The processing of RNA and the assembly of RNA-protein complexes (RNPs) are among the most important pathways in all organisms. Their key significance is underlined by the observation that a disproportionate fraction of yeast genes involved in these processes are essential for viability and highly conserved from yeast to humans. Eukaryotic pathways of RNP maturation are quite surprisingly complex when analysed in detail, and all are monitored by RNA surveillance activities. Two basic question emerge from studies of many RNP assembly and RNA processing pathways: Why are they so complicated and, given this complexity, how are “defective” particles distinguished from “normal” intermediates and selectively targeted for degradation? We are addressing these questions using a combination of biochemistry, cell biology, mathematical modeling and genetics, for which budding yeast is a particularly suitable system. Ribosome synthesis We have developed techniques for the rapid and accurate identification of protein binding sites on RNA using UV- induced cross-linking and analysis of cDNAs (CRAC). Analyses of many ribosome synthesis factors are starting to establish detailed structural models for the pre-40S and pre-60S complexes.
Pre-rRNA processing analyses have also shown that, contrary to the previous understanding in the field, the last step in maturation of the 5.8S rRNA takes place only after the pre-ribosomal particles have been exported to the cytoplasm. New techniques have allowed us to obtain and use high-resolution kinetic data to model the yeast rRNA processing pathway, yielding insights that could not readily be obtained by “traditional” analyses. Simple equations for the steps involved in ribosome synthesis, beyond RNA processing itself, led us to develop a new model based on energy-dependent “kinetic proof reading” mechanisms. These analyses suggest that the high complexity of RNP maturation may have been selected to increase overall fidelity. We have also investigated the effects of changes in the coding-sequence on gene expression in Escherichia coli. Functional analyses of ncRNAs Recent analyses have shown that non-protein-coding RNA (ncRNAs) are very numerous in the human and yeast genomes. We have analyzed the functions of specific ncRNAs and shown roles in DNA stability and the regulation of the expression of protein-coding genes via induced changes in chromatin structure. CRAC is now being used to map the binding sites for RNA surveillance factors on the ncRNA population.