Investigation of RNA polymerase III (pol III) was continued, focusing on the human La antigen. Pol III produces a variety of RNAs that include 5S rRNA and tRNAs, abundant structural RNAs essential for protein synthesis and cell proliferation. The pol III enzyme complex is assisted by ancillary transcription factors (TF) that direct accurate RNA synthesis. Certain RNAs encoded by viral genomes are also transcribed by pol III, and some viral transforming proteins modulate the activity of pol III TFs. La is a regulatory phosphoprotein that participates in the biogenesis of pol III transcripts. Patients suffering from systemic lupus erythematosous, neonatal lupus, and Sjogren=s syndrome contain antibodies to La although why La is autoantigenic in these patients is still unknown. Pol III also transcribes human Alu sequences, highly repetitive genetic elements found dispersed at nearly one million sites throughout the genome. Retroinsertion of cDNA copies of Alu RNAs into the genome causes genetic disorders. Viral infection, heat shock and other stresses induce Alu transcription yet the physiologic function of Alu RNAs and the mechanisms that regulate their expression remain largely unknown. Posttranscriptionally, Alu RNAs are bound by SRP9/14, a heterodimeric factor that binds to a variety of Alu-homologous RNAs. Complexed with 7S L RNA in the signal recognition particle, SRP9/14 contributes to the subcellular localization and translational control of certain mRNAs although the functions of other Alu RNA-SRP9/14 complexes have yet to be determined. Understanding the mechanisms by which La and other proteins function in pathways of biogenesis of pol III transcripts is a major goal of this unit.Summary of Major Findings:1. The highly-abundant, brain-specific Alu-homologous small RNA known as BC200 was shown to be tightly associated with SRP9/14 protein in brain.2. The ability of the human La antigen to control the maturation of human tRNAiMet was shown to be dependent on the phosphorylation state of La serine 366. This represents the first evidence that 5= processing of tRNA precursors by RNase P may be regulatable in eukaryotes.3. Structure-function analysis revealed a novel mode of RNA binding for La; recognition of the unique 5= triphosphate of a nascent pol III transcript by the C-terminus basic region of La.4. Biochemical studies demonstrated mechanistic links between the pol III terminator element, recycling of the pol III transcription complex, and the nascent RNA terminus-binding protein, La.5. A pol III terminator-dependent tRNA opal suppressor gene was developed for use in fission yeast. This was shown to require La for expression and represents a novel system to study the relationship between pol III termination and La function in vivo.
Showing the most recent 10 out of 28 publications
