Post-transcriptional Regulation of Heparan Sulfate Glycan Synthesis

University Of California-Irvine, Irvine CA

Investigators

Abstract

Living cells use genetic information to make proteins that carry out the essential functions necessary to survive, grow and multiply. DNA is first copied into messenger RNA (mRNA) that provides the instructions that are translated to make proteins. For most mRNAs, the cellular machinery recognizes the cap located at the beginning of the message as well as other signals to decide where to start translation. However up to 10% of cellular mRNAs and many viruses, use different, poorly understood, signals called internal ribosome entry sites (IRES) to determine where to initiate protein synthesis. This project will provide new insights into the means by which cells carry out this process and how it is regulated. Earlier studies have shown that mRNAs for several genes that synthesize heparan sulfate proteoglycans (HSPGs; proteins with long attached sugar chains) contain IRES, and that translation of their mRNAs is regulated during development. The goal of this project is to identify the signals that mark IRES, as well as genes that control the translation of these IRES-containing mRNAs. This research will use the fruitfly Drosophila, because it allows application of powerful genetic and molecular approaches that cannot be easily employed in other animals. Importantly, IRES and proteoglycans are present in all multicellular animals, so results from this work will be useful in understanding their roles in many organisms ranging from worms to humans. In addition to PhD scientists, undergraduate students will participate in the proposed research, helping them develop a deeper understanding of scientific principles as well as planning, analytical and intellectual skills. Undergraduates who have trained in the lab will serve as peer tutors for a large genetics class. The effectiveness of 'research trained' and 'regular' peer tutors will be compared to test if this approach can be used to enhance student performance in large lecture classes more broadly. HSPGs are extracellular and cell surface macromolecules found in all multicellular animals. They play key roles in development by affecting the transport, stability and signaling activity of multiple growth factors. Earlier studies showed that HSPGs are absent from early Drosophila embryos due to inhibited translation of GAG synthetic enzyme mRNAs. Further, transcripts for the Drosophila GAG chain co-polymerase Tout velu (Ttv; EXT1 in vertebrates) and the N-deacetylase/sulfotransferase (NDST) Sulfateless (Sfl) were found to contain IRESs that are negatively regulated during the first three hours of development. Homologous transcripts in other species have complex 5?UTRs, and the 5? UTR from human EXT1 also mediates temporally regulated translation in the early fly embryo suggesting a conserved regulatory mechanism. The project examines the mechanisms underlying this translational regulation by pursuing three Aims: (1) To determine whether translational regulation and/or the presence of IRES elements is a common property of other HSPG biosynthetic enzyme transcripts. (2) To identify cis elements essential for translational regulation of the HSPG synthetic enzymes, Ttv and Sfl. (3) To identify trans acting factors required for the regulation of GAG chain synthesis and IRES function through genetic and molecular screens.