Reversible chemical cross-linking and ribonuclease digestion analysis of the organization of proteins in ribonucleoprotein particles

Abstract

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.

Sex-lethal Interacts with Splicing Factors In Vitro and In Vivo

“…For this purpose, we used the reversible cross-linker DTBP. This cross-linker is specific for short-distance interactions and has been used to examine the spatial relationships among components of multimeric assemblies, including snRNPs (9,26,27). In our experiment, immunoprecipitates of nuclear extracts treated or not with DTBP were digested with RNase, and after extensive washing, the crosslinking was reversed and the remaining proteins were analyzed by Western blotting.…”

The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles.

“…Like the histones (reviewed in reference 63), the core particle proteins are transcribed from multigene families (13,20,50), and findings described here which indicate that purified C-protein tetramers bind RNA in vitro in a highly self-cooperative manner and are located along the entire length of packaged RNA. Further evidence for this binding mode in vivo is seen in chemical cross-linking studies which reveal that the C-protein tetramers exist in oligomeric arrays in isolated monoparticles (41) and in the hnRNP complexes present in splicing-competent nuclear extracts (33,34). Cooperative RNA binding has been shown for purified (A2)3B1 tetramers (7) and for purified core protein Al (21).…”

“…The (A2)3B1 and (C1)3C2 tetramers have been isolated and partially characterized (4,7). The (A1)3B2 tetramer has not been isolated, but its existence is inferred from chemical cross-linking studies which reveal that Al exists in monoparticles as homotrimers (34,41) and in a 3:1 ratio with B2. Like the histones (reviewed in reference 63), the core particle proteins are transcribed from multigene families (13,20,50), and findings described here which indicate that purified C-protein tetramers bind RNA in vitro in a highly self-cooperative manner and are located along the entire length of packaged RNA.…”

The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles

“…This distribution is consistent with the on December 11, 2013 by MAIN LIBRARY http://mcb.asm.org/ Downloaded from C-PROTEIN FUNCTION IN hnRNP ASSEMBLY 519 findings described here which indicate that purified C-protein tetramers bind RNA in vitro in a highly self-cooperative manner and are located along the entire length of packaged RNA. Further evidence for this binding mode in vivo is seen in chemical cross-linking studies which reveal that the C-protein tetramers exist in oligomeric arrays in isolated monoparticles (41) and in the hnRNP complexes present in splicing-competent nuclear extracts (33,34). Cooperative RNA binding has been shown for purified (A2)3B1 tetramers (7) and for purified core protein Al (21).…”

“…The (A2)3B1 and (C1)3C2 tetramers have been isolated and partially characterized (4, 7). The (A1)3B2 tetramer has not been isolated, but its existence is inferred from chemical cross-linking studies which reveal that Al exists in monoparticles as homotrimers (34,41) and in a 3:1 ratio with B2. Like the histones (reviewed in reference 63), the core particle proteins are transcribed from multigene families (13,20,50), and they reveal charge and nonallelic variants (14,15,20) which can be resolved in various two-parameter electrophoretic systems (11,17,35,38,46,65).…”