RESUMO
During Caenorhabditis elegans oocyte meiosis, a multi-protein ring complex (RC) localized between homologous chromosomes, promotes chromosome congression through the action of the chromokinesin KLP-19. While some RC components are known, the mechanism of RC assembly has remained obscure. We show that SUMO E3 ligase GEI-17/PIAS is required for KLP-19 recruitment to the RC, and proteomic analysis identified KLP-19 as a SUMO substrate in vivo. In vitro analysis revealed that KLP-19 is efficiently sumoylated in a GEI-17-dependent manner, while GEI-17 undergoes extensive auto-sumoylation. GEI-17 and another RC component, the kinase BUB-1, contain functional SUMO interaction motifs (SIMs), allowing them to recruit SUMO modified proteins, including KLP-19, into the RC. Thus, dynamic SUMO modification and the presence of SIMs in RC components generate a SUMO-SIM network that facilitates assembly of the RC. Our results highlight the importance of SUMO-SIM networks in regulating the assembly of dynamic protein complexes.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Posicionamento Cromossômico , Segregação de Cromossomos , Cinesinas/metabolismo , Ligases/metabolismo , Meiose , Oócitos/metabolismo , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Feminino , Genótipo , Cinesinas/genética , Ligases/genética , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Tempo , Ubiquitina-Proteína Ligases/genéticaRESUMO
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
Oocyte meiotic spindles in most species lack centrosomes and the mechanisms that underlie faithful chromosome segregation in acentrosomal meiotic spindles are not well understood. In C. elegans oocytes, spindle microtubules exert a poleward force on chromosomes that is dependent on the microtubule-stabilising protein CLS-2, the orthologue of the mammalian CLASP proteins. The checkpoint kinase BUB-1 and CLS-2 localise in the central spindle and display a dynamic localisation pattern throughout anaphase, but the signals regulating their anaphase-specific localisation remains unknown. We have shown previously that SUMO regulates BUB-1 localisation during metaphase I. Here, we found that SUMO modification of BUB-1 is regulated by the SUMO E3 ligase GEI-17 and the SUMO protease ULP-1. SUMO and GEI-17 are required for BUB-1 localisation between segregating chromosomes during early anaphase I. We also show that CLS-2 is subject to SUMO-mediated regulation; CLS-2 precociously localises in the midbivalent when either SUMO or GEI-17 are depleted. Overall, we provide evidence for a novel, SUMO-mediated control of protein dynamics during early anaphase I in oocytes.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Meiose , Oócitos/citologia , Oócitos/metabolismo , Sumoilação , Anáfase , Animais , Modelos Biológicos , Transporte Proteico , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fuso Acromático/metabolismoRESUMO
The roles of Argonaute proteins in cytoplasmic microRNA and RNAi pathways are well established. However, their implication in small RNA-mediated transcriptional gene silencing in the mammalian cell nucleus is less understood. We have recently shown that intronic siRNAs cause chromatin modifications that inhibit RNA polymerase II elongation and modulate alternative splicing in an Argonaute-1 (AGO1)-dependent manner. Here we used chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to investigate the genome-wide distribution of AGO1 nuclear targets. Unexpectedly, we found that about 80% of AGO1 clusters are associated with cell-type-specific transcriptional enhancers, most of them (73%) overlapping active enhancers. This association seems to be mediated by long, rather than short, enhancer RNAs and to be more prominent in intragenic, rather than intergenic, enhancers. Paradoxically, crossing ChIP-seq with RNA-seq data upon AGO1 depletion revealed that enhancer-bound AGO1 is not linked to the global regulation of gene transcription but to the control of constitutive and alternative splicing, which was confirmed by an individual gene analysis explaining how AGO1 controls inclusion levels of the cassette exon 107 in the SYNE2 gene.
