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1.
bioRxiv ; 2024 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-39314300

RESUMO

SAF-A is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. SAF-A has several functional domains, including an N-terminal SAP domain that binds directly to DNA. Phosphorylation of SAP domain serines S14 and S26 are important for SAF-A localization and function during mitosis, however whether these serines are involved in interphase functions of SAF-A is not known. In this study we tested for the role of the SAP domain, and SAP domain serines S14 and S26 in X chromosome inactivation, protein dynamics, gene expression, splicing, and cell proliferation. Here we show that the SAP domain serines S14 and S26 are required to maintain XIST RNA localization and polycomb-dependent histone modifications on the inactive X chromosome in female cells. In addition, we present evidence that an Xi localization signal resides in the SAP domain. We found that that the SAP domain is not required to maintain gene expression and plays only a minor role in mRNA splicing. In contrast, the SAF-A SAP domain, in particular serines S14 and S26, are required for normal protein dynamics, and to maintain normal cell proliferation. We propose a model whereby dynamic phosphorylation of SAF-A serines S14 and S26 mediates rapid turnover of SAF-A interactions with DNA during interphase.

2.
Mol Biol Cell ; 34(4): ar32, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36790906

RESUMO

Mitosis results in a dramatic reorganization of chromatin structure to promote chromosome compaction and segregation to daughter cells. Consequently, mitotic entry is accompanied by transcriptional silencing and removal of most chromatin-bound RNA from chromosomes. As cells exit mitosis, chromatin rapidly decondenses and transcription restarts as waves of differential gene expression. However, little is known about the fate of chromatin-bound RNAs following cell division. Here we explored whether nuclear RNA from the previous cell cycle is present in G1 nuclei following mitosis. We found that half of all nuclear RNA is inherited in a transcription-independent manner following mitosis. Interestingly, the snRNA U2 is efficiently inherited by G1 nuclei, while the lncRNAs NEAT1 and MALAT1 show no inheritance following mitosis. We found that the nuclear protein SAF-A, which is hypothesized to tether RNA to DNA, did not play a prominent role in nuclear RNA inheritance, indicating that the mechanism for RNA inheritance may not involve RNA chaperones that have chromatin-binding activity. Instead, we observe that the timing of RNA inheritance indicates that a select group of nuclear RNAs are reimported into the nucleus after the nuclear envelope has reassembled. Our work demonstrates that there is a fraction of nuclear RNA from the previous cell cycle that is reimported following mitosis and suggests that mitosis may serve as a time to reset the interaction of lncRNAs with chromatin.


Assuntos
RNA Longo não Codificante , RNA Nuclear , Transporte Ativo do Núcleo Celular , RNA Nuclear/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Mitose , Cromatina
3.
Trends Cell Biol ; 31(9): 760-773, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33766521

RESUMO

Beyond its originally discovered role tethering replicated sister chromatids, cohesin has emerged as a master regulator of gene expression. Recent advances in chromatin topology resolution and single-cell studies have revealed that cohesin has a pivotal role regulating highly dynamic chromatin interactions linked to transcription control. The dynamic association of cohesin with chromatin and its capacity to perform loop extrusion contribute to the heterogeneity of chromatin contacts. Additionally, different cohesin subcomplexes, with specific properties and regulation, control gene expression across the cell cycle and during developmental cell commitment. Here, we discuss the most recent literature in the field to highlight the role of cohesin in gene expression regulation during transcriptional shifts and its relationship with human diseases.


Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ciclo Celular/genética , Cromátides , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Expressão Gênica , Humanos , Coesinas
4.
J Biol Chem ; 296: 100202, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33334895

