Reversible chemical cross-linking and ribonuclease digestion analysis of the organization of proteins in ribonucleoprotein particles.
The organization of select proteins within ribonucleoprotein particles containing heterogeneous nuclear and uridine-rich small nuclear RNAs (hnRNP and UsnRNP respectively) was examined by chemical cross-linking and ribonuclease digestion using diagonal two dimensional PAGE and immunoblotting detection systems. Monoclonal antibodies specific for A2, C1 and C2 hnRNP proteins, detected these proteins at gel coordinates which suggested homotypic dimers and trimers of A2 and homotypic trimers, hexamers and larger multimers of C1 and C2. Ribonuclease digestion did not alter the cross-linking properties of hnRNP C1 and C2 proteins but did result in loss of A2 homotypic dimers and trimers. Blots simultaneously reacted with hnRNP specific monoclonal antibodies and autoimmune patient serum (RNP/Sm), or monoclonal antibodies reactive with the U1 snRNP specific 63 kDa protein and/or the UsnRNP common proteins B', B and D revealed no complexes which would indicate interactions between hnRNPs and UsnRNPs. The U1 UsnRNP specific 63 kDa protein appeared not to be cross-linked to UsnRNP common B', B and D proteins. The data also suggested that UsnRNP common protein D was cross-linkable to UsnRNP common proteins D', E and G but not to B' and B. The cross-linking properties of D were unaffected by ribonuclease digestion. In contrast, ribonuclease digestion resulted in an inability to cross-link select complexes containing either B' and B, or p63. The data suggest that both hnRNPs and UsnRNPs are comprised of RNA-dependent and RNA-independent protein-protein interactions.
Harris
S G
SG
Department of Pathology and Laboratory Medicine, University of Rochester, NY 14642.
Martin
T E
TE
Smith
H C
HC
D003432:Cross-Linking Reagents / Q000494:pharmacology*; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006801:Humans; D007096:Imidoesters / Q000494:pharmacology; D015151:Immunoblotting; D011506:Proteins / Q000032:analysis; D012260:Ribonucleases / Q000378:metabolism*; D012261:Ribonucleoproteins
D003432:Cross-Linking Reagents; D007096:Imidoesters; D011506:Proteins; D012261:Ribonucleoproteins; C007471:dimethyl dithiobispropionimidate; C005905:methyl 4-mercaptobutyrimidate; D012260:Ribonucleases