Assuntos
Processamento Alternativo/fisiologia , Proteínas Argonautas/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica/fisiologia , RNA/metabolismo , Transcrição Gênica/fisiologia , Proteínas Argonautas/genética , Linhagem Celular , Fatores de Iniciação em Eucariotos/genética , Humanos , RNA/genética , Análise de Sequência de RNARESUMO
Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a nucleocytoplasmic shuttling protein that is a key player in the p53-triggered DNA damage response, acting as a cofactor for p53 in response to DNA damage. hnRNP K is a substrate of the ubiquitin E3 ligase MDM2 and, upon DNA damage, is de-ubiquitylated. In sharp contrast with the role and consequences of the other post-translational modifications, nothing is known about the role of SUMO conjugation to hnRNP K in p53 transcriptional co-activation. In the present work, we show that hnRNP K is modified by SUMO in lysine 422 within its KH3 domain, and sumoylation is regulated by the E3 ligase Pc2/CBX4. Most interestingly, DNA damage stimulates hnRNP K sumoylation through Pc2 E3 activity, and this modification is required for p53 transcriptional activation. Abrogation of hnRNP K sumoylation leads to an aberrant regulation of the p53 target gene p21. Our findings link the DNA damage-induced Pc2 activation to the p53 transcriptional co-activation through hnRNP K sumoylation.
Assuntos
Dano ao DNA , Ribonucleoproteínas/metabolismo , Proteína SUMO-1/metabolismo , Sumoilação , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Humanos , Ligases , Proteínas do Grupo Polycomb/biossíntese , Proteínas do Grupo Polycomb/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ribonucleoproteínas/genética , Proteína SUMO-1/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genéticaRESUMO
Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína SUMO-1/metabolismo , Linhagem Celular , Resposta ao Choque Térmico , Humanos , Proteínas Nucleares/genética , Ligação Proteica , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina , Especificidade por Substrato , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Phosphorylation is a key post-translational modification that is utilised in many biological processes for the rapid and reversible regulation of protein localisation and activity. Polo-like kinase 1 (PLK-1) is essential for both mitotic and meiotic cell divisions, with key functions being conserved in eukaryotes. The roles and regulation of PLK-1 during mitosis have been well characterised. However, the discrete roles and regulation of PLK-1 during meiosis have remained obscure. Here, we used Caenorhabditis elegans oocytes to show that PLK-1 plays distinct roles in meiotic spindle assembly and/or stability, chromosome alignment and segregation, and polar body extrusion during meiosis I. Furthermore, by a combination of live imaging and biochemical analysis we identified the chromosomal recruitment mechanisms of PLK-1 during C. elegans oocyte meiosis. The spindle assembly checkpoint kinase BUB-1 directly recruits PLK-1 to the kinetochore and midbivalent while the chromosome arm population of PLK-1 depends on a direct interaction with the centromeric-associated protein CENP-CHCP-4. We found that perturbing both BUB-1 and CENP-CHCP-4 recruitment of PLK-1 leads to severe meiotic defects, resulting in highly aneuploid oocytes. Overall, our results shed light on the roles played by PLK-1 during oocyte meiosis and provide a mechanistic understanding of PLK-1 targeting to meiotic chromosomes.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Cinetocoros , Meiose , Oócitos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Quinase 1 Polo-LikeRESUMO
Rac1b is an alternatively spliced isoform of the small GTPase Rac1 that includes the 57-nucleotide exon 3b. Rac1b was originally identified through its over-expression in breast and colorectal cancer cells, and has subsequently been implicated as a key player in a number of different oncogenic signaling pathways, including tumorigenic transformation of mammary epithelial cells exposed to matrix metalloproteinase-3 (MMP-3). Although many of the cellular consequences of Rac1b activity have been recently described, the molecular mechanism by which MMP-3 treatment leads to Rac1b induction has not been defined. Here we use proteomic methods to identify heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a factor involved in Rac1 splicing regulation. We find that hnRNP A1 binds to Rac1 exon 3b in mouse mammary epithelial cells, repressing its inclusion into mature mRNA. We also find that exposure of cells to MMP-3 leads to release of hnRNP A1 from exon 3b and the consequent generation of Rac1b. Finally, we analyze normal breast tissue and breast cancer biopsies, and identify an inverse correlation between expression of hnRNP A1 and Rac1b, suggesting the existence of this regulatory axis in vivo. These results provide new insights on how extracellular signals regulate alternative splicing, contributing to cellular transformation and development of breast cancer.
