RESUMO
Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome packaging changes during infection and subsequent transcriptional activation is currently unclear. Here, we combined RNA-seq, MNase-seq, ChIP-seq, and single genome imaging during early adenovirus infection to unveil the structure- and time-resolved dynamics of viral chromatin changes as well as their correlation with gene transcription. Our MNase mapping data indicates that the adenoviral genome is arranged in precisely positioned nucleoprotein particles with nucleosome-like characteristics, that we term adenosomes. We identified 238 adenosomes that are positioned by a DNA sequence code and protect about 60-70 bp of DNA. The incoming adenoviral genome is more accessible at early gene loci that undergo additional chromatin de-condensation upon infection. Histone H3.3 containing nucleosomes specifically replaces pVII at distinct genomic sites and at the transcription start sites of early genes. Acetylation of H3.3 is predominant at the transcription start sites and precedes transcriptional activation. Based on our results, we propose a central role for the viral pVII nucleoprotein architecture, which is required for the dynamic structural changes during early infection, including the regulation of nucleosome assembly prior to transcription initiation. Our study thus may aid the rational development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy.
Assuntos
Cromatina , Nucleossomos , Nucleossomos/genética , Ativação Transcricional , Cromatina/genética , DNA/metabolismo , Montagem e Desmontagem da Cromatina , Adenoviridae/genéticaRESUMO
Live imaging of translation based on tag recognition by a single chain antibody is a powerful technique to assess translation regulation in living cells. However, especially in a multicellular organism, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the living Drosophila embryo and in mammalian cells. We tested and characterized five different Green Fluorescent Protein variants fused to the single chain fragment variable (scFv) and uncover photobleaching, aggregation and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. We introduced a new translation imaging method based on a nanobody/tag system named ALFA-array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species. Finally, we developed a largely improved RNA imaging system based on an MCP-tdStaygold fusion.
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/genéticaRESUMO
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/metabolismoRESUMO
Genome-wide chromosome conformation capture (Hi-C) has revealed the organization of chromatin into topologically associating domains (TADs) and loops, which are thought to help regulate genome functions. TADs and loops are understood as the result of DNA extrusion mediated by the cohesin complex. However, despite recent efforts, direct visualization and quantification of this process in single cells remains an open challenge. Here, we use polymer simulations and dedicated analysis methods to explore if, and under which conditions, DNA loop extrusion can be detected and quantitatively characterized by imaging pairs of fluorescently labeled loci located near loop or TAD anchors in fixed or living cells. We find that under realistic conditions, extrusion can be detected and the frequency of loop formation can be quantified from fixed cell images alone, while the lifetime of loops and the speed of extrusion can be estimated from dynamic live-cell data. Our delineation of appropriate imaging conditions and the proposed analytical methods lay the groundwork for a systematic quantitative characterization of loop extrusion in fixed or living cells.
Assuntos
Proteínas Cromossômicas não Histona , Polímeros , Proteínas Cromossômicas não Histona/genética , Cromatina , Cromossomos , DNA , Proteínas de Ciclo Celular/genéticaRESUMO
Monitoring transcription in living cells gives access to the dynamics of this complex fundamental process. It reveals that transcription is discontinuous, whereby active periods (bursts) are separated by one or several types of inactive periods of distinct lifetimes. However, decoding temporal fluctuations arising from live imaging and inferring the distinct transcriptional steps eliciting them is a challenge. We present BurstDECONV, a novel statistical inference method that deconvolves signal traces into individual transcription initiation events. We use the distribution of waiting times between successive polymerase initiation events to identify mechanistic features of transcription such as the number of rate-limiting steps and their kinetics. Comparison of our method to alternative methods emphasizes its advantages in terms of precision and flexibility. Unique features such as the direct determination of the number of promoter states and the simultaneous analysis of several potential transcription models make BurstDECONV an ideal analytic framework for live cell transcription imaging experiments. Using simulated realistic data, we found that our method is robust with regards to noise or suboptimal experimental designs. To show its generality, we applied it to different biological contexts such as Drosophila embryos or human cells.