RESUMO

Elongin A (EloA) is an essential transcription factor that stimulates the rate of RNA polymerase II (Pol II) transcription elongation in vitro. However, its role as a transcription factor in vivo has remained underexplored. Here we show that in mouse embryonic stem cells, EloA localizes to both thousands of Pol II transcribed genes with preference for transcription start site and promoter regions and a large number of active enhancers across the genome. EloA deletion results in accumulation of transcripts from a subset of enhancers and their adjacent genes. Notably, EloA does not substantially enhance the elongation rate of Pol II in vivo. We also show that EloA localizes to the nucleoli and associates with RNA polymerase I transcribed ribosomal RNA gene, Rn45s. EloA is a highly disordered protein, which we demonstrate forms phase-separated condensates in vitro, and truncation mutations in the intrinsically disordered regions (IDR) of EloA interfere with its targeting and localization to the nucleoli. We conclude that EloA broadly associates with transcribed regions, tunes RNA Pol II transcription levels via impacts on enhancer RNA synthesis, and interacts with the rRNA producing/processing machinery in the nucleolus. Our work opens new avenues for further investigation of the role of this functionally multifaceted transcription factor in enhancer and ribosomal RNA biology.


Assuntos
Elonguina/metabolismo , Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas/metabolismo , RNA/genética , Ativação Transcricional , Animais , Linhagem Celular , Elonguina/genética , Deleção de Genes , Camundongos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Sítio de Iniciação de Transcrição
5.
J Cell Biol ; 219(11)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33053167

RESUMO

During mitosis, the genome is transformed from a decondensed, transcriptionally active state to a highly condensed, transcriptionally inactive state. Mitotic chromosome reorganization is marked by the general attenuation of transcription on chromosome arms, yet how the cell regulates nuclear and chromatin-associated RNAs after chromosome condensation and nuclear envelope breakdown is unknown. SAF-A/hnRNPU is an abundant nuclear protein with RNA-to-DNA tethering activity, coordinated by two spatially distinct nucleic acid-binding domains. Here we show that RNA is evicted from prophase chromosomes through Aurora-B-dependent phosphorylation of the SAF-A DNA-binding domain; failure to execute this pathway leads to accumulation of SAF-A-RNA complexes on mitotic chromosomes, defects in metaphase chromosome alignment, and elevated rates of chromosome missegregation in anaphase. This work reveals a role for Aurora-B in removing chromatin-associated RNAs during prophase and demonstrates that Aurora-B-dependent relocalization of SAF-A during cell division contributes to the fidelity of chromosome segregation.


Assuntos
Aurora Quinase B/metabolismo , Núcleo Celular/genética , Cromatina/química , Cromossomos Humanos/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Mitose , RNA/metabolismo , Aurora Quinase B/genética , Cromatina/genética , Cromossomos Humanos/genética , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Humanos , Fosforilação , RNA/genética
6.
Mol Cell ; 78(1): 127-140.e7, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32035037

RESUMO

As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA polymerase II (Pol II) in mitosis. Here, we demonstrate that chromatin-bound cohesin is necessary to retain elongating Pol II at centromeres. We find that WAPL-mediated removal of cohesin from chromosome arms during prophase is required for the dissociation of Pol II and nascent transcripts, and failure of this process dramatically alters mitotic gene expression. Removal of cohesin/Pol II from chromosome arms in prophase is important for accurate chromosome segregation and normal activation of gene expression in G1. We propose that prophase cohesin removal is a key step in reprogramming gene expression as cells transition from G2 through mitosis to G1.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Regulação da Expressão Gênica , Mitose/genética , Transcrição Gênica , Anáfase/genética , Animais , Aurora Quinase B/análise , Ciclo Celular , Proteínas de Ciclo Celular/análise , Linhagem Celular , Centrômero/enzimologia , Segregação de Cromossomos , Fase G1/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Metáfase/genética , Prófase , RNA Polimerase II/metabolismo , Xenopus laevis , Coesinas
7.
RNA ; 26(3): 324-344, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31896558

RESUMO

Most cells change patterns of gene expression through transcriptional regulation. In contrast, oocytes are transcriptionally silent and regulate mRNA poly(A) tail length to control protein production. However, the genome-wide relationship of poly(A) tail changes to mRNA translation during vertebrate oocyte maturation is not known. We used Tail-seq and polyribosome analysis to measure poly(A) tail and translational changes during oocyte maturation in Xenopus laevis We identified large-scale poly(A) and translational changes during oocyte maturation, with poly(A) tail length changes preceding translational changes. Proteins important for completion of the meiotic divisions and early development exhibited increased polyadenylation and translation during oocyte maturation. A family of U-rich sequence elements was enriched near the polyadenylation signal of polyadenylated and translationally activated mRNAs. We propose that changes in mRNA polyadenylation are a conserved mechanism regulating protein expression during vertebrate oocyte maturation and that these changes are controlled by a spatial code of cis-acting sequence elements.


