Polyribosome Targeting to Microtubules

真核生物生活方式的标志之一是RNA在细胞间的分布不均匀。高度调控的机制存在于特定RNA分子的时间和空间分配到特定的核和细胞质结构域。复杂的细胞机制精确地对RNA进行分类、分配、靶向和锚定，使其到达最终目的地。这种RNA和蛋白质的不对称分选奠定了细胞质定位的经典胚胎学概念，即细胞命运由遗传的卵细胞质区域指定。最近的研究表明，信使RNA和蛋白质的非随机分布可能是卵细胞骨架内在产生的。同样，在分化的细胞中，细胞骨架可能为mRNA的区域分布和细胞过程（如树突、轴突或板足）的功能规范提供了不对称框架。每一种主要的细胞骨架结构——微管、中间丝和微丝——都与mRNA定位的一般过程有关。理解翻译机制如何以及为什么与细胞骨架相互作用的一种方法是用mRNA和单个细胞骨架元件生化纯化和重建核糖体和其他翻译因子。随着从海胆卵和胚胎中组装和分离出多核糖体-聚(a)+RNA-微管复合物，在实现这一目标方面取得了进展。本初步研究的目的是验证这些复合物中的mrna编码微管相关蛋白的假设，以及聚体通过在其氨基末端具有微管结合结构域的新生多肽靶向微管。在各种细胞类型（包括哺乳动物细胞、高等植物细胞和无脊椎动物细胞）中，对信使RNA与细胞骨架元件关联的观察历史悠久且有争议。由于研究这些现象所采用的方法所产生的潜在人为问题，这些观察结果很难解释。在这个试点研究中使用的方法，使用体外技术和高度纯化的成分，将允许所述的假设得到检验，同时避免一些困扰早期方法的人为因素。由于围绕一般研究领域的争议，以及该试点项目的风险程度，探索性研究小额赠款是该项目的适当资助机制。

One of the hallmarks of a eukaryotic lifestyle is that RNA is distributed unequally among cellular compartments. Highly regulated mechanisms exist for the temporal and spatial assignment of specific RNA molecules to particular nuclear and cytoplasmic domains. Intricate cellular machinery accurately sorts, distributes, targets, and anchors the RNA to its final destination. This asymmetric sorting of RNA and protein underlies the classical embryological concept of cytoplasmic localization, whereby cell fate is specified by the region of egg cytoplasm inherited. Recent studies indicate that the nonrandom distribution of messenger RNA and protein may be intrinsically generated by the egg cytoskeleton. Similarly, in differentiated cells, the cytoskeleton may provide an asymmetric framework for the regional distribution of mRNA and perhaps the functional specification of cellular processes such as dendrites, axons, or lamellipodia. Each of the major cytoskeletal structures --- microtubules, intermediate filaments, and microfilaments --- have been implicated in the general process of mRNA localization. One approach to understanding how and why the translational machinery interacts with the cytoskeleton is to biochemically purify and reconstitute ribosomes and other translational factors with mRNA and a single cytoskeletal element. Progress towards realizing this goal has been made with the in vitro assembly and isolation of a polyribosome -poly(A)+RNA- microtubule complex from sea urchin eggs and embryos. The goal of this pilot study is to test the hypotheses that the mRNAs in these complexes code for microtubule-associated proteins and that the polysomes are targeted to microtubules via nascent polypeptides with microtubule-binding domains at their amino termini. %%% There is a long and controversial history of observations of messenger RNA association with cytoskeletal elements in various cell types (including mammalian cells, higher plant cells, and invertebrate cells). These observations have been difficult to interpret due to problems of potential artifacts arising from the methods employed to study the phenomena. The approach to be used in this pilot study, using in vitro techniques and highly purified components, will allow the stated hypothesis to be tested while avoiding some of the artifacts that plagued earlier approaches to the problem. Because of the controversy surrounding the general field of study, and the degree of risk in this pilot project, a Small Grant for Exploratory Research is an appropriate funding mechanism for the project.