Assuntos
Drosophila , Transcrição Gênica , Animais , Humanos , Drosophila/genética , Regiões Promotoras GenéticasRESUMO
The conserved H/ACA RNPs consist of one H/ACA RNA and 4 core proteins: dyskerin, NHP2, NOP10, and GAR1. Its assembly requires several assembly factors. A pre-particle containing the nascent RNAs, dyskerin, NOP10, NHP2 and NAF1 is assembled co-transcriptionally. NAF1 is later replaced by GAR1 to form mature RNPs. In this study, we explore the mechanism leading to the assembly of H/ACA RNPs. We performed the analysis of GAR1, NHP2, SHQ1 and NAF1 proteomes by quantitative SILAC proteomic, and analyzed purified complexes containing these proteins by sedimentation on glycerol gradient. We propose the formation of several distinct intermediate complexes during H/ACA RNP assembly, notably the formation of early protein-only complexes containing at least the core proteins dyskerin, NOP10, and NHP2, and the assembly factors SHQ1 and NAF1. We also identified new proteins associated with GAR1, NHP2, SHQ1 and NAF1, which can be important for box H/ACA assembly or function. Moreover, even though GAR1 is regulated by methylations, the nature, localization, and functions of these methylations are not well known. Our MS analysis of purified GAR1 revealed new sites of arginine methylations. Additionally, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs, even though with less efficiency than methylated ones.
Assuntos
Proteômica , Ribonucleoproteínas , Ribonucleoproteínas/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Ligação a RNA , RNA/genéticaRESUMO
Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule RNA fluorescence in situ hybridization (smFISH) is a widely used single-cell-single-molecule imaging technique enabling quantitative studies of gene expression and its regulatory mechanisms. Today, these methods are applicable at a large scale, which in turn come with a need for adequate tools for data analysis and exploration. Here, we present FISH-quant v2, a highly modular tool accessible for both experts and non-experts. Our user-friendly package allows the user to segment nuclei and cells, detect isolated RNAs, decompose dense RNA clusters, quantify RNA localization patterns and visualize these results both at the single-cell level and variations within the cell population. This tool was validated and applied on large-scale smFISH image data sets, revealing diverse subcellular RNA localization patterns and a surprisingly high degree of cell-to-cell heterogeneity.
Assuntos
RNA , Imagem Individual de Molécula , Hibridização in Situ Fluorescente/métodos , Nanotecnologia , RNA/análise , RNA/genética , RNA Mensageiro/genética , Imagem Individual de Molécula/métodosRESUMO
DNA damage induces apoptosis and many apoptotic genes are regulated via alternative splicing (AS), but little is known about the control mechanisms. Here we show that ultraviolet irradiation (UV) affects cotranscriptional AS in a p53-independent way, through the hyperphosphorylation of RNA polymerase II carboxy-terminal domain (CTD) and a subsequent inhibition of transcriptional elongation, estimated in vivo and in real time. Phosphomimetic CTD mutants not only display lower elongation but also duplicate the UV effect on AS. Consistently, nonphosphorylatable mutants prevent the UV effect. Apoptosis promoted by UV in cells lacking p53 is prevented when the change in AS of the apoptotic gene bcl-x is reverted, confirming the relevance of this mechanism. Splicing-sensitive microarrays revealed a significant overlap of the subsets of genes that have changed AS with UV and those that have reduced expression, suggesting that transcriptional coupling to AS is a key feature of the DNA-damage response.
Assuntos
Processamento Alternativo/efeitos da radiação , RNA Polimerase II/metabolismo , Raios Ultravioleta , Apoptose , Linhagem Celular Tumoral , Dano ao DNA , Diclororribofuranosilbenzimidazol/farmacologia , Fibronectinas/genética , Fibronectinas/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Humanos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , RNA Polimerase II/química , Transcrição GênicaRESUMO
MicroRNAs silence mRNAs by guiding the RISC complex. RISC assembly occurs following cleavage of pre-miRNAs by Dicer, assisted by TRBP or PACT, and the transfer of miRNAs to AGO proteins. The R2TP complex is an HSP90 co-chaperone involved in the assembly of ribonucleoprotein particles. Here, we show that the R2TP component RPAP3 binds TRBP but not PACT. The RPAP3-TPR1 domain interacts with the TRBP-dsRBD3, and the 1.5 Å resolution crystal structure of this complex identifies key residues involved in the interaction. Remarkably, binding of TRBP to RPAP3 or Dicer is mutually exclusive. Additionally, we found that AGO(1/2), TRBP and Dicer are all sensitive to HSP90 inhibition, and that TRBP sensitivity is increased in the absence of RPAP3. Finally, RPAP3 seems to impede miRNA activity, raising the possibility that the R2TP chaperone might sequester TRBP to regulate the miRNA pathway.
