RESUMO
Nuclear bodies including nucleoli, Cajal bodies, nuclear speckles, Polycomb bodies, and paraspeckles are membraneless subnuclear organelles. They are present at steady-state and dynamically respond to basic physiological processes as well as to various forms of stress, altered metabolic conditions and alterations in cellular signaling. The formation of a specific nuclear body has been suggested to follow a stochastic or ordered assembly model. In addition, a seeding mechanism has been proposed to assemble, maintain, and regulate particular nuclear bodies. In coordination with noncoding RNAs, chromatin modifiers and other machineries, various nuclear bodies have been shown to sequester and modify proteins, process RNAs and assemble ribonucleoprotein complexes, as well as epigenetically regulate gene expression. Understanding the functional relationships between the 3D organization of the genome and nuclear bodies is essential to fully uncover the regulation of gene expression and its implications for human disease.
Assuntos
Estruturas do Núcleo Celular/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/genética , Núcleo Celular/fisiologia , Estruturas do Núcleo Celular/fisiologia , Regulação da Expressão Gênica , Humanos , Proteínas Nucleares/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Sumoilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Dynamic changes in PM PIP(2) have been implicated in the regulation of many processes that are dependent on actin polymerization and remodeling. PIP(2) is synthesized primarily by the type I phosphatidylinositol 4 phosphate 5 kinases (PIP5Ks), and there are three major isoforms, called a, b and g. There is emerging evidence that these PIP5Ks have unique as well as overlapping functions. This review will focus on the isoform-specific roles of individual PIP5K as they relate to the regulation of the actin cytoskeleton. We will review recent advances that establish PIP(2) as a critical regulator of actin polymerization and cytoskeleton/membrane linkages, and show how binding of cytoskeletal proteins to membrane PIP(2) might alter lateral or transverse movement of lipids to affect raft formation or lipid asymmetry. The mechanisms for specifying localized increase in PIP(2) to regulate dynamic actin remodeling will also be discussed.
Assuntos
Citoesqueleto de Actina/metabolismo , Células Eucarióticas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Citoesqueleto de Actina/química , Processamento Alternativo , Animais , Cálcio/metabolismo , Células Eucarióticas/citologia , Regulação da Expressão Gênica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Multimerização Proteica , Transdução de SinaisRESUMO
The MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) gene encodes a noncoding RNA that is processed into a long nuclear retained transcript (MALAT1) and a small cytoplasmic tRNA-like transcript (mascRNA). Using an RNA sequence- and structure-based covariance model, we identified more than 130 genomic loci in vertebrate genomes containing the MALAT1 3' end triple-helix structure and its immediate downstream tRNA-like structure, including 44 in the green lizard Anolis carolinensis. Structural and computational analyses revealed a co-occurrence of components of the 3' end module. MALAT1-like genes in Anolis carolinensis are highly expressed in adult testis, thus we named them testis-abundant long noncoding RNAs (tancRNAs). MALAT1-like loci also produce multiple small RNA species, including PIWI-interacting RNAs (piRNAs), from the antisense strand. The 3' ends of tancRNAs serve as potential targets for the PIWI-piRNA complex. Thus, we have identified an evolutionarily conserved class of long noncoding RNAs (lncRNAs) with similar structural constraints, post-transcriptional processing, and subcellular localization and a distinct function in spermatocytes.
Assuntos
Loci Gênicos , Genoma Humano , RNA Longo não Codificante/genética , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Humanos , Lagartos/genética , Masculino , Conformação de Ácido Nucleico , Especificidade de Órgãos/genética , RNA Longo não Codificante/química , RNA Interferente Pequeno/genética , Espermatócitos/metabolismoRESUMO
Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs) lacking protein-coding capacity. However, most lncRNAs are expressed at a very low level, and in most cases there is no genetic evidence to support their in vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1) is among the most abundant and highly conserved lncRNAs, and it exhibits an uncommon 3'-end processing mechanism. In addition, its specific nuclear localization, developmental regulation, and dysregulation in cancer are suggestive of it having a critical biological function. We have characterized a Malat1 loss-of-function genetic model that indicates that Malat1 is not essential for mouse pre- and postnatal development. Furthermore, depletion of Malat1 does not affect global gene expression, splicing factor level and phosphorylation status, or alternative pre-mRNA splicing. However, among a small number of genes that were dysregulated in adult Malat1 knockout mice, many were Malat1 neighboring genes, thus indicating a potential cis-regulatory role of Malat1 gene transcription.
