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Proc Natl Acad Sci U S A. 2013 Dec 24;110(52):E5049-58. doi: 10.1073/pnas.1315289111. Epub 2013 Dec 9.

Role of Loc1p in assembly and reorganization of nuclear ASH1 messenger ribonucleoprotein particles in yeast.

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1
Group Intracellular Transport and RNA Biology, Institute of Structural Biology, Helmholtz Zentrum München- German Research Center for Environmental Health, 85764 Neuherberg, Germany.

Abstract

Directional transport of mRNA is a universal feature in eukaryotes, requiring the assembly of motor-dependent RNA-transport particles. The cytoplasmic transport of mRNAs is preceded by the nuclear assembly of pre-messenger ribonucleoprotein particles (mRNPs). In budding yeast, the asymmetric synthesis of HO 1 (ASH1) pre-mRNP originates already cotranscriptionally and passes through the nucleolus before its nuclear export. The nucleolar localization of ASH1 mRNA protein 1 (Loc1p) is required for efficient ASH1 mRNA localization. Immunoprecipitation experiments have revealed that Loc1p forms cocomplexes with other components of the ASH1 transport complex. However, it remains unclear how Loc1p is recruited into this mRNP and why Loc1p is important for ASH1 mRNA localization. Here we demonstrate that Loc1p undergoes a direct and specific interaction with the ASH1 mRNA-binding Swi5p-dependent HO expression protein 2 (She2p). This cocomplex shows higher affinity and specificity for RNA bearing localization elements than the individual proteins. It also stabilizes the otherwise transient binding of She2p to ASH1 mRNA, suggesting that cooperative mRNA binding of Loc1p with She2p is the required nuclear function of Loc1p for ASH1 mRNA localization. After nuclear export, myosin-bound She3p joins the ASH1 mRNP to form a highly specific cocomplex with She2p and ASH1 mRNA. Because Loc1p is found only in the nucleus, it must be removed from the complex directly before or after export. In vitro and in vivo experiments indicate that the synergistic interaction of She2p and She3p displaces Loc1p from the ASH1 complex, allowing free Loc1p to rapidly reenter the nucle(ol)us. Together these findings suggest an ordered process of nuclear assembly and reorganization for the maturation of localizing ASH1 mRNPs.

KEYWORDS:

Puf6p; cotranscriptional recruitment; macromolecular complex; reconstitution experiments

PMID:
24324176
PMCID:
PMC3876240
DOI:
10.1073/pnas.1315289111
[Indexed for MEDLINE]
Free PMC Article
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