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.

Abstract

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.

Antibody reactivity to the hres‐1 endogenous retroviral element identifies a subset of patients with systemic lupus erythematosus and overlap syndromes

“…Small nuclear RNP was purified from HeLa cell nuclear extracts by immunoaffinity chromatography on an anti-2,2,7-trimethylguanosinespecific antibody-coupled Sepharose 4B column, as previously described (41,42).…”

The 5′-terminal sequence of U1 RNA complementary to the consensus 5′ splice site of hnRNA is single-stranded in intact U1 snRNP particles

“…Native Ul snRNP particles from 32P-labeled HeLa cells were isolated by immune affinity chromatography with antibodies specific for 2,2,7-trimethylguanosine (m3G) (18). Antibody-bound snRNPs were desorbed from the affinity column by competition with excess nucleoside m3G.…”

“…Owing to the presence of m3G in the cap structures of the nucleoplasmic snRNA species Ul, U2, U4 and U5, snRNP particles Ul are isolated along with the other snRNP species. Although U6 RNA lacks an m3G residue, U6 RNP is also co-isolated by this procedure, probably due to its association with one of the other snRNPs (see also references 18,26). As it has been demonstrated that snRNP species Ul exists as a discrete particle (5), the presence of the other snRNPs in the reaction mixtures should not affect the RNase H cleavage experiments.…”

“…One of the oligodeoxynucleotides employed is also complementary to a region of Ul RNA between nucleotides 132 and 136 which is part of the so-called domain A (17). The snRNPs used for this study were purified by immune affinity chromatography with antibodies specific for the 2,2,7-trimethylguanosine(m3G)-containing cap structure of the snRNAs (18 (18). The isolation of snRNPs from nuclear extract of about 9-10 x 107 cells using a 1.5 ml Protein A-Sepharose anti-m3G column was performed as described (18), except that snRNPs were desorbed from the affinity column with 2,2,7-trimethylguanosine in a buffer containing 20 mM Tris-HCl, pH 8, 10 mM MgCl2, 100 mM KCl.…”

“…The snRNPs used for this study were purified by immune affinity chromatography with antibodies specific for the 2,2,7-trimethylguanosine(m3G)-containing cap structure of the snRNAs (18 (18). The isolation of snRNPs from nuclear extract of about 9-10 x 107 cells using a 1.5 ml Protein A-Sepharose anti-m3G column was performed as described (18), except that snRNPs were desorbed from the affinity column with 2,2,7-trimethylguanosine in a buffer containing 20 mM Tris-HCl, pH 8, 10 mM MgCl2, 100 mM KCl. Column fractions containing snRNPs were either used directly for RNase H assays or were immediately submitted to phenol extraction (see below).…”