Systematic Analysis of Protein-RNA Interactions in Drosophila

Download or read book Systematic Analysis of Protein-RNA Interactions in Drosophila written by John Laver and published by . This book was released on 2016 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Post-transcriptional regulation of gene expression, through the control of mRNA splicing, polyadenylation, nuclear export, localization, translation, and stability, is essential for achieving appropriate temporal and spatial patterns of protein expression. This regulation is mediated by trans-acting factors, such as RNA-binding proteins (RBPs) and non-coding RNAs, which associate with specific mRNA targets through the recognition of sequence- or structure-based cis-elements present in the transcripts. The genomes of most organisms encode hundreds of RBPs, each of which likely associates with hundreds of mRNAs. Thus, a genome-wide view of the regulation being mediated by all trans-factors is essential for a complete understanding of post-transcriptional control. While post-transcriptional regulation is crucial in all biological systems, it has a particularly prominent role during early embryo development, as during this time there is no transcription from the zygotic genome of the embryo, and, thus, gene expression and development is controlled entirely post-transcriptionally. In this thesis, I describe my efforts towards obtaining a global understanding of post-transcriptional regulation in early Drosophila melanogaster embryos, through the development and use of synthetic antibodies as tools to identify, genome-wide, RBP-mRNA interactions. First, I demonstrated that synthetic antibodies generated against RBPs can be used as tools to identify RBP-associated mRNAs through immunoprecipitation-based approaches, or, conversely, to disrupt RBP-mRNA interactions. I then used synthetic antibodies to identify the entire complement of mRNAs associated with 3 developmentally-important RBPs: the double-stranded RBP Staufen, the TRIM-NHL protein Brain Tumor, and the PUF protein Pumilio. Computational analyses of these mRNAs revealed: (1) novel cis-elements likely mediating the mRNA-binding activity of Staufen and Brain Tumor; (2) that, unexpectedly, Brain Tumor and Pumilio function largely independently of each other in early embryos; and, (3) a novel role for Brain Tumor in promoting mRNA decay, which was demonstrated through a transcriptome-wide analysis of mRNA levels in brain tumor mutant embryos. To facilitate a truly genome-wide analysis of RBP-mRNA interactions, we developed a high-throughput pipeline for production of synthetic antibodies, and used this pipeline to generate 279 antibodies against 61 RBPs. In future this pipeline and the antibodies generated will allow for global studies of post-transcriptional regulation.