Assuntos
Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Neuropeptídeos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Células Epiteliais , Feminino , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Glândulas Mamárias Animais , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteômica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
Serine/arginine-rich (SR) proteins are among the most studied splicing regulators. They constitute a family of evolutionarily conserved proteins that, apart from their initially identified and deeply studied role in splicing regulation, have been implicated in genome stability, chromatin binding, transcription elongation, mRNA stability, mRNA export and mRNA translation. Remarkably, this list of SR protein activities seems far from complete, as unexpected functions keep being unraveled. An intriguing aspect that awaits further investigation is how the multiple tasks of SR proteins are concertedly regulated within mammalian cells. In this article, we first discuss recent findings regarding the regulation of SR protein expression, activity and accessibility. We dive into recent studies describing SR protein auto-regulatory feedback loops involving different molecular mechanisms such asunproductive splicing, microRNA-mediated regulation and translational repression. In addition, we take into account another step of regulation of SR proteins, presenting new findings about a variety of post-translational modifications by proteomics approaches and how some of these modifications can regulate SR protein sub-cellular localization or stability. Towards the end, we focus in two recently revealed functions of SR proteins beyond mRNA biogenesis and metabolism, the regulation of micro-RNA processing and the regulation of small ubiquitin-like modifier (SUMO) conjugation.
Assuntos
Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Sequência Conservada , Retroalimentação Fisiológica , Humanos , MicroRNAs , Proteínas Nucleares/química , Proteínas Nucleares/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismoRESUMO
In search for physiological pathways affecting alternative splicing through its kinetic coupling with transcription, we found that membrane depolarization of neuronal cells triggers the skipping of exon 18 from the neural cell adhesion molecule (NCAM) mRNA, independently of the calcium/calmodulin protein kinase IV pathway. We show that this exon responds to RNA polymerase II elongation, because its inclusion is increased by a slow polymerase II mutant. Depolarization affects the chromatin template in a specific way, by causing H3K9 hyper-acetylation restricted to an internal region of the NCAM gene surrounding the alternative exon. This intragenic histone hyper-acetylation is not paralleled by acetylation at the promoter, is associated with chromatin relaxation, and is linked to H3K36 tri-methylation. The effects on acetylation and splicing fully revert when the depolarizing conditions are withdrawn and can be both duplicated and potentiated by the histone deacetylase inhibitor trichostatin A. Our results are consistent with a mechanism involving the kinetic coupling of splicing and transcription in response to depolarization through intragenic epigenetic changes on a gene that is relevant for the differentiation and function of neuronal cells.
Assuntos
Processamento Alternativo , Cromatina/genética , Epigênese Genética , Potenciais da Membrana/fisiologia , Moléculas de Adesão de Célula Nervosa/genética , Neurônios/fisiologia , Acetilação , Animais , Éxons , Histonas/metabolismo , Neurônios/citologia , RNA Polimerase II/metabolismo , RatosRESUMO
It has been reported that expression of tumor necrosis factor superfamily members occur at the onset of the mammary gland post-lactational involution. One of these proteins, tumor necrosis factor alpha (TNFalpha), is a major mediator of inflammation that is able to induce expression of several cytokines. Leukemia inhibitory factor (LIF) is an inflammatory cytokine that is induced and plays a fundamental role during post-lactational involution of the mammary gland. Therefore, our goal was to determine whether TNFalpha activity in the mammary epithelium might include regulation of LIF expression. This biological role would increase the significance of TNFalpha expression at the end of lactation. Our results show that TNFalpha was able to induce LIF transcription through ERK1/2 activation in a non-tumorigenic mouse mammary epithelial cell line, SCp2. We found that activation of TNFalpha receptor-2 (TNFR2) was specifically involved in triggering this signaling pathway. In addition, our data suggest the participation of AP-1 transcription factor family members in this pathway. We determined that TNFalpha treatment induced c-fos transcription, and blocking AP-1 activity resulted in a significant inhibition of TNFalpha-induced LIF expression. Finally, we found that TNFalpha was also able to trigger LIF expression and ERK1/2 activation in the mouse mammary gland in vivo. Therefore, our data suggest that TNFalpha may contribute to mammary gland involution by, among other activities, eliciting LIF expression through ERK1/2 and AP1 activation.
