5'-terminal caps of snRNAs are accessible for reaction with 2,2,7-trimethylguanosine-specific antibody in intact snRNPs.
Immune precipitation assays with antibodies specific for 2,2,7-trimethylguanosine (m2,2,7(3)G) have been used to study the accessibility of the 5'-terminal m2,2,7(3)G-containing caps of eucaryotic small nuclear RNAs (snRNAs) either as naked RNAs or in intact small nuclear ribonucleoprotein (snRNPs). The antibody selectively precipitates snRNA species U1a, U1b, U2, U4, and U5 from total deproteinized RNA isolated from Ehrlich ascites cells. Binding by the antibody occurs via the m2,2,7(3)G moiety of the snRNAs' caps, since complex formation with the antibody can be completely abolished by excess nucleoside m2,2,7(3)G. The specificity of the antibody is further demonstrated by the complete absence of reaction with deproteinized snRNA species U6, the 5' terminus of which does not contain m2,2,7(3)G. Most importantly, the cap structures of the snRNAs U1a, U1b, U2, U4, and U5 are also accessible for anti-m2,2,7(3)G IgGs when intact snRNPs are reacted with the antibody. In this case, snRNP species U6 is coprecipitated, suggesting that there are intermolecular interactions between this and other snRNPs. Our data demonstrate that the 5'-terminal regions of the above snRNAs are not protected by the snRNP proteins. This finding is of special interest for snRNP species U1, and is discussed in terms of a model which proposes that the 5'-terminal region of U1 participates in the proper alignment of splice junctions in eucaryotic pre-mRNAs (Lerner, M. R., Boyle, J.A., Mount, S.M., Wolin, S.L., and Steitz, J. A. (1980) Nature (Lond.) 283, 220-224).
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002286:Carcinoma, Ehrlich Tumor / Q000032:analysis*; D006151:Guanosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology; D007118:Immunoassay; D051379:Mice; D008970:Molecular Weight; D009698:Nucleoproteins / Q000032:analysis*; D012313:RNA / Q000032:analysis* / Q000276:immunology; D012315:RNA Caps / Q000032:analysis*; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins / Q000032:analysis* / Q000276:immunology; D017411:Ribonucleoproteins, Small Nuclear
D000906:Antibodies; D000936:Antigen-Antibody Complex; D009698:Nucleoproteins; D012315:RNA Caps; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D006151:Guanosine; C017778:N(2),N(2),7-trimethylguanosine; D012313:RNA