The long-term goal of our research is to understand, in detail, the mechanism and function of internal modifications of spliceosomal snRNAs and mRNA/pre-mRNA. A thorough understanding of the nature of these modifications will contribute greatly to our knowledge of gene regulation (in other words, how protein expression is regulated in the cell). Toward this goal, we propose three specific aims. (1) Identification and characterization of enzymatic activities for spliceosomal snRNA modifications. Using affinity chromatography, we will isolate spliceosomal snRNA-specific modifying enzymes (sno/scaRNPs) from Xenopus oocyte nuclear extracts. Given that each sno/scaRNP contains a set of common proteins and a unique guide RNA, we will purify the RNA components and identify them through the construction of an RNA library. We will also screen the yeast GST-ORF protein library for modifying activities (sno/scaRNPs and/or protein-only enzymes) catalyzing yeast spliceosomal snRNA modifications. Through systematic characterization, a complete set of spliceosomal snRNA-specific modifying activities will likely be identified, thus enhancing our understanding of the mechanism of spliceosomal snRNA modifications. (2) Functional analysis of U2 snRNA modifications. Using Xenopus oocytes, we will first dissect and uncouple the pseudouridines (in the U2 branch site recognition region) important for snRNP assembly from those important for pre-mRNA splicing. We will then further analyze those pseudouridines important for splicing using SELEX to allow a detailed understanding of how they contribute to the branch site recognition during splicing. We will also carry out deletional analysis to establish the importance of U2 pseudouridines in yeast. Vertebrate U2 contains three more pseudouridines in the branch site recognition region compared with yeast U2, and thus we will introduce extra pseudouridines into yeast U2 to mimic the vertebrate U2 modifications and test, using the yeast ACT1-CUP1 reporter gene system, whether more pseudouridines can tolerate the diverse branch site sequences found in vertebrate pre-mRNAs. This analysis should complement the above SELEX analysis in Xenopus oocytes. (3) Identification and characterization of RNA-guided mRNA/pre-mRNA modifications. Essentially nothing is known about mRNA/pre-mRNA modifications. Our preliminary experiments suggest that mRNA and perhaps pre-mRNA contain 2'-O-methylated nucleotides and pseudouridines. We will use the available sensitive methods, including those based on nuclease-TLC, primer-extension, site-specific RNase H cleavage, and hybridization using modified oligonucleotide probes, to systematically characterize and possibly quantify 2'-O-methylation and pseudouridylation in mRNA/pre-mRNA. Identification of mRNA/pre- mRNA modifications will form the basis for future functional analyses. ? ? ?
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