This proposal concerns the structure, organization and function of hnRNP particles from HeLa cells. These nucleoprotein complexes are formed on nascent transcripts in the nuclei of eukaryotic cells and are the sites where functional mRNAs are generated. A group of about 6 related and evolutionarily conserved core proteins accounts for most of the protein mass of hnRNP. These proteins exhibit an enzymatic activity and appear to be the structural components of hnRNP. The proposed approaches involve: 1. Structure: a) analysis of hydrodynamic properties and electron microscopy of complexes formed between core proteins and polynucleotides of defined lengths to describe the basic architecture of hnRNP, b) determination of the topography of RNA within the native and reconstituted hnRNP particles by electron spectroscopic imaging, c) in vitro reconstitution of an hnRNP particle containing an unprocessed transcript of a defined gene in order to i) investigate the effect of snRNP on reconstitution, and ii) explore the possible sequence-specific organization of hnRNP. 2. Function: a) investigating the effect of anti-core protein antibodies on the synthesis of hnRNA and on mRNA splicing using several crude but accurate cell-free transcription systems, b) use of these antibodies to assess the effect of core proteins on the transport of mRNA in permeabilized cells, c) investigating the fate of hnRNP proteins upon inhibition of transcription, d) examining hnRNP complexes in heat-shocked cells. 3. Continuation of previous work on Artemia salina hnRNP: a) completion of the sequencing of protein HD40, the major hnRNP protein of Artemia, from the sequence of available HD40 cDNA clones, and generating a map of RNA binding domains of HD40 from the established partial amino acid sequences of RNA binding peptides of HD40, b) reconstruction of 3-D images of, and localization of RNA within, native Artemia hnRNP and model complexes formed by protein HD40 and polynucleotides.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023705-16
Application #
3271851
Study Section
Molecular Biology Study Section (MBY)
Project Start
1976-06-01
Project End
1991-08-31
Budget Start
1988-09-01
Budget End
1989-08-31
Support Year
16
Fiscal Year
1988
Total Cost
Indirect Cost
Name
New York University
Department
Type
Schools of Medicine
DUNS #
004514360
City
New York
State
NY
Country
United States
Zip Code
10012
Szer, I S; Sierakowska, H; Szer, W (1994) A novel autoantibody to the putative oncoprotein DEK in pauciarticular onset juvenile rheumatoid arthritis. J Rheumatol 21:2136-42
Sierakowska, H; Williams, K R; Szer, I S et al. (1993) The putative oncoprotein DEK, part of a chimera protein associated with acute myeloid leukaemia, is an autoantigen in juvenile rheumatoid arthritis. Clin Exp Immunol 94:435-9
Szer, W; Sierakowska, H; Szer, I S (1991) Antinuclear antibody profile in juvenile rheumatoid arthritis. J Rheumatol 18:401-8
Khan, F A; Jaiswal, A K; Szer, W (1991) Cloning and sequence analysis of a human type A/B hnRNP protein. FEBS Lett 290:159-61
Zuklys, K L; Szer, I S; Szer, W (1991) Autoantibodies to DNA topoisomerase II in juvenile rheumatoid arthritis. Clin Exp Immunol 84:245-9
Kumar, A; Sierakowska, H; Szer, W (1987) Purification and RNA binding properties of a C-type hnRNP protein from HeLa cells. J Biol Chem 262:17126-37
Kumar, A; Williams, K R; Szer, W (1986) Purification and domain structure of core hnRNP proteins A1 and A2 and their relationship to single-stranded DNA-binding proteins. J Biol Chem 261:11266-73
Cruz-Alvarez, M; Szer, W; Pellicer, A (1985) Cloning of cDNA sequences for an Artemia salina hnRNP protein: evidence for conservation through evolution. Nucleic Acids Res 13:3917-30