Here, we use high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging reveals active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysome transport mediated by nascent proteins.


Assuntos
Centrossomo/metabolismo , Polirribossomos/metabolismo , Transporte de RNA , Animais , Proteínas de Ciclo Celular/metabolismo , Centrossomo/efeitos dos fármacos , Cicloeximida/farmacologia , Drosophila/genética , Células HeLa , Humanos , Mitose/efeitos dos fármacos , Fases de Leitura Aberta/genética , Polirribossomos/efeitos dos fármacos , Puromicina/farmacologia , Transporte de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo
11.
Nat Commun ; 12(1): 1351, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649372

RESUMO

Exon junction complexes (EJCs) mark untranslated spliced mRNAs and are crucial for the mRNA lifecycle. An imbalance in EJC dosage alters mouse neural stem cell (mNSC) division and is linked to human neurodevelopmental disorders. In quiescent mNSC and immortalized human retinal pigment epithelial (RPE1) cells, centrioles form a basal body for ciliogenesis. Here, we report that EJCs accumulate at basal bodies of mNSC or RPE1 cells and decline when these cells differentiate or resume growth. A high-throughput smFISH screen identifies two transcripts accumulating at centrosomes in quiescent cells, NIN and BICD2. In contrast to BICD2, the localization of NIN transcripts is EJC-dependent. NIN mRNA encodes a core component of centrosomes required for microtubule nucleation and anchoring. We find that EJC down-regulation impairs both pericentriolar material organization and ciliogenesis. An EJC-dependent mRNA trafficking towards centrosome and basal bodies might contribute to proper mNSC division and brain development.


Assuntos
Centrossomo/metabolismo , Cílios/metabolismo , Éxons/genética , Transporte de RNA , RNA Mensageiro/metabolismo , Animais , Autoantígenos/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo
12.
Nucleic Acids Res ; 49(2): 1094-1113, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33367824

RESUMO

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/biossíntese , Trifosfato de Adenosina/metabolismo , Proteínas do Olho/metabolismo , Técnicas de Inativação de Genes , Genes Reporter , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Complexos Multiproteicos , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteômica/métodos , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
RNA Biol ; : 1-17, 2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-33111627

RESUMO

Transport of mRNAs is an important step of gene expression, which brings the genetic message from the DNA in the nucleus to a precise cytoplasmic location in a regulated fashion. Perturbation of this process can lead to pathologies such as developmental and neurological disorders. In this review, we discuss recent advances in the field of mRNA transport made using single molecule fluorescent imaging approaches. We present an overview of these approaches in fixed and live cells and their input in understanding the key steps of mRNA journey: transport across the nucleoplasm, export through the nuclear pores and delivery to its final cytoplasmic location. This review puts a particular emphasis on the coupling of mRNA transport with translation, such as localization-dependent translational regulation and translation-dependent mRNA localization. We also highlight the recently discovered translation factories, and how cellular and viral RNAs can hijack membrane transport systems to travel in the cytoplasm.

14.
iScience ; 23(9): 101529, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-33083759

RESUMO

Nuclear factor (NF)-κB controls the transcriptional response to inflammatory signals by translocating into the nucleus, but we lack a single-cell characterization of the resulting transcription dynamics. Here we show that upon tumor necrosis factor (TNF)-α transcription of NF-κB target genes is heterogeneous in individual cells but results in an average nascent transcription profile that is prompt (i.e., occurs almost immediately) and sharp (i.e., increases and decreases rapidly) compared with NF-κB nuclear localization. Using an NF-κB-controlled MS2 reporter we show that the single-cell nascent transcription is more heterogeneous than NF-κB translocation dynamics, with a fraction of synchronized "first responders" that shape the average transcriptional profile and are more prone to respond to multiple TNF-α stimulations. A mathematical model combining NF-κB-mediated gene activation and a gene refractory state is able to reproduce these features. Our work shows how the expression of target genes induced by transcriptional activators can be heterogeneous across single cells and yet time resolved on average.

15.
Dev Cell ; 54(6): 773-791.e5, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32783880

RESUMO

Local translation allows spatial control of gene expression. Here, we performed a dual protein-mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. 32 mRNAs displayed specific cytoplasmic localizations with local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope, and centrosomes, the latter being cell-cycle-dependent. Automated classification of mRNA localization patterns revealed a high degree of intercellular heterogeneity. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For ß-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.


Assuntos
Regulação da Expressão Gênica/fisiologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , RNA/metabolismo , Linhagem Celular , Centrossomo/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Polirribossomos/genética , Polirribossomos/metabolismo , Biossíntese de Proteínas/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , RNA Mensageiro/genética
16.
Methods Mol Biol ; 2166: 121-144, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32710406

RESUMO

Live imaging of single RNA from birth to death brought important advances in our understanding of the spatiotemporal regulation of gene expression. These studies have provided a comprehensive understanding of RNA metabolism by describing the process step by step. Most of these studies used for live imaging a genetically encoded RNA-tagging system fused to fluorescent proteins. One of the best characterized RNA-tagging systems is derived from the bacteriophage MS2 and it allows single RNA imaging in real-time and live cells. This system has been successfully used to track the different steps of mRNA processing in many living organisms. The recent development of optimized MS2 and MCP variants now allows the labeling of endogenous RNAs and their imaging without modifying their behavior. In this chapter, we discuss the improvements in detecting single mRNAs with different variants of MCP and fluorescent proteins that we tested in yeast and mammalian cells. Moreover, we describe protocols using MS2-MCP systems improved for real-time imaging of single mRNAs and transcription dynamics in S. cerevisiae and mammalian cells, respectively.


