She2p Is a Novel RNA Binding Protein with a Basic Helical Hairpin Motif

Laboratories of Molecular Biophysics and The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Cell (Impact Factor: 32.24). 11/2004; 119(4):491-502. DOI: 10.1016/j.cell.2004.10.018
Source: PubMed


Selective transport of mRNAs in ribonucleoprotein particles (mRNP) ensures asymmetric distribution of information within and among eukaryotic cells. Actin-dependent transport of ASH1 mRNA in yeast represents one of the best-characterized examples of mRNP translocation. Formation of the ASH1 mRNP requires recognition of zip code elements by the RNA binding protein She2p. We determined the X-ray structure of She2p at 1.95 A resolution. She2p is a member of a previously unknown class of nucleic acid binding proteins, composed of a single globular domain with a five alpha helix bundle that forms a symmetric homodimer. After demonstrating potent, dimer-dependent RNA binding in vitro, we mapped the RNA binding surface of She2p to a basic helical hairpin in vitro and in vivo and present a mechanism for mRNA-dependent initiation of ASH1 mRNP complex assembly.

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    • "Although our ChIP results provide evidence for the formation of a multiprotein complex on the ASH1 E3 element during transcription, these data do not prove that such complexes really assemble. In order to determine whether a Puf6-Loc1-She2 ternary complex can be formed on the ASH1 E3 RNA in vitro, pull-down experiments were performed in presence of a 118 nt E3 localization element RNA using physiological concentration of each protein: 230 nM of She2 (19), 200 nM of Loc1 and 1.2 μM of His-tagged Puf6 (20). The E3 localization element RNA was first incubated with She2, then added to the Puf6-His:Loc1 complex bound to Ni-NTA agarose beads. "
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    ABSTRACT: Messenger RNA (mRNA) localization is coupled to the translational repression of transcripts during their transport. It is still unknown if this coupling depends on physical interactions between translational control and mRNA localization machineries, and how these interactions are established at the molecular level. In yeast, localization of transcripts like ASH1 to the bud depends on the RNA-binding protein She2. During its transport, ASH1 mRNA translation is repressed by Puf6. Herein, we report that She2 recruits Puf6 on ASH1 co-transcriptionally. The recruitment of Puf6 depends on prior co-transcriptional loading of Loc1, an exclusively nuclear protein. These proteins form a ternary complex, in which Loc1 bridges Puf6 to She2, that binds the ASH1 3′UTR. Using a genome-wide ChIP-chip approach, we identified over 40 novel targets of Puf6, including several bud-localized mRNAs. Interestingly, the co-transcriptional recruitment of Puf6 on genes coding for these bud-localized mRNAs is also She2- and Loc1-dependent. Our results suggest a coordinated assembly of localization and translational control machineries on localized mRNAs during transcription, and underline the importance of co-transcriptional events in establishing the cytoplasmic fate of mRNAs.
    Nucleic Acids Research 07/2014; 42(13). DOI:10.1093/nar/gku597 · 9.11 Impact Factor
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    • "Quite unexpectedly, 4/1 proteins showed the greatest similarity in their folding to yeast protein She2p (246 amino acids in length). This protein is a member of a class of nucleic acid binding proteins that contain a single globular domain with a five alpha helix bundle and form a symmetric homodimer (Niessing et al., 2004; Figure 2A). In yeast, this protein is involved in directional transport and localization of a specific mRNA (ASH1 mRNA) that regulates gene expression on a temporal and spatial level (Jansen and Niessing, 2012). "
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    ABSTRACT: Originally isolated as a result of its ability to interact with the movement protein of Tomato spotted wilt virus in a yeast two-hybrid system, the 4/1 protein is proving to be an excellent tool for studying intracellular protein trafficking and intercellular communication. Expression of 4/1 in vivo is tightly regulated, first appearing in the veins of the cotyledon and later in the vasculature of the leaf and stem in association with the xylem parenchyma and phloem parenchyma. Structural studies indicate that 4/1 proteins contain as many as five coiled-coil (CC) domains; indeed, the highest level of sequence identity among 4/1 proteins involves their C-terminal CC domains, suggesting that protein-protein interaction is important for biological function. Recent data predict that the tertiary structure of this C-terminal CC domain is strikingly similar to that of yeast protein She2p; furthermore, like She2p, 4/1 protein exhibits RNA-binding activity, and mutational analysis has shown that the C-terminal CC domain is responsible for RNA binding. The 4/1 protein contains a nuclear export signal. Additional microscopy studies involving leptomycin and computer prediction suggest the presence of a nuclear localization signal as well.
    Frontiers in Plant Science 02/2014; 5:26. DOI:10.3389/fpls.2014.00026 · 3.95 Impact Factor
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    • "Efficient mRNA binding and localization require the formation of She2p tetramers (Müller et al., 2009; Chung and Takizawa, 2010). As different stoichiometric ratios have been suggested for She2p RNA complexes (Niessing et al., 2004; Olivier et al., 2005), we determined the molecular mass of bacterially expressed She2p in complex with a 77-nucleotide ASH1-E3 zip code RNA fused to tRNA (ASH1-E3-77-tRNA) by SLS. The tRNA tag was used to produce large amounts of stable RNA (Ponchon and Dardel, 2007). "
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    ABSTRACT: The assembly and composition of ribonucleic acid (RNA)-transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.
    The Journal of Cell Biology 12/2013; 203(6):971-84. DOI:10.1083/jcb.201302095 · 9.83 Impact Factor
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