Our specific aims are (1) to study the possibility that RNA-primed transcription is of general significance in eukaryotic cells and viruses. Further studies on primed transcription in influenza virus infected HeLA cell extracts and their uninfected counterpartts will be conducted, with particular emphasis on the origin of a particular sub-population of RNA polymerase II products recently discovered in HeLa subcellular extracts. These may represent the first example of cellular transcripts whose synthesis is stimulated by addition of capped RNA's capable of serving as primers in the influenza virus transcription system. Two sorts of control experiments will be carried out in parallel to the above studies: (a) the mechanics of influenza viral RNA primed transcription in the novel HeLa cell extract system recently worked out in this laboratory will continue to be ascertained; and (b) a series of collaborative studies on what may be another authentic case of cellular RNA-primed transcription--that occurring in the trypanosome system--will be continued to provide another example of such a system. (2) A second aim is to study regulatory RNA in HeLa cells by focusing on two RNA's which carry out such a role--(a) 5S RNA, which regulates its own transcription and for which we have recently worked out an assay system correlating RNA structure and function; this assay will be applied to specific complexes between 5S rRNA and its transcriptional control complex with factor IIIA. (b) The RNa subunit of an RNA processing enzyme, RNase P, concentrating on the specific way in which this RNA (in HeLa cells and elsewhere) can promote the specific cleavage of a whole set of differing tRNA precursors. Methods to be used include in vitro transcription of RNA and DNA templates; purification of RNA populations by affinity and hybridization methods; two-dimensional gel electrophoretic RNA structural characterization; formation of specific UV-induced derivatives of RNA molecules in which a single, specific crosslink is introduced at a site of biologically active local tertiary structure; and conventional RNA and DNA fingerprinting and sequencing methods. The role of RNA signals in gene regulation will thus be further analyzed, and the extension of methods and pathways first worked out in viral systems will be extended to cellular populations, both normal and transformed.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028294-11
Application #
3275591
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1988-12-01
Project End
1990-07-31
Budget Start
1989-08-01
Budget End
1990-07-31
Support Year
11
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Type
Schools of Medicine
DUNS #
201373169
City
New York
State
NY
Country
United States
Zip Code
10065
Mellits, K H; Pe'ery, T; Manche, L et al. (1990) Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector. Nucleic Acids Res 18:5401-6
Branch, A D; Benenfeld, B J; Baroudy, B M et al. (1989) An ultraviolet-sensitive RNA structural element in a viroid-like domain of the hepatitis delta virus. Science 243:649-52
Hannon, G J; Maroney, P A; Branch, A et al. (1989) Accurate processing of human pre-rRNA in vitro. Mol Cell Biol 9:4422-31
Nicholson, A W; Niebling, K R; McOsker, P L et al. (1988) Accurate in vitro cleavage by RNase III of phosphorothioate-substituted RNA processing signals in bacteriophage T7 early mRNA. Nucleic Acids Res 16:1577-91
Freistadt, M S; Cross, G A; Robertson, H D (1988) Discontinuously synthesized mRNA from Trypanosoma brucei contains the highly methylated 5' cap structure, m7GpppA*A*C(2'-O)mU*A. J Biol Chem 263:15071-5
Freistadt, M S; Cross, G A; Branch, A D et al. (1987) Direct analysis of the mini-exon donor RNA of Trypanosoma brucei: detection of a novel cap structure also present in messenger RNA. Nucleic Acids Res 15:9861-79
Nicholson, A W; Frankfort, H M; Davis, N G et al. (1986) Direct characterization of influenza viral NS1 mRNA and related sequences from infected HeLa cells and a cell-free transcription system. Biochim Biophys Acta 868:153-63
Branch, A D; Benenfeld, B J; Robertson, H D (1985) Unusual properties of two branched RNA's with circular and linear components. Nucleic Acids Res 13:4889-903