Assuntos
Fator Inibidor de Leucemia/metabolismo , Glândulas Mamárias Humanas/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Western Blotting , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Ativação Enzimática , Humanos , Imuno-Histoquímica , Glândulas Mamárias Humanas/citologia , Camundongos , Camundongos Endogâmicos BALB C , Fator de Transcrição AP-1/metabolismoRESUMO
Serine/arginine-rich (SR) proteins are important regulators of mRNA splicing. Several postsplicing activities have been described for a subset of shuttling SR proteins, including regulation of mRNA export and translation. Using the fibronectin gene to study the links between signal-transduction pathways and SR protein activity, we show that growth factors not only modify the alternative splicing pattern of the fibronectin gene but also alter translation of reporter messenger RNAs in an SR protein-dependent fashion, providing two coregulated levels of isoform-specific amplification. These effects are inhibited by specific small interfering RNAs against SR proteins and are mediated by the AKT kinase, which elicits opposite effects to those evoked by overexpressing SR protein kinases Clk and SRPK. These results show how SR protein activity is modified in response to extracellular stimulation, leading to a concerted regulation of splicing and translation.
Assuntos
Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Biossíntese de Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Splicing de RNA , Sequência de Aminoácidos , Animais , Núcleo Celular/química , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Fibronectinas/genética , Substâncias de Crescimento/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/genética , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA , Fatores de Processamento de Serina-Arginina , Transdução de SinaisRESUMO
The role of the kinetochore during meiotic chromosome segregation in C. elegans oocytes has been a matter of controversy. Danlasky et al. (2020. J. Cell. Biol.https://doi.org/10.1083/jcb.202005179) show that kinetochore proteins KNL-1 and KNL-3 are required for early stages of anaphase during female meiosis, suggesting a new kinetochore-based model of chromosome segregation.
Assuntos
Proteínas de Caenorhabditis elegans , Segregação de Cromossomos , Anáfase , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Feminino , Cinetocoros , Meiose , Proteínas Associadas aos Microtúbulos/genética , OócitosRESUMO
The insulin/IGF signalling pathway impacts lifespan across distant taxa, by controlling the activity of nodal transcription factors. In the nematode Caenorhabditis elegans, the transcription regulators DAF-16/FOXO and SKN-1/Nrf function to promote longevity under conditions of low insulin/IGF signalling and stress. The activity and subcellular localization of both DAF-16 and SKN-1 is further modulated by specific posttranslational modifications, such as phosphorylation and ubiquitination. Here, we show that ageing elicits a marked increase of SUMO levels in C. elegans. In turn, SUMO fine-tunes DAF-16 and SKN-1 activity in specific C. elegans somatic tissues, to enhance stress resistance. SUMOylation of DAF-16 modulates mitochondrial homeostasis by interfering with mitochondrial dynamics and mitophagy. Our findings reveal that SUMO is an important determinant of lifespan, and provide novel insight, relevant to the complexity of the signalling mechanisms that influence gene expression to govern organismal survival in metazoans.
Assuntos
Envelhecimento/fisiologia , Caenorhabditis elegans/fisiologia , Mitocôndrias/metabolismo , Proteína SUMO-1/fisiologia , Envelhecimento/metabolismo , Animais , Western Blotting , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Clonagem Molecular , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Homeostase/fisiologia , Longevidade/fisiologia , Mitocôndrias/fisiologia , Consumo de Oxigênio , Proteína SUMO-1/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore-microtubule attachments in yeast and mouse meiosis. In Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Cromossomos/fisiologia , Meiose/fisiologia , Oócitos/fisiologia , Proteína Fosfatase 2/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Oócitos/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Post-splicing activities have been described for a subset of shuttling serine/arginine-rich splicing regulatory proteins, among them SF2/ASF. We showed that growth factors activate a Ras-PI 3-kinase-Akt/PKB signaling pathway that not only modifies alternative splicing of the fibronectin EDA exon, but also alters in vivo translation of reporter mRNAs containing the EDA binding motif for SF2/ASF, providing two co-regulated levels of isoform-specific amplification. Translation of most eukaryotic mRNAs is initiated via the scanning mechanism, which implicates recognition of the m7G cap at the mRNA 5'-terminus by the eIF4F protein complex. Several viral and cellular mRNAs are translated in a cap-independent manner by the action of cis-acting mRNA elements named internal ribosome entry sites that direct internal ribosome binding to the mRNA. Here we use bicistronic reporters that generate mRNAs carrying two open reading frames, one translated in a cap-dependent manner while the other by internal ribosome entry site-dependent initiation, to show that in vivo over-expression of SF2/ASF increases the ratio between cap-dependent and internal ribosome entry site-dependent translation. Consistently, knocking-down of SF2/ASF causes the opposite effect. Changes in expression levels of SF2/ASF also affect alternative translation of an endogenous mRNA, that one coding for fibroblast growth factor-2. These results strongly suggest a role for SF2/ASF as a regulator of alternative translation, meaning the generation of different proteins by the balance among these two translation initiation mechanisms, and expand the known potential of SF2/ASF to regulate proteomic diversity to the translation field.