Assuntos
Oogênese/genética , Poliadenilação/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento
8.
J Biol Chem ; 293(32): 12593-12605, 2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-29903915

RESUMO

RNA-binding proteins (RBP) are critical regulators of gene expression. Recent studies have uncovered hundreds of mRNA-binding proteins that do not contain annotated RNA-binding domains and have well-established roles in other cellular processes. Investigation of these nonconventional RBPs is critical for revealing novel RNA-binding domains and may disclose connections between RNA regulation and other aspects of cell biology. The endosomal sorting complex required for transport II (ESCRT-II) is a nonconventional RNA-binding complex that has a canonical role in multivesicular body formation. ESCRT-II was identified previously as an RNA-binding complex in Drosophila oocytes, but whether its RNA-binding properties extend beyond Drosophila is unknown. In this study, we found that the RNA-binding properties of ESCRT-II are conserved in Xenopus eggs, where ESCRT-II interacted with hundreds of mRNAs. Using a UV cross-linking approach, we demonstrated that ESCRT-II binds directly to RNA through its subunit, Vps25. UV cross-linking and immunoprecipitation (CLIP)-Seq revealed that Vps25 specifically recognizes a polypurine (i.e. GA-rich) motif in RNA. Using purified components, we could reconstitute the selective Vps25-mediated binding of the polypurine motif in vitro Our results provide insight into the mechanism by which ESCRT-II selectively binds to mRNA and also suggest an unexpected link between endosome biology and RNA regulation.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Óvulo/metabolismo , Purinas/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Xenopus laevis/metabolismo , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Feminino , Subunidades Proteicas , Purinas/química , RNA/química , RNA/genética , Proteínas de Ligação a RNA/genética , Xenopus laevis/genética
9.
Mol Cell Biol ; 38(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29941491

RESUMO

Accurate chromosome segregation is a fundamental process in cell biology. During mitosis, chromosomes are segregated into daughter cells through interactions between centromeres and microtubules in the mitotic spindle. Centromere domains have evolved to nucleate formation of the kinetochore, which is essential for establishing connections between chromosomal DNA and microtubules during mitosis. Centromeres are typically formed on highly repetitive DNA that is not conserved in sequence or size among organisms and can differ substantially between individuals within the same organism. However, transcription of repetitive DNA has emerged as a highly conserved property of the centromere. Recent work has shown that both the topological effect of transcription on chromatin and the nascent noncoding RNAs contribute to multiple aspects of centromere function. In this review, we discuss the fundamental aspects of centromere transcription, i.e., its dual role in chromatin remodeling/CENP-A deposition and kinetochore assembly during mitosis, from a cell cycle perspective.


Assuntos
Centrômero/genética , Centrômero/metabolismo , Transcrição Gênica , Animais , Aurora Quinase B/metabolismo , Proteína Centromérica A/metabolismo , Montagem e Desmontagem da Cromatina , Segregação de Cromossomos , DNA/genética , DNA/metabolismo , Humanos , Cinetocoros/metabolismo , Mitose , Modelos Genéticos , RNA Polimerase II/metabolismo , RNA Nuclear/genética , RNA Nuclear/metabolismo
10.
Dev Cell ; 42(3): 201-202, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28787584

RESUMO

Centromeric transcription is a common eukaryotic centromere feature, yet it is unclear how transcription is linked to underlying repetitive satellite sequences. In this issue of Developmental Cell, McNulty et al. (2017) show for human centromeres that all α-satellite sequences are transcribed into chromatin-bound RNAs and are required for centromere assembly.


