An Exclusively Nuclear RNA-Binding Protein Affects Asymmetric Localization of <i>ASH1</i> mRNA and Ash1p in Yeast

Long, Roy; Gu, Wei; Meng, Xiuhua; Gonsalvez, Graydon B.; Singer, Robert H.; Chartrand, Pascal

doi:10.1083/jcb.153.2.307

Cited by 84 publications

(115 citation statements)

References 43 publications

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“…Unlike She3p, which is exclusively located in the cytoplasm, She2p contains a nuclear localization signal and exhibits both a nuclear and a cytoplasmic lifestyle. In the nucleus, She2p is thought to associate primarily with zipcoded mRNA with the nuclear protein Loc1p and Puf6p (an mRNA-binding protein that binds to 3′ UTR) (18)(19)(20)(21). However, most nuclear messenger ribonucloprotein particles are remodeled by helicases either within the nucleus or, during nuclear export, by helicase Dbp5p sitting on guard at the cytoplasmic aspect of the nuclear pore complex (22).…”

Section: Discussionmentioning

confidence: 99%

“…3). For quantitative pull-down assay (16) between MBP-She3pep and She2p, She2p (200 μL, 0.5 mg/mL in buffer D) was titrated with varying amount of MBP-She3pep-bound amylose beads (10,20,25,30,35,40,80,160, and 320 μL; 2 mg/mL in buffer D) in a final volume of 520 μL. MBPShe3pep-bound amylose beads were incubated with She2p for 30 min and then spinned down for 5 min at 15,000 × g. Supernatant was collected for each sample, and concentration was estimated by UV 280 .…”

Section: Methodsmentioning

confidence: 99%

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Hooking She3p onto She2p for myosin-mediated cytoplasmic mRNA transport

Singh

Blobel

Shi

2014

Proc. Natl. Acad. Sci. U.S.A.

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The segregation of approximately two dozen distinct mRNAs from yeast mother to daughter cell cytoplasm is a classical paradigm for eukaryotic mRNA transport. The information for transport resides in an mRNA element 40–100 nt in length, known as “zipcode.” Targeted transport requires properly positioned actin filaments and cooperative loading of mRNA cargo to myosin. Cargo loading to myosin uses myosin 4 protein (Myo4p), swi5p-dependent HO expression 2 protein (She2p) and 3 protein (She3p), and zipcode. We previously determined a crystal structure of Myo4p and She3p, their 1:2 stoichiometry and interactome; we furthermore showed that the motor complex assembly requires two Myo4p⋅She3p heterotrimers, one She2p tetramer, and at least a single zipcode to yield a stable complex of [Myo4p⋅She3p⋅She2p⋅zipcode] in 2:4:4:1 stoichiometry in vitro. Here, we report a structure at 2.8-Å resolution of a cocrystal of a She2p tetramer bound to a segment of She3p. In this crystal structure, the She3p segment forms a striking hook that binds to a shallow hydrophobic pocket on the surface of each She2p subunit of the tetramer. Both She3p hook and cognate She2p binding pocket are composed of highly conserved residues. We also discovered a highly conserved region of She3p upstream of its hook region. Because this region consists of basic and aromatic residues, it likely represents part of She3p’s binding activity for zipcode. Because She2p also exhibits zipcode-binding activity, we suggest that “hooking” She3p onto She2p aligns each of their zipcode-binding activities into a high-affinity site, thereby linking motor assembly to zipcode.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Hooking She3p onto She2p for myosin-mediated cytoplasmic mRNA transport

Singh

Blobel

Shi

2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Such coupling is supported by the finding that She2p coimmunoprecipitates with Puf6p and Loc1p, suggesting an interaction between these proteins in vivo. The mechanism by which She2p promotes the recruitment of Loc1p and Puf6p on the ASH1 mRNA in vivo is not known, because all three proteins can bind the 3ЈUTR of this transcript independently in vitro (Bohl et al, 2000;Long et al, 2001;Gu et al, 2004). One possibility is that, being in the nucleolus, Puf6p and Loc1p are spatially restricted from polyA ϩ mRNAs.…”

Section: Nuclear She2p Couples Mrna Localization and Translational Rementioning

confidence: 99%

Nuclear Shuttling of She2p Couples ASH1 mRNA Localization to its Translational Repression by Recruiting Loc1p and Puf6p

Shen

Paquin

Forget

et al. 2009

MBoC

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The transport and localization of mRNAs results in the asymmetric synthesis of specific proteins. In yeast, the nucleocytoplasmic shuttling protein She2 binds the ASH1 mRNA and targets it for localization at the bud tip by recruiting the She3p-Myo4p complex. Although the cytoplasmic role of She2p in mRNA localization is well characterized, its nuclear function is still unclear. Here, we show that She2p contains a nonclassical nuclear localization signal (NLS) that is essential for its nuclear import via the importin ␣ Srp1p. Exclusion of She2p from the nucleus by mutagenesis of its NLS leads to defective ASH1 mRNA localization and Ash1p sorting. Interestingly, these phenotypes mimic knockouts of LOC1 and PUF6, which encode for nuclear RNA-binding proteins that bind the ASH1 mRNA and control its translation. We find that She2p interacts with both Loc1p and Puf6p and that excluding She2p from the nucleus decreases this interaction. Absence of nuclear She2p disrupts the binding of Loc1p and Puf6p to the ASH1 mRNA, suggesting that nuclear import of She2p is necessary to recruit both factors to the ASH1 transcript. This study reveals that a direct coupling between localization and translation regulation factors in the nucleus is required for proper cytoplasmic localization of mRNAs.

