title: A single molecule approach to mRNA transport by a class V myosin.
abstract: mRNA 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 microtubule-based motors.
authors.affiliation: a Department of Molecular Physiology & Biophysics ; University of Vermont ; Burlington , VT USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Sladewski
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Trybus
mesh_terms: D008841:Actin Cytoskeleton / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000235:genetics / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism; D024701:Myosin Type V / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D034443:RNA Transport / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C098733:ASH1 protein, S cerevisiae; D000199:Actins; C098732:Myo4 protein, S cerevisiae; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D012261:Ribonucleoproteins; C471175:SHE2 protein, S cerevisiae; C471176:SHE3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C026452:messenger ribonucleoprotein; D024701:Myosin Type V; D018995:Myosin Heavy Chains
keywords: ASH1 mRNA; Myo4p; She2p; She3p; mRNA localization; mRNA transport; mRNP, messenger ribonucleoprotein; messenger ribonucleoprotein