The long range objective of the proposed study is to characterize the roles of small nucleolar RNAs (snoRNAs) in ribosomal RNA (rRNA) processing in the parasitic protozoan, Trypanosoma brucei. Studies with this evolutionarily ancient organism should elucidate both general and distinct aspects of eukaryotic rRNA processing. Maturation of rRNAs in higher eukaryotes requires participation of snoRNAs, found in association with the nucleolar protein, fibrillarin. The abundant U3 snoRNA appears to be required for primary cleavage of the precursor rRNA transcript within the 5' external transcribed spacers (ETS), as well as for other events. The T. brucei U3 homologs, RNA B, has been characterized and it is of atypical small size and sequence, yet RNA B is similar to other snoRNAs in its association with fibrillarin and pre-rRNAs. The immediate objectives of these studies will be: 1. Examination of the structure of RNA b. RNase digestion and chemical mapping of naked RNA B and of RNA B within RNP forms will assess the unique structure of this U3 snoRNA homolog and will identify protein- protected RNA B sequences. RNA B structural information will be compared with that for other U3 snoRNAs to evaluate similar and divergent features. 2. Assessment of RNA B functions in rRNA processing. Chemical cross- links between RNA B and substrate pre-rRNAs will be produced in vivo and in vitro to identify sites of interaction. RNA B-dependent rRNA processing events will be identified by depletion of RNA B in situ and in vivo. Pre-rRNA sequences implicated in interaction s with RNA B will be mutagenized and their involvement in processing studied in vivo. U3- dependent cleavage of the 5-ETS may commence rRNA processing in all eukaryotes; conservation of this event will be examined initially in T. brucei. Genetic complementation studies may be used to test the hypothesis that RNA B interacts with ETS sequences by hydrogen bonding. 3. Characterization of additional RNA components which may participate in ribosome biogenesis. Pre-rRNA sequences required for rRNA maturation, in addition to sequences which interaction with RNA B, will be identified. Several T. brucei small RNAs found associated with fibrillarin will be sequenced and characterized as analogs of known snoRNAs or as unique snoRNAs. Homologs of other snaRNAs functionally important in higher eukaryotes, such as U14 snoRNA, will be identified through sequence and/or important in higher eukaryotes, such as U14 snoRNA, will be identified through sequence and/or structural homology. Potentially, candidate snoRNA will be identified through sequence and/or structural homology. Potentially, candidate snoRNAs will be identified by crosslinking either with RNA B or pre-rRNAs. Putative roles of identified snoRNAs in rRHA processing will be examined as for RNA B. This study will provide insight into fundamental mechanisms of ribosome biogenesis. Features unique to parasitic trypanosomatids may prove useful for drug development.
