Processos de extrema complexidade até em levedura

quinta-feira, abril 21, 2011

A Cytoplasmic Complex Mediates Specific mRNA Recognition and Localization in Yeast

Marisa Müller1,2#, Roland Gerhard Heym1,2#, Andreas Mayer2, Katharina Kramer3, Maria Schmid2¤, Patrick Cramer2,4, Henning Urlaub3, Ralf-Peter Jansen2,5, Dierk Niessing1,2*

1 Institute of Structural Biology, Helmholtz Zentrum München–German Research Center for Environmental Health, München, Germany, 2 Gene Center and Department of Biochemistry, Ludwig-Maximilians-University, München, Germany, 3 Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, 4 Center for Integrated Protein Science CIPSM, München, Germany, 5 Interfaculty Institute for Biochemistry, University of Tübingen, Tübingen, Germany

Abstract

In eukaryotes, hundreds of mRNAs are localized by specialized transport complexes. For localization, transcripts are recognized by RNA-binding proteins and incorporated into motor-containing messenger ribonucleoprotein particles (mRNPs). To date, the molecular assembly of such mRNPs is not well understood and most details on cargo specificity remain unresolved. We used ASH1-mRNA transport in yeast to provide a first assessment of where and how localizing mRNAs are specifically recognized and incorporated into mRNPs. By using in vitro–interaction and reconstitution assays, we found that none of the implicated mRNA-binding proteins showed highly specific cargo binding. Instead, we identified the cytoplasmic myosin adapter She3p as additional RNA-binding protein. We further found that only the complex of the RNA-binding proteins She2p and She3p achieves synergistic cargo binding, with an at least 60-fold higher affinity for localizing mRNAs when compared to control RNA. Mutational studies identified a C-terminal RNA-binding fragment of She3p to be important for synergistic RNA binding with She2p. The observed cargo specificity of the ternary complex is considerably higher than previously reported for localizing mRNAs. It suggests that RNA binding for mRNP localization generally exhibits higher selectivity than inferred from previous in vitro data. This conclusion is fully consistent with a large body of in vivo evidence from different organisms. Since the ternary yeast complex only assembles in the cytoplasm, specific mRNA recognition might be limited to the very last steps of mRNP assembly. Remarkably, the mRNA itself triggers the assembly of mature, motor-containing complexes. Our reconstitution of a major portion of the mRNA-transport complex offers new and unexpected insights into the molecular assembly of specific, localization-competent mRNPs and provides an important step forward in our mechanistic understanding of mRNA localization in general.

Author Summary 

In eukaryotes, the majority of cells are asymmetric and a way to establish such polarity is directional transport of macromolecules along cytoskeletal filaments. Among the cargoes transported, mRNAs play an essential role, as their localized translation contributes significantly to the generation of asymmetry. To date, hundreds of asymmetrically localized mRNAs in various organisms have been identified. These mRNAs are recognized by RNA-binding proteins and incorporated into large motor-containing messenger ribonucleoprotein particles (mRNPs) whose molecular assembly is poorly understood. In this study, we used the well-characterized process ofASH1-mRNA transport in Saccharomyces cerevisiae to address the question of how localizing mRNAs are recognized and specifically incorporated into mRNPs. Surprisingly, we found that the previously implicated mRNA-binding proteins She2p and Puf6p do not bind to cargo mRNAs with high specificity. Instead, the cytoplasmic motor-adapter protein She3p is responsible for synergistic cargo binding with She2p and for the stable incorporation of specific localizing mRNA into the transport complex. We propose that the specific recognition of localizing mRNAs happens at the very last step of cytoplasmic mRNP maturation. Other organisms might employ similar mechanisms to establish cellular polarity.

Citation: Müller M, Heym RG, Mayer A, Kramer K, Schmid M, et al. (2011) A Cytoplasmic Complex Mediates Specific mRNA Recognition and Localization in Yeast. PLoS Biol 9(4): e1000611. doi:10.1371/journal.pbio.1000611

Academic Editor: Simon Bullock, Cancer Research UK, United Kingdom

Received: July 14, 2010; Accepted: March 10, 2011; Published: April 19, 2011

Copyright: © 2011 Müller et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the Helmholtz Association (VG-NH 142 to D.N.), Deutsche Forschungsgemeinschaft (FOR855 to M.M. and D.N.; SFB646 to M.S., P.C., R.-P.J. and D.N.), and the Boehringer Ingelheim Fonds (to M.M.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; IP, immunoprecipitated; mRNP, messenger ribonucleoprotein particle; ORF, open reading frame


# These authors contributed equally to this work.

¤ Current address: Roche Diagnostics GmbH, Penzberg, Germany

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NOTA DESTE BLOGGER:

Repare que a extrema complexidade dos processos aqui descritos foram encontrados em levedura. Imagine, só imagine, como é muito mais complexo os processos celulares nos organismos eucariotos complexos - um elefante, por exemplo.

Os autores usaram 68 vezes a palavra 'zip code' para descrever o processo celular complexo (outro nível de controle de qualidade) em levedura. 

E se Darwin não explica a origem e a evolução do flagelo bacteriano, como explicar a origem e evolução da diversidade e complexidade das espécies??? O fato, Fato, FATO da evolução está muito mal explicado e os cientistas precisam reconhecer isso: Darwin, já era!!!