Assuntos
Centrômero , Cromatina , DNA Satélite , Humanos , Sequências Repetitivas de Ácido Nucleico
11.
Elife ; 62017 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-28346135

RESUMO

The synaptonemal complex (SC) is an ultrastructurally conserved proteinaceous structure that holds homologous chromosomes together and is required for the stabilization of pairing interactions and the completion of crossover (CO) formation between homologs during meiosis I. Here, we identify a novel role for a central region component of the SC, SYP-4, in negatively regulating formation of recombination-initiating double-strand breaks (DSBs) via a feedback loop triggered by crossover designation in C. elegans. We found that SYP-4 is phosphorylated dependent on Polo-like kinases PLK-1/2. SYP-4 phosphorylation depends on DSB formation and crossover designation, is required for stabilizing the SC in pachytene by switching the central region of the SC from a more dynamic to a less dynamic state, and negatively regulates DSB formation. We propose a model in which Polo-like kinases recognize crossover designation and phosphorylate SYP-4 thereby stabilizing the SC and making chromosomes less permissive for further DSB formation.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Quebras de DNA de Cadeia Dupla , Retroalimentação Fisiológica , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Fosforilação
12.
RNA ; 23(4): 504-520, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28031481

RESUMO

Piwi proteins utilize small RNAs (piRNAs) to recognize target transcripts such as transposable elements (TE). However, extensive piRNA sequence diversity also suggests that Piwi/piRNA complexes interact with many transcripts beyond TEs. To determine Piwi target RNAs, we used ribonucleoprotein-immunoprecipitation (RIP) and cross-linking and immunoprecipitation (CLIP) to identify thousands of transcripts associated with the Piwi proteins XIWI and XILI (Piwi-protein-associated transcripts, PATs) from early stage oocytes of X. laevis and X. tropicalis Most PATs associate with both XIWI and XILI and include transcripts of developmentally important proteins in oogenesis and embryogenesis. Only a minor fraction of PATs in both frog species displayed near perfect matches to piRNAs. Since predicting imperfect pairing between all piRNAs and target RNAs remains intractable, we instead determined that PAT read counts correlate well with the lengths and expression levels of transcripts, features that have also been observed for oocyte mRNAs associated with Drosophila Piwi proteins. We used an in vitro assay with exogenous RNA to confirm that XIWI associates with RNAs in a length- and concentration-dependent manner. In this assay, noncoding transcripts with many perfectly matched antisense piRNAs were unstable, whereas coding transcripts with matching piRNAs were stable, consistent with emerging evidence that Piwi proteins both promote the turnover of TEs and other RNAs, and may also regulate mRNA localization and translation. Our study suggests that Piwi proteins play multiple roles in germ cells and establishes a tractable vertebrate system to study the role of Piwi proteins in transcript regulation.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , RNA Interferente Pequeno/genética , Transcriptoma , Proteínas de Xenopus/genética , Xenopus/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Bioensaio , Elementos de DNA Transponíveis , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Oócitos/crescimento & desenvolvimento , Oogênese/genética , Filogenia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Xenopus/classificação , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo
13.
Open Biol ; 6(4): 150218, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27248654

RESUMO

Endocytosis and local protein synthesis (LPS) act coordinately to mediate the chemotropic responses of axons, but the link between these two processes is poorly understood. The endosomal sorting complex required for transport (ESCRT) is a key regulator of cargo sorting in the endocytic pathway, and here we have investigated the role of ESCRT-II, a critical ESCRT component, in Xenopus retinal ganglion cell (RGC) axons. We show that ESCRT-II is present in RGC axonal growth cones (GCs) where it co-localizes with endocytic vesicle GTPases and, unexpectedly, with the Netrin-1 receptor, deleted in colorectal cancer (DCC). ESCRT-II knockdown (KD) decreases endocytosis and, strikingly, reduces DCC in GCs and leads to axon growth and guidance defects. ESCRT-II-depleted axons fail to turn in response to a Netrin-1 gradient in vitro and many axons fail to exit the eye in vivo These defects, similar to Netrin-1/DCC loss-of-function phenotypes, can be rescued in whole (in vitro) or in part (in vivo) by expressing DCC. In addition, ESCRT-II KD impairs LPS in GCs and live imaging reveals that ESCRT-II transports mRNAs in axons. Collectively, our results show that the ESCRT-II-mediated endocytic pathway regulates both DCC and LPS in the axonal compartment and suggest that ESCRT-II aids gradient sensing in GCs by coupling endocytosis to LPS.