Assuntos
Proteínas do Capsídeo/genética , Técnicas de Cultura de Células/métodos , Hibridização in Situ Fluorescente/métodos , Levivirus/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , Análise de Célula Única/métodos , Animais , Linhagem Celular , Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Razão Sinal-Ruído
17.
Nat Commun ; 10(1): 3178, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320644

RESUMO

mRNA transport restricts translation to specific subcellular locations, which is the basis for many cellular functions. However, the precise process of mRNA sorting to synapses in neurons remains elusive. Here we use Rgs4 mRNA to investigate 3'-UTR-dependent transport by MS2 live-cell imaging. The majority of observed RNA granules display 3'-UTR independent bidirectional transport in dendrites. Importantly, the Rgs4 3'-UTR causes an anterograde transport bias, which requires the Staufen2 protein. Moreover, the 3'-UTR mediates dynamic, sustained mRNA recruitment to synapses. Visualization at high temporal resolution enables us to show mRNA patrolling dendrites, allowing transient interaction with multiple synapses, in agreement with the sushi-belt model. Modulation of neuronal activity by either chemical silencing or local glutamate uncaging regulates both the 3'-UTR-dependent transport bias and synaptic recruitment. This dynamic and reversible mRNA recruitment to active synapses would allow translation and synaptic remodeling in a spatially and temporally adaptive manner.


Assuntos
Regiões 3' não Traduzidas/genética , Dendritos/genética , Hipocampo/metabolismo , Transporte de RNA/fisiologia , RNA Mensageiro/genética , Sinapses/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Proteínas RGS/genética , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley
18.
Nat Commun ; 9(1): 4584, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389932

RESUMO

RNA localization is a crucial process for cellular function and can be quantitatively studied by single molecule FISH (smFISH). Here, we present an integrated analysis framework to analyze sub-cellular RNA localization. Using simulated images, we design and validate a set of features describing different RNA localization patterns including polarized distribution, accumulation in cell extensions or foci, at the cell membrane or nuclear envelope. These features are largely invariant to RNA levels, work in multiple cell lines, and can measure localization strength in perturbation experiments. Most importantly, they allow classification by supervised and unsupervised learning at unprecedented accuracy. We successfully validate our approach on representative experimental data. This analysis reveals a surprisingly high degree of localization heterogeneity at the single cell level, indicating a dynamic and plastic nature of RNA localization.


Assuntos
Simulação por Computador , RNA/metabolismo , Algoritmos , Animais , Células HeLa , Humanos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo
19.
Sci Rep ; 8(1): 12901, 2018 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-30150655

RESUMO

The RNA exosome fulfills important functions in the processing and degradation of numerous RNAs species. However, the mechanisms of recruitment to its various nuclear substrates are poorly understood. Using Epstein-Barr virus mRNAs as a model, we have discovered a novel function for the splicing factor SRSF3 in the quality control of nuclear mRNAs. We have found that viral mRNAs generated from intronless genes are particularly unstable due to their degradation by the nuclear RNA exosome. This effect is counteracted by the viral RNA-binding protein EB2 which stabilizes these mRNAs in the nucleus and stimulates both their export to the cytoplasm and their translation. In the absence of EB2, SRSF3 participates in the destabilization of these viral RNAs by interacting with both the RNA exosome and its adaptor complex NEXT. Taken together, our results provide direct evidence for a connection between the splicing machinery and mRNA decay mediated by the RNA exosome. Our results suggest that SRSF3 aids the nuclear RNA exosome and the NEXT complex in the recognition and degradation of certain mRNAs.


Assuntos
Exossomos/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Western Blotting , Núcleo Celular/genética , Núcleo Celular/metabolismo , Exossomos/genética , Células HeLa , Humanos , Imunoprecipitação , Splicing de RNA/genética , Splicing de RNA/fisiologia , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina/genética
20.
Mol Cell ; 71(3): 468-480, 2018 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-30075145

RESUMO

The spatiotemporal regulation of gene expression is key to many biological processes. Recent imaging approaches opened exciting perspectives for understanding the intricate mechanisms regulating RNA metabolism, from synthesis to decay. Imaging techniques allow their observation at high spatial and temporal resolution, while keeping cellular morphology and micro-environment intact. Here, we focus on approaches for imaging single RNA molecules in cells, tissues, and embryos. In fixed cells, the rapid development of smFISH multiplexing opens the way to large-scale single-molecule studies, while in live cells, gene expression can be observed in real time in its native context. We highlight the strengths and limitations of these methods, as well as future challenges. We present how they advanced our understanding of gene expression heterogeneity and bursting, as well as the spatiotemporal aspects of splicing, translation, and RNA decay. These insights yield a dynamic and stochastic view of gene expression in single cells.


Assuntos
Imagem Individual de Molécula/métodos , Análise de Célula Única/métodos , Expressão Gênica/genética , Hibridização in Situ Fluorescente/métodos , Biossíntese de Proteínas/genética , RNA/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
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