RESUMO
Ribonucleoprotein (RNP) granules are membraneless compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are localized as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here we demonstrate that RNPs containing vegetally localized RNAs represent a new class of cytoplasmic RNP granule, termed localization-bodies (L-bodies). We show that L-bodies contain a dynamic protein-containing phase surrounding a nondynamic RNA-containing phase. Our results support a role for RNA as a critical component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.
Assuntos
Condensados Biomoleculares/metabolismo , Grânulos Citoplasmáticos/metabolismo , Oócitos/metabolismo , RNA Mensageiro/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Transporte Biológico , Condensados Biomoleculares/química , Organelas/metabolismo , Ribonucleoproteínas/química , Xenopus laevisRESUMO
RNA localization in the Xenopus oocyte is responsible for the establishment of polarity during oogenesis as well as the specification of germ layers during embryogenesis. However, the inability to monitor mRNA localization in live vertebrate oocytes has posed a major barrier to understanding the mechanisms driving directional transport. Here we describe a method for imaging MS2 tagged RNA in live Xenopus oocytes to study the dynamics of RNA localization. We also focus on methods for implementing and analyzing FRAP data. This protocol is optimized for imaging of the RNAs in stage II oocytes but it can be adapted to study dynamics of other molecules during oogenesis. Using this approach, mobility can be measured in different regions of the oocyte, enabling the direct observation of molecular dynamics throughout the oocyte.
Assuntos
Recuperação de Fluorescência Após Fotodegradação/métodos , Oócitos/ultraestrutura , RNA Mensageiro/química , Imagem Individual de Molécula/métodos , Xenopus laevis/genética , Animais , Feminino , Corantes Fluorescentes/química , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , Oogênese/genética , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo , Globinas beta/genética , Globinas beta/metabolismoRESUMO
Cytoplasmic RNA localization is a key biological strategy for establishing polarity in a variety of organisms and cell types. However, the mechanisms that control directionality during asymmetric RNA transport are not yet clear. To gain insight into this crucial process, we have analyzed the molecular machinery directing polarized transport of RNA to the vegetal cortex in Xenopus oocytes. Using a novel approach to measure directionality of mRNA transport in live oocytes, we observe discrete domains of unidirectional and bidirectional transport that are required for vegetal RNA transport. While kinesin-1 appears to promote bidirectional transport along a microtubule array with mixed polarity, dynein acts first to direct unidirectional transport of RNA towards the vegetal cortex. Thus, vegetal RNA transport occurs through a multistep pathway with a dynein-dependent directional cue. This provides a new framework for understanding the mechanistic basis of cell and developmental polarity.
Assuntos
Polaridade Celular , Dineínas/metabolismo , Transporte de RNA , Animais , Padronização Corporal , Núcleo Celular/metabolismo , Cinesinas/metabolismo , Microscopia de Fluorescência , Oócitos/metabolismo , RNA Mensageiro/metabolismo , Análise de Célula Única , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
The biogenesis of a localization-competent mRNP begins in the nucleus. It is thought that the coordinated action of nuclear and cytoplasmic components of the localization machinery is required for the efficient export and subsequent subcellular localization of these mRNAs in the cytoplasm. Using quantitative poly(A)(+) and transcript-specific fluorescent in situ hybridization, we analyzed different nonessential nucleoporins and nuclear pore-associated proteins for their potential role in mRNA export and localization. We found that Nup60p, a nuclear pore protein located on the nucleoplasmic side of the nuclear pore complex, was required for the mRNA localization pathway. In a Δnup60 background, localized mRNAs were preferentially retained within the nucleus compared to nonlocalized transcripts. However, the export block was only partial and some transcripts could still reach the cytoplasm. Importantly, downstream processes were also affected. Localization of ASH1 and IST2 mRNAs to the bud was impaired in the Δnup60 background, suggesting that the assembly of a localization competent mRNP ("locasome") was inhibited when NUP60 was deleted. These results demonstrate transcript specificity of a nuclear mRNA retention defect and identify a specific nucleoporin as a functional component of the localization pathway in budding yeast.
