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.
