In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation

Heym, Roland G.; Zimmermann, Dennis; Edelmann, F.T.; Israel, Lars; Ökten, Zeynep; Kovar, David R.; Niessing, Dierk

doi:10.1083/jcb.201302095

Cited by 27 publications

(50 citation statements)

References 59 publications

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“…26 In addition, metal-shadowed electron microscopic images obtained with equimolar concentrations of mRNA, Myo4p and She2p, showed that zip codes are required for recruitment of Myo4p to localizing transcripts, and that up to eight motor heads can bind to the native four zip code ASH1 mRNA. 15 In agreement, Heym et al 20 also conclude that the vast majority of zip code elements in the cell are bound to motor complexes. Multiple zip codes are not essential for cellular mRNA localization, but their presence offers advantages by enhancing both the percentage of cells with correct mRNA localization, and the quality of localization at the bud tip.…”

Section: Conflicting Viewpointssupporting

confidence: 56%

“…Subsequent to our publication, 15 a single molecule study by Heym et al 20 drew several conclusions contradictory to ours. They concluded that She2p alone is necessary for Myo4p to undergo processive motion, and that neither mRNA nor zip codes are necessary.…”

Section: Conflicting Viewpointscontrasting

confidence: 43%

“…Heym et al 20 suggested several reasons other than ionic strength for these differing conclusions, but we do not believe that these other factors are responsible. They suggested that mRNP run length increases with zip code number because we used high stoichiometric ratios of myosin over mRNA.…”

Section: Conflicting Viewpointsmentioning

confidence: 88%

“…She2p is thus capable of recruiting two single-headed Myo4p-She3p motors, resulting in a stoichiometry of two Myo4p heavy chains: two She3p dimers: one She2p tetramer. 19,20 Importantly, this two-headed motor complex, recruited via She2p, steps processively on actin filaments with a 72 nm step-size and hand-over-hand stepping pattern identical to that observed with myosin Va (Fig. 2A).…”

Section: Two Motor Heads Are Needed For Processivitymentioning

confidence: 95%

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A single molecule approach to mRNA transport by a class V myosin

Sladewski

Trybus

2014

RNA Biology

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m RNA localization ensures correct spatial and temporal control of protein synthesis in the cell. We show that an in vitro single molecule approach, using purified recombinant full-length proteins and synthesized mRNA, provides insight into the mechanism by which localizing mRNAs are carried to their destination. A messenger ribonucleoprotein (mRNP) complex was reconstituted from a budding yeast class V myosin motor complex (Myo4p-She3p), an mRNA-binding adaptor protein (She2p), and a localizing mRNA (ASH1). The motion of the mRNP was tracked with high spatial (»10 nm) and temporal (70 ms) resolution. Using this "bottom-up" methodology, we show that mRNA triggers the assembly of a high affinity double-headed motor-mRNA complex that moves continuously for long distances on actin filaments at physiologic ionic strength. Without mRNA, the myosin is monomeric and unable to move continuously on actin. This finding reveals an elegant strategy to ensure that only cargo-bound motors are activated for transport. Increasing the number of localization elements ("zip codes") in the mRNA enhanced both the frequency of motile events and their run length, features which likely enhance cellular localization. Future in vitro reconstitution of mRNPs with kinesin and dynein motors should similarly yield mechanistic insight into mRNA transport by microtubulebased motors.

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Section: Conflicting Viewpointssupporting

confidence: 56%

Section: Conflicting Viewpointscontrasting

confidence: 43%

Section: Conflicting Viewpointsmentioning

confidence: 88%

Section: Two Motor Heads Are Needed For Processivitymentioning

confidence: 95%

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A single molecule approach to mRNA transport by a class V myosin

Sladewski

Trybus

2014

RNA Biology

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“…Dimerization is regulated by the availability of cargo (4)(5)(6). However, cargo does not bind directly to Myo4p.…”

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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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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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mRNA transport in fungal top models

et al. 2017

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Eukaryotic cells rely on the precise determination of when and where proteins are synthesized. Spatiotemporal expression is supported by localization of mRNAs to specific subcellular sites and their subsequent local translation. This holds true for somatic cells as well as for oocytes and embryos. Most commonly, mRNA localization is achieved by active transport of the molecules along the actin or microtubule cytoskeleton. Key factors are molecular motors, adaptors, and RNA-binding proteins that recognize defined sequences or structures in cargo mRNAs. A deep understanding of this process has been gained from research on fungal model systems such as Saccharomyces cerevisiae and Ustilago maydis. Recent highlights of these studies are the following: (1) synergistic binding of two RNA-binding proteins is needed for high affinity recognition; (2) RNA sequences undergo profound structural rearrangements upon recognition; (3) mRNA transport is tightly linked to membrane trafficking; (4) mRNAs and ribosomes are transported on the cytoplasmic surface of endosomes; and (5) heteromeric protein complexes are, most likely, assembled co-translationally during endosomal transport. Thus, the study of simple fungal model organisms provides valuable insights into fundamental mechanisms of mRNA transport boosting the understanding of similar events in higher eukaryotes.

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In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation

Abstract: The budding yeast SHE mRNA-transport complex dimerizes to activate processive RNA transport, irrespective of the presence of RNA cargo, and multimerizes upon binding RNAs with multiple localization elements.

Cited by 27 publications

References 59 publications

A single molecule approach to mRNA transport by a class V myosin

A single molecule approach to mRNA transport by a class V myosin

Hooking She3p onto She2p for myosin-mediated cytoplasmic mRNA transport

mRNA transport in fungal top models

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