CIS AND TRANS REGULATION OF GERM PLASM mRNA LOCALIZATION IN DROSOPHILA

Abstract

ABSTRACT

Subcellular mRNA localization is an efficacious and pervasive strategy for generating the asymmetric protein distributions necessary for cellular and developmental polarity. During early Drosophila development, RNA localization is critically important for specification and maintenance of germline fate. Primordial germ cell development requires molecular determinants contained within complex ribonucleoprotein (RNP) granules (polar granules), that define a specialized cytoplasmic domain at the oocyte posterior called the germ plasm. Within the polar granules, localized mRNA species are organized into distinct multi-copy homotypic clusters.

Germ plasm enriched mRNAs localize through a diffusion and entrapment mechanism, whereby single transcripts associate with nascent polar granules and subsequently initiate a recruitment phase, during which additional like-transcripts are incorporated to generate homotypic clusters. How this process is directed is poorly understood. Only for the most extensively studied germ plasm RNA, nanos (nos), have cis acting sequences (zipcodes) or trans acting protein factors that mediate the localization mechanism been identified.

To determine how germ plasm transcripts are incorporated into polar granules, I sought to identify zipcodes that mediate this process. By dissecting the 3' untranslated regions of two polar granule transcripts, germ cell-less (gcl) and polar granule component (pgc), I showed that like nos, these localization signals are composed of multiple partially functionally redundant zipcodes. Moreover, I demonstrated that docking and self-recruitment of pgc are mediated by distinct classes of zipcodes. In addition, perturbation or multimerization of enrichment zipcodes directly influences the average mRNA content of homotypic clusters, without affecting the propensity with which they develop. Together, these data support a model in which enrichment zipcode modularity is necessary for homotypic cluster growth. I also discovered that germ plasm mRNAs often form multiple independent homotypic clusters within a polar granule, contributing to the overall efficacy of germ plasm transcript enrichment.

Through RNA affinity isolation I identified numerous proteins that associate with the pgc 3'UTR. In a targeted RNAi screen, I isolated 6 candidate localization factors and present a preliminary characterization of their roles in germ plasm mRNA enrichment. In sum, this thesis sheds light on the critical cis and trans molecular factors that mediate an understudied RNA localization mechanism.