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

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.M essenger RNA localization is a universal feature of eukaryotes (1-3). By complementing transcriptional control (4), it fulfills a variety of functions, including the establishment of cell polarity and specialization of subcellular regions. In recent years, the directional transport of asymmetric synthesis of HO 1 (ASH1) mRNA in budding yeast has emerged as a particularly well-suited model to study mechanistic principles of RNA localization. Here, comparably few proteins participate in the directional transport of ASH1 mRNA and about 30 other transcripts (5, 6).Chromatin-immunoprecipitaton experiments revealed that the dedicated RNA-binding Swi5p-dependent HO expression protein 2 (She2p) binds already cotranscriptionally to nascent ASH1 mRNA (7, 8). Two additional RNA-binding proteins, pumiliohomology domain family protein 6 (Puf6p) and heterogeneous nuclear RNP K-like protein 1 (Khd1p), are also present in the nucleus, bind to ASH1 mRNA, and act in the cytoplasm as translational repressors during ASH1 transport (9-12). A fourth nuclear factor, termed localization of ASH1 mRNA protein 1 (Loc1p), has been implicated in the assembly of nuclear premessenger ribonucleoprotein particles (mRNPs). Like Puf6p, Loc1p is a nuclear protei...

Co-transcriptional recruitment of Puf6 by She2 couples translational repression to mRNA localization

“…Our results are in agreement with a recent study published while this manuscript was in revision, which also report a direct interaction between She2 and Loc1 in vitro (26). However, no direct interaction between recombinant Puf6 and Loc1 was detected in this study, and the authors did not observe the formation of a Puf6:Loc1:She2 ternary complex on the E3 RNA zipcode in vitro .…”

Structure of a myosin•adaptor complex and pairing by cargo

“…5A). These data argue strongly against a model in which a complex of two molecules of Myo4p and an unidentified number of She3p molecules can form in an unassisted fashion in the cytoplasm (25,36).…”

A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

“…She2 associates in vivo with Puf6 or Loc1 in an RNA-independent manner; thus, She2 or Loc1 may enhance Puf6 specificity. However, no interaction between Puf6 and She2 or Loc1 was detected in vitro with purified components (28,29). This inconsistency may indicate that other factors are necessary for Puf6 to associate with the She2/Loc1 complex on ASH1 mRNA.…”