Sladewski Thomas E, Trybus Kathleen M
a Department of Molecular Physiology & Biophysics ; University of Vermont ; Burlington , VT USA.
RNA Biol. 2014;11(8):986-91. doi: 10.4161/rna.29947. Epub 2014 Oct 31.
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.
信使核糖核酸（mRNA）定位可确保细胞中蛋白质合成在空间和时间上得到正确控制。我们发现，一种体外单分子方法，即使用纯化的重组全长蛋白和合成的mRNA，能够深入了解定位mRNA被转运至其目的地的机制。从出芽酵母V类肌球蛋白运动复合体（Myo4p-She3p）、一种mRNA结合衔接蛋白（She2p）和一种定位mRNA（ASH1）中重构了信使核糖核蛋白（mRNP）复合体。以高空间分辨率（约10纳米）和时间分辨率（70毫秒）追踪mRNP的运动。使用这种“自下而上”的方法，我们发现mRNA触发了一种高亲和力双头运动蛋白-mRNA复合体的组装，该复合体在生理离子强度下能在肌动蛋白丝上持续长距离移动。没有mRNA时，肌球蛋白呈单体状态，无法在肌动蛋白上持续移动。这一发现揭示了一种巧妙的策略，可确保只有与货物结合的运动蛋白被激活用于运输。增加mRNA中定位元件（“邮政编码”）的数量可提高运动事件的频率及其运行长度，这些特性可能增强细胞定位。未来用驱动蛋白和动力蛋白对mRNP进行体外重构，同样应能深入了解基于微管的运动蛋白对mRNA的运输机制。