Assuntos
Axônios/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Biossíntese de Proteínas , Receptores de Superfície Celular/metabolismo , Retina/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Axônios/efeitos dos fármacos , Receptor DCC , Endocitose/efeitos dos fármacos , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/metabolismo , Fatores de Crescimento Neural/farmacologia , Netrina-1 , Fenótipo , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Proteínas Supressoras de Tumor/farmacologia , Proteínas de Xenopus/genética , Xenopus laevis
14.
Cell Rep ; 15(8): 1624-33, 2016 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-27184843

RESUMO

Centromeric transcription is widely conserved; however, it is not clear what role centromere transcription plays during mitosis. Here, I find that centromeres are transcribed in Xenopus egg extracts into a long noncoding RNA (lncRNA; cen-RNA) that localizes to mitotic centromeres, chromatin, and spindles. cen-RNAs bind to the chromosomal passenger complex (CPC) in vitro and in vivo. Blocking transcription or antisense inhibition of cen-RNA leads to a reduction of CPC localization to the inner centromere and misregulation of CPC component Aurora-B activation independently of known centromere recruitment pathways. Additionally, transcription is required for normal bipolar attachment of kinetochores to the mitotic spindle, consistent with a role for cen-RNA in CPC regulation. This work demonstrates that cen-RNAs promote normal kinetochore function through regulation of the localization and activation of the CPC and confirm that lncRNAs are components of the centromere.


Assuntos
Aurora Quinase B/metabolismo , Centrômero/genética , Transcrição Gênica , Animais , Extratos Celulares , Centrômero/metabolismo , Cromatina/metabolismo , Ativação Enzimática , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Óvulo/metabolismo , Transporte Proteico , RNA/metabolismo , RNA Antissenso/metabolismo , Xenopus laevis
15.
Methods Mol Biol ; 1413: 303-24, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27193857

RESUMO

RNAs associate with the mitotic spindle in a variety of organisms, where they can spatially regulate protein production, ensure their proper segregation during cell division, or perform translation-independent roles in spindle formation. The identification of spindle-associated RNAs is an important first step in understanding the biological consequences of this phenomenon. In this chapter, we describe a method to use Xenopus laevis egg extracts to assemble and isolate mitotic spindles and to identify the spindle-associated RNAs. The method described here can be used in combination with immunodepletions, the addition of inhibitors, or other perturbations to investigate factors that affect RNA localization to the spindle. Finally, we describe a method to assess the consequences of ablating RNA in the extract on spindle formation.


Assuntos
Mitose , RNA/genética , RNA/metabolismo , Fuso Acromático/metabolismo , Transcrição Gênica , Animais , Extratos Celulares , Biologia Computacional/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Microscopia de Fluorescência/métodos , Mitose/genética , Oócitos/metabolismo , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Ribonuclease Pancreático/metabolismo , Tubulina (Proteína)/metabolismo , Xenopus laevis
16.
Elife ; 5: e12039, 2016 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-26920220

RESUMO

Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Troca Genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Sistema de Sinalização das MAP Quinases , Meiose , Processamento de Proteína Pós-Traducional , Complexo Sinaptonêmico/metabolismo , Animais , Caenorhabditis elegans , Linhagem Celular , Proteína Quinase 1 Ativada por Mitógeno/metabolismo
17.
Cell Mol Life Sci ; 73(1): 79-94, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26433683

RESUMO

The endoplasmic reticulum (ER) is a large, dynamic structure that serves many roles in the cell including calcium storage, protein synthesis and lipid metabolism. The diverse functions of the ER are performed by distinct domains; consisting of tubules, sheets and the nuclear envelope. Several proteins that contribute to the overall architecture and dynamics of the ER have been identified, but many questions remain as to how the ER changes shape in response to cellular cues, cell type, cell cycle state and during development of the organism. Here we discuss what is known about the dynamics of the ER, what questions remain, and how coordinated responses add to the layers of regulation in this dynamic organelle.


