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.
