Investigation of the RNA polymerase III (pol III) system of eukaryotes was continued, focusing on transcription termination and the function of the human La antigen. The human La antigen is a prevelant target of autoantibodies in patients suffering from rheumatic disorders such as neonatal lupus, systemic lupus erythematosus and Sjogrens syndrome. Pol III produces small RNAs, including the 5S rRNA and tRNAs essential for protein synthesis, as well as certain virus-encoded transcripts. Pol III is a complex enzyme composed of seventeen subunits with multiple catalytic activities. Human La antigen is a regulatory phosphoprotein that has been shown to serve as a termination factor for pol III, stimulating transcription and directing the posttranscripional maturation of the transcripts, the latter of which includes end-processing, intron removal, base modification, and proper RNA folding. The La phosphoprotein interacts with pol III transcripts by recognizing their 3 terminal UUU-OH motifs (which result from transcription termination), found at the ends of all newly synthesized pol III transcripts. Nonphosphorylated La is localized in the cytoplasm where it interacts with certain cellular and viral mRNAs including HIV RNA, hepatitis C RNA. poliovirus mRNA and others. La interacts with the internal ribosome entry sites (IRES) of viral and cellular mRNAs to modulate their translation. Some viral-encoded factors, including the adenovirus E1A protein, modulate pol III activity. Poliovirus protease-3 protein cleaves the phosphorylation site and nuclear localization signals away from the body of the La antigen, leading to a mainly cytoplasmic localization that facilitates La-mediated translation of poliovirus mRNA. We are interested in the tRNA anticodon modifications that impact the codon-usage specific translation of specific mRNAs involved in growth and development. Understanding the mechanisms by which La and other pol III subunits function in RNA production pathways, and how these pathways are controlled during normal development and cellular proliferation, are major goals of this Section. Summary of Major Findings: 1. The gene encoding the La antigen is required for mouse development beyond the blastocyst stage of growth. 2. The La antigen is required for the establishment of mouse embryonic stem (ES) cell lines in the laboratory. 3. The pre-tRNA chaperone activity and RNA 3 end protection activity of the human La antigen have been distinguished by different tRNA alleles and by point mutants that show these activities to map to different motifs in the La protein. 3. Human La antigen serves two distinct functions in tRNA maturation. 4. The interaction between the pol III subunits, Rpc11p and Rpc2p, is important for digestive tract development during zebra fish embryogenesis. We extended our focus to La-related proteins (LARPs), including human LARP4, a mRNA-associated protein.
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