Chemical cross-linking of Sm and RNP antigenic proteins

Abstract

Nuclear extracts, competent for in vitro premessenger RNA splicing, were chemically cross-linked with thiol-reversible reagents in order to study the organization of proteins within ribonucleoprotein particles (RNPs) containing uridine-rich small nuclear RNAs (UsnRNPs). The distribution of select UsnRNP antigens within cross-linked complexes was determined by Western blotting of diagonal two-dimensional gels. On the basis of calculations from the molecular weights of cross-linked complexes containing UsnRNP common proteins B', B, and D, it is proposed that each of these proteins was associated with UsnRNP common proteins E and G. In addition, D' is proposed to be positioned close to D. The spatial distribution of UsnRNP common proteins was such that B' and B could not be cross-linked to D. The data also suggested that the 63-kDa U1 snRNP specific protein was cross-linked to other U1-specific proteins, particularly C, but not to the UsnRNP common proteins. We propose that part of the UsnRNP core of common proteins contains at least two asymmetrical copies of B':B:D:D':E:G with stoichiometries of 2:1:1:1:1:1 and 1:2:1:1:1:1.