A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact, class U, small nuclear ribonucleoproteins as well as with 7-methylguanosine-capped RNAs.
A hybridoma secreting a monoclonal antibody (H-20) that recognizes the 2,2,7-trimethylguanosine(m3G)-containing cap structure of U snRNAs was derived from a mouse which was immunized with a m3G-containing human serum albumin conjugate. The antibody specifically reacts with intact small nuclear ribonucleoprotein particles, U snRNPs, and allows the snRNPs U1 to U6 to be isolated in one step from nuclear extracts of eucaryotic cells by affinity chromatography on a preparative scale. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess of the cross-reactive nucleoside 7-methylguanosine (m7G), which guarantees maintenance of their native structure. The 20 affinity column also allows the snRNPs U1, U2 and U5 to be separated from U4/U6 RNPs by sequential elution of the particles with m7G under differential salt concentrations. As determined by competitive radioimmunoassay and protein-A--Sepharose immunoprecipitation, mAb H-20 crossreacts with intact m7G cap structures. In particular we could show that non-denatured m7G-capped SP6/beta-globin RNA was precipitated efficiently by the antibody while GpppG-capped or non-capped RNAs did not react. Thus the monoclonal antibody H-20 should have a wide application, not only for studying the molecular biology and immunology of the U snRNPs from diverse organisms, but also for the characterization and isolation of m7G-capped transcripts.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D006151:Guanosine / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007118:Immunoassay; D051379:Mice; D008807:Mice, Inbred BALB C; D012315:RNA Caps / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011863:Radioimmunoassay; D012261:Ribonucleoproteins / Q000276:immunology*; D017411:Ribonucleoproteins, Small Nuclear; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D012315:RNA Caps; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D006151:Guanosine; C017778:N(2),N(2),7-trimethylguanosine