The specific aims of this proposal are (1) to study the involvement of HeLa cell components in primed influenza mRNA transcription--these studies will include characterization of capped cellular RNA segments transferred onto flu NS mRNA during cell-free synthesis; characterization of the 5 feet terminal RNA segments cleaved by the flu-associated endonuclease in vitro from HeLa cell mRNA and hnRNA populations; seeking activities capable of cleaving capped RNA's to form flu mRNA primers in HeLa nuclear and cytoplasmic subcellular fractions prepared from normal and flu-infected cells; and to carry out flu mRNA transcription in a HeLa cell extract capable of cellular mRNA synthesis and processing. (2) To seek primed transcription by HeLa cell RNA polymerase II, using various cloned cellular DNA genes and adenovirus, using cleaved RNA segments active in the flu system as primers; mixed HeLA cell DNA templates will also be utilized, and the extent of uptake and specificity of any primer RNA populations ascertained. (3) To study RNA signals for specific RNA processing, including characterizing the RNA subunit of HeLA cell RNase P; isolating RNA's from HeLa subcellular fractions capable of specific flu mRNA processing; characterizing the two RNA co-factors involved in removing Alu family transcripts from HeLa cell hnRNA: 2 feet 5 feet oligo(A) and double-stranded RNA regions of the hnRNA substrate; and to study RNA ligation and circularization to seek both protein-level and RNA-level reactions. Methods to be used include in vitro transcription and oligo (dT) column and DNA cellulose affinity column chromatography of viral and cellular mRNA transcripts; 32P in vivo labeling and 125I-labeling in vitro; RNA fingerprinting and sequence analysis of labeled RNA primer segments; and cellulose CF11 and polyacrylamide gel electrophoretic isolation of specific RNA's; E. coli RNase III and specific subcellular fractions of HeLa cells capable of RNA processing reactions will also be employed. These studies support our long term goal of assessing the role of RNA signals in gene regulation in two ways: first, the specific reactions of the primed flu mRNA transcription system allow us to develop assays to find out once and for all whether such reactions occur in the cell; and second, RNA subunits and co-factors inspecific RNA processing reactions comprise and unprecedented opportunity to study gene regulation at this level.
