Abstract

In vivo analyses of RNA processing are impeded by two major problems: 1) The kinetics of the process must generally be inferred from steady-state levels of intermediates, so rapid processing events are essentially invisible, particularly if they occur cotranscriptionally. 2) The structures of RNA -protein complexes, within which almost all RNA processing and RNP assembly steps occur, generally remain poorly characterized. These limitations are particularly acute for low abundance members of complex populations, such as pre-mRNAs or ncRNAs. We have therefore been prioritizing techniques to overcome these limitations. Fast kinetic labeling supported by mathematical modeling to analyses rapid processing. UV crosslinking (CRAC) to determine sites of RNA -protein interaction. Crosslinking, ligation and sequencing of hybrids (CLASH) to experimentally identify RNA -RNA interactions. These techniques were developed to understand fundamental RNA metabolism in budding yeast – but are being applied in systems from pathogenic E.coli to virus-infected mammalian cells.