Assuntos
Crescimento e Desenvolvimento/genética , RNA Longo não Codificante/fisiologia , Sequências Reguladoras de Ácido Ribonucleico/fisiologia , Transcrição Gênica , Fatores Etários , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Biológicos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Sequências Reguladoras de Ácido Ribonucleico/genética , Transcrição Gênica/genéticaRESUMO
The cell nucleus is a highly compartmentalized organelle harbouring a variety of dynamic membraneless nuclear bodies. How these subnuclear domains are established and maintained is not well understood. Here, we investigate the molecular mechanism of how one nuclear body, the paraspeckle, is assembled and organized. Paraspeckles are discrete ribonucleoprotein bodies found in mammalian cells and implicated in nuclear retention of hyperedited mRNAs. We developed a live-cell imaging system that allows for the inducible transcription of Men É/ß (also known as Neat1; ref. 12) noncoding RNAs (ncRNAs) and the direct visualization of the recruitment of paraspeckle proteins. Using this system, we demonstrate that Men É/ß ncRNAs are essential to initiate the de novo assembly of paraspeckles. These newly formed structures effectively harbour nuclear-retained mRNAs confirming that they are bona fide functional paraspeckles. By three independent approaches, we show that it is the act of Men É/ß transcription, but not ncRNAs alone, that regulates paraspeckle maintenance. Finally, fluorescence recovery after photobleaching (FRAP) analyses supported a critical structural role for Men É/ß ncRNAs in paraspeckle organization. This study establishes a model in which Men É/ß ncRNAs serve as a platform to recruit proteins to assemble paraspeckles.
Assuntos
Núcleo Celular/metabolismo , Corpos de Inclusão Intranuclear/metabolismo , RNA não Traduzido/metabolismo , Transcrição Gênica , Animais , Linhagem Celular , Recuperação de Fluorescência Após Fotodegradação , Humanos , Hibridização in Situ Fluorescente/métodos , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Here we investigate the role of Phosphatidylinositol (4,5) bisphosphate (PIP(2)) in the physiological activation of primary murine T cells by antigen presenting cells (APC) by addressing two principal challenges in PIP(2) biology. First, PIP(2) is a regulator of cytoskeletal dynamics and a substrate for second messenger generation. The relative importance of these two processes needs to be determined. Second, PIP(2) is turned over by multiple biosynthetic and metabolizing enzymes. The joint effect of these enzymes on PIP(2) distributions needs to be determined with resolution in time and space. We found that T cells express four isoforms of the principal PIP(2)-generating enzyme phosphatidylinositol 4-phosphate 5-kinase (PIP5K) with distinct spatial and temporal characteristics. In the context of a larger systems analysis of T cell signaling, these data identify the T cell/APC interface and the T cell distal pole as sites of differential PIP(2) turnover. Overexpression of different PIP5K isoforms, as corroborated by knock down and PIP(2) blockade, yielded an increase in PIP(2) levels combined with isoform-specific changes in the spatiotemporal distributions of accessible PIP(2). It rigidified the T cell, likely by impairing the inactivation of Ezrin Moesin Radixin, delayed and diminished the clustering of the T cell receptor at the cellular interface, reduced the efficiency of T cell proximal signaling and IL-2 secretion. These effects were consistently more severe for distal PIP5K isoforms. Thus spatially constrained cytoskeletal roles of PIP(2) in the control of T cell rigidity and spatiotemporal organization dominate the effects of PIP(2) on T cell activation.