Assuntos
Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Animais , Cálcio/metabolismo , Retículo Endoplasmático/química , Fertilização , Humanos , Metabolismo dos Lipídeos , Mitose , Biossíntese de Proteínas , Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Transdução de Sinais , Resposta a Proteínas não Dobradas
18.
RNA ; 21(2): 279-95, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25519486

RESUMO

ADAR (adenosine deaminase acting on RNA) is an RNA-editing enzyme present in most metazoans that converts adenosines in double-stranded RNA targets into inosines. Although the RNA targets of ADAR-mediated editing have been extensively cataloged, our understanding of the cellular function of such editing remains incomplete. We report that long, double-stranded RNA added to Xenopus laevis egg extract is incorporated into an ADAR-containing complex whose protein components resemble those of stress granules. This complex localizes to microtubules, as assayed by accumulation on meiotic spindles. We observe that the length of a double-stranded RNA influences its incorporation into the microtubule-localized complex. ADAR forms a similar complex with endogenous RNA, but the endogenous complex fails to localize to microtubules. In addition, we characterize the endogenous, ADAR-associated RNAs and discover that they are enriched for transcripts encoding transcriptional regulators, zinc-finger proteins, and components of the secretory pathway. Interestingly, association with ADAR correlates with previously reported translational repression in early embryonic development. This work demonstrates that ADAR is a component of two, distinct ribonucleoprotein complexes that contain different types of RNAs and exhibit diverse cellular localization patterns. Our findings offer new insight into the potential cellular functions of ADAR.


Assuntos
Adenosina Desaminase/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Oócitos/enzimologia , Transporte de RNA , Ribonucleoproteínas/metabolismo , Fuso Acromático/metabolismo , Xenopus laevis
19.
J Cell Biol ; 207(1): 41-57, 2014 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-25287301

RESUMO

How cells shape and remodel organelles in response to cellular signals is a poorly understood process. Using Xenopus laevis egg extract, we found that increases in cytosolic calcium lead to the activation of an endogenous ribonuclease, XendoU. A fraction of XendoU localizes to the endoplasmic reticulum (ER) and is required for nuclear envelope assembly and ER network formation in a catalysis-dependent manner. Using a purified vesicle fusion assay, we show that XendoU functions on the surface of ER membranes to promote RNA cleavage and ribonucleoprotein (RNP) removal. Additionally, RNA removal from the surface of vesicles by RNase treatment leads to increased ER network formation. Using human tissue culture cells, we found that hEndoU localizes to the ER, where it promotes the formation of ER tubules in a catalysis-dependent manner. Together, these results demonstrate that calcium-activated removal of RNA from membranes by XendoU promotes and refines ER remodeling and the formation of tubular ER.


Assuntos
Retículo Endoplasmático/metabolismo , Endorribonucleases/metabolismo , Membrana Nuclear/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Células HeLa , Humanos , Óvulo/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Ribonucleoproteínas/metabolismo , Transdução de Sinais/genética , Xenopus laevis/genética
20.
Proc Natl Acad Sci U S A ; 111(24): 8985-90, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24889638

RESUMO

The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits--mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)--and two soluble regulatory components--MICU1 and its paralog MICU2. The minimal components sufficient for in vivo uniporter activity are unknown. Here we consider Dictyostelium discoideum (Dd), a member of the Amoebazoa outgroup of Metazoa and Fungi, and show that it has a highly simplified uniporter machinery. We show that D. discoideum mitochondria exhibit membrane potential-dependent calcium uptake compatible with uniporter activity, and also that expression of DdMCU complements the mitochondrial calcium uptake defect in human cells lacking MCU or EMRE. Moreover, expression of DdMCU in yeast alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Having established yeast as an in vivo reconstitution system, we then reconstituted the human uniporter. We show that coexpression of MCU and EMRE is sufficient for uniporter activity, whereas expression of MCU alone is insufficient. Our work establishes yeast as a powerful in vivo reconstitution system for the uniporter. Using this system, we confirm that MCU is the pore-forming subunit, define the minimal genetic elements sufficient for metazoan and nonmetazoan uniporter activity, and provide valuable insight into the evolution of the uniporter machinery.


Assuntos
Canais de Cálcio/química , Cálcio/química , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Cálcio/metabolismo , Linhagem Celular , Dictyostelium , Técnicas Genéticas , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo
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