Biochemical and mass spectrometric analysis of interactions in Drosophila mRNA localization

Abstract

mRNA localization is a common mechanism of gene regulation, involved in a broad range of biological processes including embryonic patterning, asymmetric cell division and cell migration. In Drosophila oocytes, asymmetric deposition of maternal oskar (osk), nanos, gurken and bicoid mRNAs defines the future embryonic axes. This differential targeting of mRNAs in a defined spatio-temporal manner requires several trans-acting factors which assemble with the mRNA into messenger ribonucleoprotein particles (mRNPs). Many trans-acting factors are RNA-binding proteins (RBPs) that recognize specific cis-acting elements in the RNA, and often function both in the localization and translational regulation of the transcript. Although individual RBPs have been identified and extensively studied for their role in mRNA localization, less is known about their interaction network. Often the same RBPs bind to differentially localized transcripts and it is unclear how transcript specificity and differential targeting is achieved. A possibility is that while these RBPs form the core of the mRNP, a higher level of transcript-specific regulation comes from the regulatory partners that interact directly with them. To gain further insights into the functional components of an mRNP and possibly understand the regulation of RBPs, I performed co-purification studies of both a localizing mRNP and the RBPs associated with localizing transcripts in Drosophila. In the first part of the project, using a tandem affinity purification approach, I established a protocol to biochemically purify osk mRNP using the MS2 system. In the second part of the project, I immunoprecipitated six tagged RBPs that are known to regulate localization of one or more maternal mRNAs at different developmental stages. By employing mass spectrometry and subsequent statistical analysis, I identified proteins significantly enriched with each tagged RBP and constructed an interactome. By using co-immunoprecipitation assay in cultured HEK cells, I was able to validate several interactions identified in the mass spectrometric data, including 26 novel interactions of potential RBP regulators. This work presents the foundation for in vivo functional and co-localization studies, as well as in vitro structural characterization of the identified interactants, to fully understand the relevance of these interactions in the regulation of mRNA localization.