Purification of snRNPs U1, U2, U4, U5 and U6 with 2,2,7-trimethylguanosine-specific antibody and definition of their constituent proteins reacting with anti-Sm and anti-(U1)RNP antisera.
Small nuclear ribonucleoprotein particles (snRNPs) of the U-snRNP class from Ehrlich ascites tumor cells were purified in a one-step procedure by affinity chromatography with antibodies specific for 2,2,7-trimethylguanosine (m23.2.7G), which is part of the 5'-terminal cap structure of snRNAs U1-U5. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess nucleoside m23.2.7G; this guarantees maintenance of their native structure. The snRNPs U1, U2, U4, U5 and U6 can be recovered quantitatively from nuclear extracts by this procedure. Co-isolation of U6 snRNP must be due to interactions between this and other snRNPs, as anti-m23.2.7G antibodies do not react with deproteinized U6 snRNA. We have so far defined nine proteins of approximate mol. wts. 10 000, 12 000, 13 000, 16 000, 21 000, 28 000, 32 000, 34 000 and 75 000. Purified snRNPs react with anti-(U1)RNP and with anti-Sm antisera from patients with mixed connective tissue disease and from MRL/l mice. As determined by the protein blotting technique, six of the snRNP polypeptides, characterized by apparent mol. wts. 13 000, 16 000, 21 000, 28 000, 34 000 and 75 000, bear antigenic determinants for one or the other of the above autoantibody classes. This suggests strongly that the U-snRNPs produced by the procedure described here are indeed representative of the snRNPs in the cell. With highly purified snRNPs available, investigation of possible enzymic functions of the particles may now be undertaken.
D000818:Animals; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001324:Autoantigens; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism; D002846:Chromatography, Affinity; D006151:Guanosine / Q000031:analogs & derivatives / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification; D012313:RNA / Q000378:metabolism; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins / Q000276:immunology / Q000302:isolation & purification*; D017411:Ribonucleoproteins, Small Nuclear; D055517:snRNP Core Proteins
D000941:Antigens; D001324:Autoantigens; D011506:Proteins; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D055517:snRNP Core Proteins; D006151:Guanosine; C017778:N(2),N(2),7-trimethylguanosine; D012313:RNA