RESUMO
Asymmetric ASH1 mRNA transport during mitosis of budding yeast constitutes one of the best-studied examples of mRNA localization. Recently, 2 studies used in vitro motility assays to prove that motile ASH1 mRNA-transport complexes can be reconstituted entirely from recombinant factors. Both studies, however, differed in their conclusions on whether cargo RNA itself is required for particle assembly and thus activation of directional transport. Here we provide direct evidence that stable complexes do assemble in absence of RNA at physiologic conditions and even at ionic strengths above cellular levels. These results directly confirm the previous notion that the ASH1 transport machinery is not activated by the cargo RNA itself, but rather through protein-protein interactions.
Assuntos
Regulação Fúngica da Expressão Gênica , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mitose , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Concentração Osmolar , Fosforilação , Multimerização Proteica , Transporte de RNA , RNA Fúngico/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
mRNA localization and localized translation is a common mechanism that contributes to cell polarity and cellular asymmetry. In metazoan, mRNA transport participates in embryonic axis determination and neuronal plasticity. Since the mRNA localization process and its molecular machinery are rather complex in higher eukaryotes, the unicellular yeast Saccharomyces cerevisiae has become an attractive model to study mRNA localization. Although the focus has so far been on the mechanism of ASH1 mRNA transport, it has become evident that mRNA localization also assists in protein sorting to organelles, as well as in polarity establishment and maintenance. A diversity of different pathways has been identified that targets mRNA to their destination site, ranging from motor protein-dependent trafficking of translationally silenced mRNAs to co-translational targeting, in which mRNAs hitch-hike to organelles on ribosomes during nascent polypeptide chain elongation. The presence of these diverse pathways in yeast allows a systemic analysis of the contribution of mRNA localization to the physiology of a cell.
Assuntos
Transporte de RNA/genética , RNA Mensageiro/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Polaridade Celular/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Asymmetric, motor-protein dependent transport of mRNAs and subsequent localized translation is an important mechanism of gene regulation. Due to the high complexity of such motile particles, our mechanistic understanding of mRNA localization is limited. Over the last two decades, ASH1 mRNA localization in budding yeast has served as comparably simple and accessible model system. Recent advances have helped to draw an increasingly clear picture on the molecular mechanisms governing ASH1 mRNA localization from its co-transcriptional birth to its delivery at the site of destination. These new insights help to better understand the requirement of initial nuclear mRNPs, the molecular basis of specific mRNA-cargo recognition via cis-acting RNA elements, the different stages of RNP biogenesis and reorganization, as well as activation of the motile activity upon cargo binding. We discuss these aspects in context of published findings from other model organisms.
Assuntos
Transporte de RNA/genética , Proteínas Repressoras/genética , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Complexos Multiproteicos/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
mRNA localization ensures correct spatial and temporal control of protein synthesis in the cell. We show that an in vitro single molecule approach, using purified recombinant full-length proteins and synthesized mRNA, provides insight into the mechanism by which localizing mRNAs are carried to their destination. A messenger ribonucleoprotein (mRNP) complex was reconstituted from a budding yeast class V myosin motor complex (Myo4p-She3p), an mRNA-binding adaptor protein (She2p), and a localizing mRNA (ASH1). The motion of the mRNP was tracked with high spatial (â¼10 nm) and temporal (70 ms) resolution. Using this "bottom-up" methodology, we show that mRNA triggers the assembly of a high affinity double-headed motor-mRNA complex that moves continuously for long distances on actin filaments at physiologic ionic strength. Without mRNA, the myosin is monomeric and unable to move continuously on actin. This finding reveals an elegant strategy to ensure that only cargo-bound motors are activated for transport. Increasing the number of localization elements ("zip codes") in the mRNA enhanced both the frequency of motile events and their run length, features which likely enhance cellular localization. Future in vitro reconstitution of mRNPs with kinesin and dynein motors should similarly yield mechanistic insight into mRNA transport by microtubule-based motors.