Assuntos
Processamento Alternativo , Proteínas Nucleares/fisiologia , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas/genética , Proteoma/genética , Linhagem Celular , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Fases de Leitura Aberta , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA , Ribossomos/metabolismo , Fatores de Processamento de Serina-ArgininaRESUMO
Alternative splicing of messenger RNA precursors is an extraordinary source of protein diversity and the regulation of this process is crucial for diverse cellular functions in both physiological and pathological situations. For many years, several signaling pathways have been implicated in alternative splicing regulation. Recent work has begun to unravel the molecular mechanisms by which extracellular stimuli activate signaling cascades that modulate the activity of the splicing machinery and therefore the splicing pattern of many different target messenger RNA precursors. These experiments are revealing unexpected aspects of the mechanism that control splicing and the consequences of the regulated splicing events. We summarize here the current knowledge about signal-induced alternative splicing regulation of Slo, NR1, CD44, CD45 and fibronectin genes, and also discuss the importance of some of these events in determination of cellular fate. Furthermore, we highlight the relevance of signal-induced changes in phosphorylation state and subcellular distribution of splicing factors as a way of regulating the splicing process. Lastly, we explore new and unexpected findings about regulated splicing in anucleated cells.
Assuntos
Processamento Alternativo/genética , Transdução de Sinais , Animais , Núcleo Celular/genética , Humanos , Modelos Genéticos , Precursores de RNA , Frações Subcelulares/metabolismoRESUMO
The cell biology of sumoylation has mostly been studied using transformed cultured cells and yeast. In recent years, genetic analysis has demonstrated important roles for sumoylation in the biology of C. elegans. Here, we expand the existing set of tools making it possible to address the role of sumoylation in the nematode C. elegans using a combination of genetics, imaging, and biochemistry. Most importantly, the dynamics of SUMO conjugation and deconjugation can be followed very precisely both in space and time within living worms. Additionally, the biochemistry of SUMO conjugation and deconjugation can be addressed using recombinant purified components of the C. elegans sumoylation machinery, including E3 ligases and SUMO proteases. These tools and reagents will be useful to gain insights into the biological role of SUMO in the context of a multicellular organism.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cisteína Endopeptidases/metabolismo , Ligases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Imagem com Lapso de Tempo/métodos , Enzimas de Conjugação de Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Divisão Celular , Cromossomos/metabolismo , Cromossomos/ultraestrutura , Cisteína Endopeptidases/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Ligases/antagonistas & inibidores , Ligases/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genética , Proteína Vermelha FluorescenteRESUMO
The small ubiquitin-like modifier (SUMO), initially characterized as a suppressor of a mutation in the gene encoding the centromeric protein MIF2, is involved in many aspects of cell cycle regulation. The dynamics of conjugation and deconjugation and the role of SUMO during the cell cycle remain unexplored. Here we used Caenorhabditis elegans to establish the contribution of SUMO to a timely and accurate cell division. Chromatin-associated SUMO conjugates increase during metaphase but decrease rapidly during anaphase. Accumulation of SUMO conjugates on the metaphase plate and proper chromosome alignment depend on the SUMO E2 conjugating enzyme UBC-9 and SUMO E3 ligase PIAS(GEI-17). Deconjugation is achieved by the SUMO protease ULP-4 and is crucial for correct progression through the cell cycle. Moreover, ULP-4 is necessary for Aurora B(AIR-2) extraction from chromatin and relocation to the spindle mid-zone. Our results show that dynamic SUMO conjugation plays a role in cell cycle progression.