Dr. Paul Macdonald proposes a study of translational control in Drosophila development. Specifically, he is interested in the mechanisms by which translational regulation occurs and the roles that this level of control plays in embryonic patterning. Mechanisms for maintaining maternal messages in a translationally silent state have long been studied, mostly in developmental systems not amenable to genetic analysis. In most cases, these studies are directed at general mechanisms. Evidence has mounted, primarily in C. elegans and Drosophila for translational control of specific mRNAs as an aspect of regulation of patterning and other critical developmental mechanisms. Dr. Macdonald has been investigating the patterning gene oskar (osk) and has learned that osk function depends both on transcript localization and on translational regulation. osk functions in the targeting of posterior axis and germ cell determinants to the posterior end of the embryo. osk mRNA is itself localized posteriorly, and its activity is confined to that region. Mutations in osk lead to the mislocalization of the posterior axis determinant, nanos. In previous work on this system, the Macdonald lab learned that there is an interval between osk mRNA synthesis and its movement to a posterior position. During this interval, osk protein is not expressed. The protein only becomes detectable after localization of the message has occurred. The most reasonable explanation for this observation is that translation is repressed, probably via bound proteins. In spite of extensive studies of posterior group genes, none had surfaced that are likely candidates for this type of repressor function. Therefore, a biochemical search was conducted using a uv cross-linking assay. An 80 kD protein was identified, obp80, which binds specifically to three regions in the 3' UTR of osk mRNA. The three binding regions are related in sequence. Small changes in this sequence destroyed binding capacity. The level of obp 80 was found to be normal in all other posterior group mutants tested, confirming that no likely candidates for this function were known. Transgenes lacking the binding elements express RNAs that are localized normally, but which are translated prematurely, thereby separating these two elements of patterning gene expression. This proposal initially proposed alternate strategies for obtaining the obp 80 gene and pure protein. However, since submission of the proposal the gene was isolated via screens of expression libraries and sequenced and antibodies have been made. The protein obtained is consistent with predicted size, has the expected binding properties and contains three RNP binding domains, each different in sequence. The distinct binding domains result in a prediction that each is likely to have distinct binding preferences and further that osk is likely to be only one of several RNA targets.
The specific aims of this portion of the grant are therefore revised in part.
The aims are to correlate the binding of the obp 80 protein, now renamed bruno and with changes in poly A tail lenth, cap dependence. Plans are described for exploring further the parameters of binding, with emphasis now changed to address the capacities of the three different bruno binding domains. in vitro systems are described to explore the mechanism of translational repression in the absence of complicating developmental processes. in vitro translation systems and cell cutures will be tested for their suitability for manipulating bruno regulation of translation. Genetic analysi of bruno has been given high priority. A cytological locaton will be determined, follwed by a search of deficiencies and known mutations in the region. If such mutations are not found, full scale mutagenesis is planned. The second part of this proposal concerns the gene aubergine. The Macdonald lab isolated two new alleles of this previously discovered gene in a screen for the suppression of a bicaudal phenotype caused by mislocalization of osk RNA to the anterior end. Characterization of the phenotypes of these mutants revealed patterning defects along the dorsal-ventral axis. Working on the hypothesis that the gene functions in anterior-posterior and dorsovetral patterning, the effects of aub mutations on genes involved in one or the other patterning pathway were studied. Most localized messages were unaffected. However, osk and gerken proteins are absent and bcd is reduced in aub mutants. Aub therefore appears to be necessary for protein expression. An osk trangene with a bcd 3' UTR produces osk protein equally in wildtype and aub mutant backgrounds, showing that aub does not act by affecting protein stability, but that it does require 3' sequence elements. Experiments planned for aub are directed towards a more detailed description of aub action. Elements of osk and grk RNA required for aub function will be mapped. Experiments will be conducted to determine if aub protein interacts directly with these RNAs. Plans are described for cloning and characterizing the gene.
Showing the most recent 10 out of 29 publications
