Small nuclear ribonucleoprotein particles (snRNPs) from eucaryotic cells can be fractionated on affinity columns prepared with antibodies of high affinity for 2,2,7-trimethylguanosine (m3G), which is present in the 5'-terminal caps of the snRNAs. While the snRNPs Ul, U2 and U5 are eluted with the nucleoside m3G in the presence of 0.1 M salt, the snRNP species U4 and U6 are only desorbed when the salt concentration is increased. The same fractionation pattern was likewise observed for snRNPs from HeLa or Ehrlich ascites tumor cells. Since U6 RNA lacks the m3G residue and its RNA does not react with anti-m3G, its co-chromatography with U4 RNP on anti-m3G affinity columns suggests either that discrete snRNPs U4 and U6 are intimately associated in nuclear extracts or that both RNAs are organized in one ribonucleoprotein particle. Further evidence for a U4/U6 RNP particle is obtained by sedimentation studies with purified snRNPs in sucrose gradients. Gel fractionation of RNAs shows identical distributions of snRNAs U4 and U6 in the gradient, and the U4/U6 RNP particle sediments faster than the snRNPs Ul or U2. Physical association between snRNPs U4 and U6 during sedimentation is shown by their co-precipitation with anti-m3G IgG from the gradient fractions. Finally, experimental evidence is provided that snRNAs U4 and U6 are associated by intermolecular base pairing in the U4/U6 RNP particle, as demonstrated by our finding that anti-m3G IgG co-precipitates U6 RNA with U4 RNA following phenolization of U4/U6 RNPs at 0°C. When the phenolization is performed at 65°C the two RNAs dissociate and anti-m3G IgG solely precipitates U4 RNA. In the particle sedimentation studies U5 RNP was found to be the slowest sedimenting snRNP species, which indicates that it is not associated with one of the other RNPs and therefore exists as a discrete RNP particle. Key words: small nuclear ribonucleoprotein/fractionation of snRNPs/snRNP sedimentation studies/U4/U6 RNP particle/base pairing between snRNPs U4 and U6 ably do not function as naked RNA molecules in the cell but rather as ribonucleoprotein particles (Raj et al., 1975;Lerner and Steitz, 1979). Lerner and Steitz (1979) observed that immune precipitates obtained after reaction of anti-RNP or anti-Sm sera from patients with lupus erythematosus and nuclear extracts contained either only Ul RNA or all the snRNAs Ul, U2, U4, U5 and U6, respectively, and that the antigenic determinants reacting with both classes of antibodies were located on the protein part of the snRNPs. These results showed clearly that at least Ul RNA was not in the same physical particle as U2, U4, U5 and U6 RNAs. Together with the finding that RNP complexes containing the snRNAs Ul, U2 and U4 to U6 display sedimentation coefficients of -lOS, the data suggested further that each snRNA molecule exists as a distinct snRNPI complex.Various attempts have been made ever since to purify the snRNPs (Hinterberger et al., 1983;Kinlaw et al., 1983;Bringmann et al., 1983a;Billings and Hoch, 1983). U2 RNP has been separated f...
Anti‐RNP sera were used to isolate a cDNA clone for the largest polypeptide of the U1 snRNP, a protein of mol. wt 70 kd designated 70K, from a human liver cDNA library constructed in the expression vector pEX1. The cro‐beta‐galactosidase‐70K fusion protein reacted with various anti‐RNP patient sera, a rabbit anti‐70K antiserum, as well as with a monoclonal antibody specific for this protein. The sequences of four 70K peptides were determined and they match parts of the deduced amino acid sequence of the 1.3 kb insert of p70.1 indicating that it is a genuine 70K cDNA. Screening of a new cDNA library constructed from polysomal mRNA of HeLa cells with the p70.1 clone yielded an overlapping clone, FL70K, which was 2.7 kb long and covered the complete coding and 3′‐untranslated sequence of the 70K protein in addition to 680 nucleotides upstream of the putative initiation codon, The predicted mol. wt of the encoded protein is approximately 70 kd. Amino acid analysis of the purified HeLa 70K protein yielded values close or identical to those deduced from the nucleotide sequence of the full‐length cDNA. The 70K protein is rich in arginine (20%) and acidic amino acids (18%). Extremely hydrophilic regions containing mixed‐charge amino acid clusters have been identified at the carboxyl‐terminal half of the protein, which may function in RNA binding. A sequence comparison with two recently cloned RNA binding proteins revealed homology with one region in the U1 RNP 70K protein. This domain may also be responsible for RNA binding.
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