The purpose of this proposal is to continue and extend an investigation of the involvement of nucleolar ribonucleases in ribosomal RNA processing. Nucleoli, prepared from Ehrlich ascites carcinoma cells, provide a relatively pure source of ribosomal RNA precursors and processing products as well as the starting material from which to isolate enzymic components. These enzymic components, in particular ribonucleases, will be dissociated and purified from the nucleolar system in order to characterize their enzymic and physical properties. Preribosomal 45S RNA and its 80S ribonucleoprotein particle will be labeled and purified from nucleoli to be used as substrates to study the patterns of cleavage by nucleolar ribonucleases. Cloned 18S and 28S mouse ribosonal DNA hybridization probes will be used to distinguish the sequences represented in the ribonuclease patterns of 45S preribosonal RNA. S1 nuclease mapping studies will be carried out with specific ribosomal DNA restriction fragments in order to precisely define the ribonuclease cleavage positions. In addition, it is the intent of this proposal to develop an in vitro system to examine ribosomal RNA processing and, in particular, the enzymology of the cleavage reaction. To this purpose, in vitro processing will be studied in a soluble system with an easily obtainable precursor ribosomal RNA as substrate, and with a rapid and unambiguous assay for the processed products. This system will allow for the biochemical separation of the components involved in the processing reaction as well as their identification by reconstitution experiments. Taken together, this information will be used to establish in vivo function and the importance of these degradative processes in affecting RNA metabolism in mammalian tissue.