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“…In addition to the cis-acting localization elements, efficient ASH1 mRNA localization also requires several trans-acting factors: She1-5p, Loc1p, Khd1p, Bud6p, Puf5p, Puf6p, Scp160p, and a polarized actin cytoskeleton (11,12,(15)(16)(17)(18)(19)(20). The proteins apparently most directly involved with the localization of ASH1 mRNA are Myo4p-She1p, She2p, and She3p.…”

mentioning

confidence: 99%

“…The unequal segregation of Ash1p results from the localization of ASH1 mRNA at the distal tip of budding yeast cells during anaphase of the cell cycle (10 -12). Localization of ASH1 mRNA depends upon four cis-acting localization elements, named E1, E2A, E2B, and E3, and each element is sufficient for localizing a heterologous reporter RNA to daughter cells (10,13,14).In addition to the cis-acting localization elements, efficient ASH1 mRNA localization also requires several trans-acting factors: She1-5p, Loc1p, Khd1p, Bud6p, Puf5p, Puf6p, Scp160p, and a polarized actin cytoskeleton (11,12,(15)(16)(17)(18)(19)(20). The proteins apparently most directly involved with the localization of ASH1 mRNA are Myo4p-She1p, She2p, and She3p.…”

mentioning

confidence: 99%

ASH1 mRNA Anchoring Requires Reorganization of the Myo4p-She3p-She2p Transport Complex

Gonsalvez

Little

Long

2004

Journal of Biological Chemistry

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One mechanism by which cells post-transcriptionally regulate gene expression is via intercellular and intracellular sorting of mRNA. In Saccharomyces cerevisiae, the localization of ASH1 mRNA to the distal tip of budding cells results in the asymmetric sorting of Ash1p to daughter cell nuclei. Efficient localization of ASH1 mRNA depends upon the activity of four cisacting localization elements and also upon the activity of trans-factors She2p, She3p, and Myo4p. She2p, She3p, and Myo4p have been proposed to form an ASH1 mRNA localization particle. She2p directly and specifically binds each of the four ASH1 cis-acting localization elements, whereas She3p has been hypothesized to function as an adaptor by recruiting the She2p-mRNA complex to Myo4p, a type V myosin. The Myo4p-She3p-She2p heterotrimeric protein complex has been proposed to localize mRNA to daughter cells using polarized actin cables. Here we demonstrate that whereas the predicted Myo4p-She3p-She2p heterotrimeric complex forms in vivo, it represents a relatively minor species compared with the Myo4p-She3p complex. Furthermore, contrary to a prediction of the heterotrimeric complex model for ASH1 mRNA localization, ASH1 mRNA artificially tethered to She2p is not localized. Upon closer examination, we found that mRNA tightly associated with She2p is transported to daughter cells but is not properly anchored at the bud tip. These results are consistent with a model whereby anchoring of ASH1 mRNA requires molecular remodeling of the Myo4p-She3p-She2p heterotrimeric complex, a process that is apparently altered when mRNA is artificially tethered to She2p.The establishment and maintenance of cellular polarity is a salient feature of eukaryotic cells and involves the asymmetric sorting of proteins to distinct compartments or regions of the cell. One mechanism by which proteins can be sorted in a variety of eukaryotic cell types is via mRNA localization, a process by which mRNA is specifically localized to a particular region of a cell (1-3). A primary step in the process of mRNA localization is the identification of the localization substrate by trans-acting factors, resulting in the formation of a localization mRNP. 1 The mRNP localization complexes are dynamic structures undergoing molecular reorganization at various stages of the localization pathway (4 -6). RNA localization can result from a number of distinct mechanisms involving direct transport of the mRNA to the site of localization, generalized degradation of the mRNA with localized protection or random diffusion followed by entrapment of the mRNA at the site of localization (1, 2). Ultimately, translation of the mRNA at the site of localization leads to the asymmetric distribution of the protein in the cell.The asymmetric sorting of Ash1p in Saccharomyces cerevisiae is a paradigm for investigating the asymmetric segregation of proteins via mRNA localization. Ash1p is a transcriptional repressor that is asymmetrically sorted to daughter cells, resulting in differential gene expression between mother...

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An Exclusively Nuclear RNA-Binding Protein Affects Asymmetric Localization of ASH1 mRNA and Ash1p in Yeast

Cited by 84 publications

References 43 publications

Hooking She3p onto She2p for myosin-mediated cytoplasmic mRNA transport

Hooking She3p onto She2p for myosin-mediated cytoplasmic mRNA transport

Nuclear Shuttling of She2p Couples ASH1 mRNA Localization to its Translational Repression by Recruiting Loc1p and Puf6p

ASH1 mRNA Anchoring Requires Reorganization of the Myo4p-She3p-She2p Transport Complex

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