Assuntos
Ativação Linfocitária/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Linfócitos T/metabolismo , Actinas , Animais , Células Cultivadas , Interleucina-2/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 4,5-Difosfato/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has many essential functions and its homeostasis is highly regulated. We previously found that hypertonic stress increases PIP(2) by selectively activating the beta isoform of the type I phosphatidylinositol phosphate 5-kinase (PIP5Kbeta) through Ser/Thr dephosphorylation and promoting its translocation to the plasma membrane. Here we report that hydrogen peroxide (H(2)O(2)) also induces PIP5Kbeta Ser/Thr dephosphorylation, but it has the opposite effect on PIP(2) homeostasis, PIP5Kbeta function, and the actin cytoskeleton. Brief H(2)O(2) treatments decrease cellular PIP(2) in a PIP5Kbeta-dependent manner. PIP5Kbeta is tyrosine phosphorylated, dissociates from the plasma membrane, and has decreased lipid kinase activity. In contrast, the other two PIP5K isoforms are not inhibited by H(2)O(2). We identified spleen tyrosine kinase (Syk), which is activated by oxidants, as a candidate PIP5Kbeta kinase in this pathway, and mapped the oxidant-sensitive tyrosine phosphorylation site to residue 105. The PIP5KbetaY105E phosphomimetic is catalytically inactive and cytosolic, whereas the Y105F non-phosphorylatable mutant has higher intrinsic lipid kinase activity and is much more membrane associated than wild type PIP5Kbeta. These results suggest that during oxidative stress, as modeled by H(2)O(2) treatment, Syk-dependent tyrosine phosphorylation of PIP5Kbeta is the dominant post-translational modification that is responsible for the decrease in cellular PIP(2).
Assuntos
Regulação para Baixo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estresse Oxidativo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Tirosina Quinases/genética , Quinase SykRESUMO
Phosphatidylinositol 4-kinases play essential roles in cell signaling and membrane trafficking. They are divided into type II and III families, which have distinct structural and enzymatic properties and are essentially unrelated in sequence. Mammalian cells express two type II isoforms, phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) and IIbeta (PI4KIIbeta). Nearly all of PI4KIIalpha, and about half of PI4KIIbeta, associates integrally with membranes, requiring detergent for solubilization. This tight membrane association is because of palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domains of both type II isoforms. Deletion of this motif from PI4KIIalpha converts the kinase from an integral to a tightly bound peripheral membrane protein and abrogates its catalytic activity ( Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Sudhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708 ). Here we identify the first two cysteines in the CCPCC motif as the principal sites of palmitoylation under basal conditions, and we demonstrate the importance of the central proline for enzymatic activity, although not for membrane binding. We further show that palmitoylation is critical for targeting PI4KIIalpha to the trans-Golgi network and for enhancement of its association with low buoyant density membrane fractions, commonly termed lipid rafts. Replacement of the four cysteines in CCPCC with a hydrophobic residue, phenylalanine, substantially restores catalytic activity of PI4KIIalpha in vitro and in cells without restoring integral membrane binding. Although this FFPFF mutant displays a perinuclear distribution, it does not strongly co-localize with wild-type PI4KIIalpha and associates more weakly with lipid rafts.
Assuntos
1-Fosfatidilinositol 4-Quinase/química , Lipoilação , 1-Fosfatidilinositol 4-Quinase/metabolismo , Motivos de Aminoácidos , Animais , Células COS , Catálise , Membrana Celular/metabolismo , Chlorocebus aethiops , Insetos , Microdomínios da Membrana/química , Modelos Biológicos , Prolina/química , Ratos , Proteínas Recombinantes/química , Rede trans-Golgi/metabolismoRESUMO
The actin cytoskeleton is dynamically remodeled during Fcgamma receptor (FcgammaR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP(2))-dependent manner. We investigated the role of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) gamma and alpha isoforms, which synthesize PIP(2), during phagocytosis. PIP5K-gamma-/- bone marrow-derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcgammaR clustering. Delivery of exogenous PIP(2) rescued these defects. PIP5K-gamma knockout BMM also have more RhoA and less Rac1 activation, and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5K-gamma by RNA interference inhibits particle attachment. In contrast, PIP5K-alpha knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein activation, and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5K-gamma but not PIP5K-alpha is transiently activated by spleen tyrosine kinase-mediated phosphorylation. We propose that PIP5K-gamma acts upstream of Rac/Rho and that the differential regulation of PIP5K-gamma and -alpha allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.
Assuntos
Fagocitose , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de IgG/metabolismo , Actinas/metabolismo , Animais , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de IgG/genéticaRESUMO
Phosphatidylinositol (4,5)-bisphosphate (PIP(2)) is an important lipid mediator that has multiple regulatory functions. There is now increasing evidence that the phosphatidylinositol 4-phosphate 5 kinases (PIP5Ks), which synthesize PIP(2), are regulated spatially and temporally and that they have isoform-specific functions and regulations. This review will summarize the highlights of recent developments in understanding how the three major PIP5K isoforms regulate the actin cytoskeleton and other important cellular processes.