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。高度调节的机制存在的时间和空间分配的特定RNA分子的特定的核和胞质结构域。复杂的细胞机器准确地分类，分配，靶向和锚定RNA到其最终目的地。这种RNA和蛋白质的不对称分类是细胞质定位的经典胚胎学概念的基础，细胞的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由卵细胞骨架内在产生的。类似地，在分化的细胞中，细胞骨架可以为mRNA的区域分布提供不对称的框架，并且可能为细胞过程（例如树突、轴突或板状伪足）的功能规范提供不对称的框架。每一个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。实现这一目标的进展已经取得了多聚核糖体-聚（A）+RNA-微管复合物的体外组装和分离，从海胆卵和胚胎。这项初步研究的目的是测试的假设，这些复合物中的mRNA编码微管相关蛋白，并通过新生的多肽与微管结合结构域在其氨基末端的多聚核糖体的微管为目标。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）中观察信使RNA与细胞骨架元件的关系有着悠久而有争议的历史。这些观察结果一直难以解释，由于问题的潜在文物所采用的方法来研究的现象。在这项试点研究中使用的方法，使用体外技术和高度纯化的成分，将允许所陈述的假设进行测试，同时避免一些困扰早期方法的问题。由于围绕一般研究领域的争议，以及这一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供资机制。

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.