Control of Pre-mRNA Metabolism in Drosophila

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Intellectual Merit: Eukaryotic protein-coding genes are expressed through a multi-step process that involves transcription of the DNA sequences of genes into precursor mRNAs (pre-mRNAs) that are processed to a mature form (mRNAs) before being exported from the nucleus to the cytoplasm for translation into proteins. To ensure the efficient synthesis of functional proteins and, thus, that cells are able to function normally, RNA quality is monitored at each stage of gene expression by RNA surveillance components, whose role is to detect defective or otherwise problematic RNAs and stimulate their rapid degradation. Most of the research on nuclear RNA surveillance to date has been performed using yeast, a single-celled eukaryote, and relatively little is known about this process in more complex organisms. The goal of this project is to investigate this process in the fruit fly Drosophila melanogaster, a popular animal model system. Recent evidence from this lab indicates that Su(s), an RNA-binding protein in Drosophila, is a nuclear RNA quality control cofactor that stimulates the degradation of aberrant nuclear pre-mRNA and noncoding RNA derived from transposable DNA elements. This research will further explore the mechanism involved. Since several different RNA quality control systems operate in eukaryotic cells, it is important to determine the specific pathway and stage of gene expression where Su(s) protein acts. The first aim will address this point by identifying the other proteins that function in conjunction with Su(s). Two different approaches will be used to do this. First, Su(s) will be immunoaffinity-purified, and the polypeptides in the Su(s) complex will be identified by mass spectroscopy analysis. In addition, an RNA interference (RNAi)-based screen will be performed in cultured Drosophila cells. This screen is based on the expectation that RNAi-mediated depletion of proteins that function in the same cellular process as Su(s) will lead to increased accumulation of RNAs that are normally targeted for degradation by Su(s), and this will be evaluated experimentally. The second aim of this project is to define the RNA sequences that are bound by Su(s) in living cells. Two approaches, both involving cultured cells, will be employed to accomplish this aim. An established reporter gene assay will be used for precise mapping of a Su(s) target site in a heat-shock induced RNA. In addition, a large number of new Su(s) target sites will be identified by UV crosslinking and immunoaffinity-purification of Su(s)-RNA complexes followed by high-throughput sequencing of the bound RNAs. Subsequently, computational analysis will be performed to define the consensus sequences and to determine if Su(s) binding sites are enriched in a particular region of RNAs, e.g. coding versus noncoding sequences. The results of this research will provide new insights into nuclear RNA quality control mechanisms in multicellular organisms and help to clarify how defective RNAs are identified by the nuclear surveillance machinery. The presence of multiple RNA quality control systems and the high degree of similarity in the general components of these systems indicate that this is a vitally important process for most, if not all, eukaryotic organisms.

Broader Impacts: In addition to advancing the understanding of RNA quality control mechanisms in higher organisms, this research is making a significant contribution to the development of human resources in science. This ongoing project has supported the training of numerous graduate and undergraduate students. In addition, throughout her career the PI has been active in efforts at multiple levels, within the university and beyond, to promote the academic success of undergraduate science majors and their participation in undergraduate research. The PI is also committed to activities that enhance the participation and success of underrepresented groups in scientific research and academia.