RESUMO
In oligodendrocytes and neurons genetic information is transmitted from the nucleus to dendrites in the form of RNA granules. Here we describe how transport of multiple different RNA molecules in individual granules is analogous to the process of multiplexing in telecommunications. In both cases multiple messages are combined into a composite signal for transmission on a single carrier. Multiplexing provides a mechanism to coordinate local expression of ensembles of genes in myelin in oligodendrocytes and at synapses in neurons.
Assuntos
Neurônios/metabolismo , Oligodendroglia/metabolismo , Transporte de RNA/fisiologia , RNA/metabolismo , Animais , Núcleo Celular/metabolismo , Dendritos/metabolismo , HumanosRESUMO
Tumor overexpressed gene (TOG) protein, encoded by cytoskeleton-associated protein CKAP5, is a microtubule-associated protein that binds to heterogeneous nuclear ribonucleoprotein (hnRNP) A2. hnRNP A2 is an RNA trafficking factor that associates with myelin basic protein (MBP) mRNA. In oligodendrocytes, TOG, hnRNP A2, and MBP mRNA colocalize in granules that assemble in the perikaryon and are transported to the peripheral network of processes that extends from it. MBP accumulates preferentially in the membrane of the medial and distal portions of these cellular processes. MBP expression was reduced when TOG level was lowered by short-hairpin (sh) RNA. The reduction in TOG did not affect overall cell morphology or the assembly, transport, localization, or number of MBP mRNA-containing granules. Reduced levels of TOG did not affect another oligodendrocyte-specific component, myelin oligodendrocyte glycoprotein, which is expressed at the same time as MBP but translated from mRNA localized in the cell body. Expression in a neural cell line of a green fluorescent protein (GFP)-MBP fusion protein derived from a construct containing GFP and the full-length cDNA for the rat 14 kDa MBP was reduced when TOG level was lowered by shRNA treatment. Expression of GFP, derived from GFP mRNA containing the hnRNP A2 binding element of MBP mRNA, was similarly reduced in cells with low TOG levels. These data indicate that TOG is necessary for efficient translation of MBP mRNA and suggest that this role is mediated by its interaction with hnRNP A2.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Básica da Mielina/metabolismo , Oligodendroglia/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/biossíntese , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Hibridização in Situ Fluorescente/métodos , Proteínas Associadas aos Microtúbulos/genética , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/farmacologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Tempo , Transfecção/métodosRESUMO
Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Elementos de Resposta/genética , Adulto , Animais , Biotina , Células Cultivadas , Reagentes de Ligações Cruzadas , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a DNA , Humanos , Imunoprecipitação , Masculino , Camundongos , Oligodendroglia/citologia , Ligação Proteica , Transporte Proteico , Transporte de RNA , Proteínas de Ligação a RNA , Ratos , Espectrometria de Fluorescência , Técnicas do Sistema de Duplo-HíbridoRESUMO
In neural cells, such as oligodendrocytes and neurons, transport of certain RNAs along microtubules is mediated by the cis-acting heterogeneous nuclear ribonucleoprotein A2 response element (A2RE) trafficking element and the cognate trans-acting heterogeneous nuclear ribonucleoprotein (hnRNP) A2 trafficking factor. Using a yeast two-hybrid screen, we have identified a microtubule-associated protein, tumor overexpressed gene (TOG)2, as an hnRNP A2 binding partner. The C-terminal third of TOG2 is sufficient for hnRNP A2 binding. TOG2, the large protein isoform of TOG, is the only isoform detected in oligodendrocytes in culture. TOG coimmunoprecipitates with hnRNP A2 present in the cytoskeleton (CSK) fraction of neural cells, and both coprecipitate with microtubule stabilized pellets. Staining with anti-TOG reveals puncta that are localized in proximity to microtubules, often at the plus ends. TOG is colocalized with hnRNP A2 and A2RE-mRNA in trafficking granules that remain associated with CSK-insoluble tissue. These data suggest that TOG mediates the association of hnRNP A2-positive granules with microtubules during transport and/or localization.
