In recent years a tremendous amount of excitement has surrounded the finding that regulated proteolysis is intricately involved in controlling the cell cycle in yeast and mammals. We have found that regulated ribonuclease digestion also plays a crucial part in controlling the cell cycle in yeast. The major objective for this proposal is to determine the sites, mechanism and regulation of mRNA destabilization by the ribonucleoprotein endoribonuclease RNase MRP in late telophase of the cell division cycle. We have discovered that mutations of different components of the RNase MRP enzyme complex lead to a cell division cycle (CDC) arrest. Divided nuclei and an elongated spindle characterize this late cell cycle arrest, in which cells fail to disassemble their spindle or to undergo cytokinesis. This M to G1 arrest is classically defined by the CDC5, DBF2 group of mutations. These M to G1 mutants lead to the inability to turn off the Clb1&2/Cdc28 cyclin/kinase. Inactivation of the kinase is required for disassembly of the spindle, cytokinesis and the ability to enter G1. Results indicate that the cell division cycle arrest in RNase MRP mutants is the result of a failure to rapidly degrade the CLB2 mRNA. Increased CLB2 mRNA leads to increased CIb2 protein, associated kinase activity and cell cycle arrest. The specific goals during the project period will include characterizing the pathway of CLB2 mRNA degradation and determining direct targets of RNase MRP in addition to CLB2 mRNA. Genome technology will be used to identify all of the potential RNase MRP substrates in a cell. A series of biochemical and genetic analyses will identify instability elements and the direct sites of RNase MRP action. Lastly, we will investigate the regulation of RNase MRP during the cell cycle and determine the means of that regulation. The significance of the role of RNase MRP in the M to G1 transition cannot be overstated. Regulated degradation of specific mRNAs may play as large a part in cell cycle control as regulated proteolysis. Many of these late acting genes have human homologues (PTEN/MMAC1, PIk, Polo, p55CDC) that are intimately involved in regulating the cell cycle and cell proliferation. Humans also have an RNase MRP complex with RNA and protein subunits similar to the yeast enzyme. Mutations in human RNase MRP cause a pleotropic disease, Cartilage Hair Hypoplasia. The manifestations of this disease are caused by a general cell proliferation defect, similar to what we see in yeast. This will allow us to use a simple genetic model system to lend insights into human disease.