Assuntos
MicroRNAs , Complexo de Inativação Induzido por RNA , Inativação Gênica , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Coativadores de Receptor Nuclear/química , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
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.
Assuntos
Vesículas Extracelulares , MicroRNAs , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , RNA Helicases DEAD-box , Endocitose , Vesículas Extracelulares/metabolismo , Células HeLa , Humanos , MicroRNAs/genética , Ribonuclease IIIRESUMO
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.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Extensões da Superfície Celular/metabolismo , Citoplasma/metabolismo , Cinesinas/metabolismo , Transporte de RNA , RNA Mensageiro/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Células HeLa , Humanos , Cinesinas/genética , RNA Mensageiro/genéticaRESUMO
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/metabolismoRESUMO
The Rvb1-Rvb2-Tah1-Pih1/prefoldin-like (R2TP/PFDL) complex is a unique chaperone that provides a platform for the assembly and maturation of many key multiprotein complexes in mammalian cells. Here, we propose to rename R2TP/PFDL as PAQosome (particle for arrangement of quaternary structure) to more accurately represent its unique function.
Assuntos
Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Estrutura Quaternária de Proteína , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Humanos , Complexos Multiproteicos/biossínteseRESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Biossíntese de Proteínas , Transporte de RNA , RNA Mensageiro/metabolismo , Transcrição Gênica , Animais , Núcleo Celular/genética , Citoplasma/genética , Humanos , RNA Mensageiro/genéticaRESUMO
In S. cerevisiae cells debilitated in mRNA nuclear export, transcripts are retained in nuclear foci ("dots"). The ultimate fate of dot-mRNA has remained elusive. Here, we use single molecule counting microscopy and (35)S-methionine pulse-labeling assays to quantify cytoplasmic HSP104 RNA levels and estimate HSP104 RNA translation status. HSP104 transcripts, retained in dots as a consequence of the mex67-5 mutation, are slowly released over time for cytoplasmic translation. Thus, dot-mRNA retains function. However, forcing its nuclear export, by overexpressing the Sub2p mRNA export factor, does not elevate Hsp104p protein levels but is instead paralleled by growth deficiency. Nuclear export and growth phenotypes are both counteracted by coexpressing the nuclear RNA quality control factor Rrp6p. Thus, prematurely released dot-mRNA is translationally inactive and possibly toxic. Accordingly, nuclear retention of mRNA may serve a precautionary role during stressful situations such as, e.g., decreased mRNA maturation competence.
Assuntos
RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Genes Fúngicos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Mutação , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Termination of transcription is important for establishing gene punctuation marks. It is also critical for suppressing many of the pervasive transcription events occurring throughout eukaryotic genomes and coupling their RNA products to efficient decay. In human cells, the ARS2 protein has been implicated in such function as its depletion causes transcriptional read-through of selected gene terminators and because it physically interacts with the ribonucleolytic nuclear RNA exosome. Here, we study the role of ARS2 on transcription and RNA metabolism genome wide. We show that ARS2 depletion negatively impacts levels of promoter-proximal RNA polymerase II at protein-coding (pc) genes. Moreover, our results reveal a general role of ARS2 in transcription termination-coupled RNA turnover at short transcription units like snRNA-, replication-dependent histone-, promoter upstream transcript- and enhancer RNA-loci. Depletion of the ARS2 interaction partner ZC3H18 mimics the ARS2 depletion, although to a milder extent, whereas depletion of the exosome core subunit RRP40 only impacts RNA abundance post-transcriptionally. Interestingly, ARS2 is also involved in transcription termination events within first introns of pc genes. Our work therefore establishes ARS2 as a general suppressor of pervasive transcription with the potential to regulate pc gene expression.
Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Nucleares/fisiologia , RNA Polimerase II/metabolismo , Terminação da Transcrição Genética , Imunoprecipitação da Cromatina , Complexo Multienzimático de Ribonucleases do Exossomo/fisiologia , Células HeLa , Humanos , Íntrons , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA/fisiologiaRESUMO
Coupling between transcription and RNA processing is key for gene regulation. Using live-cell photobleaching techniques, we investigated the factor TCERG1, which coordinates transcriptional elongation with splicing. We demonstrate that TCERG1 is highly mobile in the nucleoplasm and that this mobility is slightly decreased when it is associated with speckles. Dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) but not α-amanitin treatment reduced the mobility of TCERG1, which suggests interaction with paused transcription elongation complexes. We found that TCERG1 mobility is rapid at the transcription site (TS) of a reporter that splices post-transcriptionally and that TCERG1 is recruited to the active TS independent of the CTD of RNAPII, thus excluding phosphorylated CTD as a requirement for recruiting this factor to the TS. Importantly, the mobility of TCERG1 is reduced when the reporter splices cotranscriptionally, which suggests that TCERG1 forms new macromolecular complexes when splicing occurs cotranscriptionally. In this condition, spliceostatin A has no effect, indicating that TCERG1 rapidly binds and dissociates from stalled spliceosomal complexes and that the mobility properties of TCERG1 do not depend on events occurring after the initial spliceosome formation. Taken together, these data suggest that TCERG1 binds independently to elongation and splicing complexes, thus performing their coupling by transient interactions rather than by stable association with one or the other complexes. This finding has conceptual implications for understanding the coupling between transcription and RNA processing.
Assuntos
Splicing de RNA , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição/fisiologia , Núcleo Celular/metabolismo , Genes Reporter , Células HEK293 , HIV-1/genética , Humanos , Transporte ProteicoRESUMO
Transcription and mRNA maturation are interdependent events. Although stimulatory connections between these processes within the same round of transcription are well described, functional coupling between separate transcription cycles remains elusive. Comparing time-resolved transcription profiles of single-copy integrated ß-globin gene variants, we demonstrate that a polyadenylation site mutation decreases transcription initiation of the same gene. Upon depletion of the 3' end processing and transcription termination factor PCF11, endogenous genes exhibit a similar phenotype. Readthrough RNA polymerase II (RNAPII) engaged on polyadenylation site-mutated transcription units sequester the transcription initiation/elongation factors TBP, TFIIB and CDK9, leading to their depletion at the promoter. Additionally, high levels of TBP and TFIIB appear inside the gene body, and Ser2-phosphorylated RNAPII accumulates at the promoter. Our data demonstrate that 3' end formation stimulates transcription initiation and suggest that coordinated recycling of factors from a gene terminator back to the promoter is essential for sustaining continued transcription.
Assuntos
Processamento de Terminações 3' de RNA/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Sequência de Bases , Quinase 9 Dependente de Ciclina/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Fenótipo , Fosforilação , Fosfosserina/metabolismo , Mutação Puntual/genética , Poli A/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Splicing de RNA/genética , RNA Mensageiro/genética , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Tempo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Posttranslational SUMO modification is an important mechanism of regulating protein function, especially in the cell nucleus. The nucleolus is the subnuclear organelle responsible for rRNA synthesis, processing, and assembly of the large and small ribosome subunits. Here, we have used SILAC-based quantitative proteomics to identify nucleolar SUMOylated proteins. This reveals a role for SUMOylation in the biogenesis and/or function of small nucleolar ribonucleoprotein complexes (snoRNPs) via the targeting of Nhp2 and Nop58. Using combined in vitro and in vivo approaches, both Nhp2 and Nop58 (also known as Nop5) are shown to be substrates for SUMOylation. Mutational analyses revealed the sites of modification on Nhp2 as K5, and on Nop58 as K467 and K497. Unlike Nop58 and Nhp2, the closely related Nop56 and 15.5K proteins appear not to be SUMO targets. SUMOylation is essential for high-affinity Nop58 binding to snoRNAs. This study provides direct evidence linking SUMO modification with snoRNP function.