We will continue our studies on eukaryotic ribosomal RNA (rRNA), an essential component of ribosomes found in all cells, to further understand basic mechanisms in both healthy and diseased cells. Previously, we determined the first primary sequence and secondary structure of 285 rRNA in a multicellular eukaryote. Using this as a foundation, we will test how other RNPs (U3 small nucleolar RNA = snoRNA and signal recognition particle = SRP) interact with ribosomes. Since no in vitro system exists for the complete processing of pre-rRNA, we will use Xenopus oocytes for our U3 snoRNA studies. Previously, we determined the primary sequence and secondary structure of Xenopus U3 snoRNP, and provided the first evidence, done in vivo, that U3 snoRNA plays a role in ribosomal RNA (rRNA) processing. Components that complex with U3 snoRNA during rRNA processing will be identified by using a biotinylated 2'-OMe oligonucleotide complementary to the U3 hinge region to pellet U3 high molecular weight complexes after sucrose gradient fractionation. The RNA components of these fractions will be identified on gels. Binding sites on U3 for association with components of the rRNA processing complex (""""""""processome"""""""") will be assayed using cross-linking with 4-thio-uridine. The cross-linked pre-RNAs and other snoRNAs will be identified by Northern blots and the position of the cross-links will be mapped. Also, proteins cross-linked to U3 snoRNA will be identified. Association with fibrillarin will be tested by immunoprecipitation of the U3 complexes. The functional importance of specific sites will be tested by oligo injection into oocytes to destroy intact endogenous U3 snoRNA, followed by a rescue with injected in vitro synthesized U3 RNA carrying mutations in each site to be tested. These multifaceted approaches should determine what components are complexed with U3 snoRNA, what their binding sites are and the functional roles of regions within U3 for rRNA processing. The internal transcribed spacer 2 (ITS 2) will be mutated to assay the effect this has on rRNA processing. If a suppressor U3 carrying compensatory changes in Box C to the ITS 2 mutation can rescue the processing phenotype, it will provide functional support for the proposed interaction between Box C of U3 and the ITS 2 of pre-rRNA. DMS modification of 7SL RNA in SRP will ascertain which nucleotides are critical for binding or release from ribosomes, and site directed mutagenesis will be used to support these deductions. 4-thio-uridine cross-links will identify positions in rRNA that associate with 7SL RNA and may play important roles for nascent peptide elongation (a process blocked by SRP binding to ribosomes).
