Small ribonucleic acid (RNA)-protein complexes precipitated by anti-Ro and anti-La antibodies from lupus patients have been examined with emphasis on their RNA components. In both ribonucleoprotein (RNP)
Abstract.A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (,,~7S) and contains most of the nonribosome-associated 5S RNA molecules in HeLa cells. The 5S RNA in the particle is matured at its 3' end, consistent with the results of in vivo pulse-chase experiments which indicate that this RNP represents a later step in 5S biogenesis than a previously described 5S*/La protein complex. The protein moiety of the 5S RNP has been identified as ribosomal protein L5, which is known to be released from ribosomes in a complex with 5S after various treatments of the 60S subunit. Indirect immunofluorescence indicates that the L5/5S complex is concentrated in the nucleolus. L5 may therefore play a role in delivering 5S rRNA to the nucleolus for assembly into ribosomes.T HE biosynthesis of ribosomes in mammalian cells involves the assembly of rRNAs synthesized by RNA polymerase I (18S, 28S, 5.8S) and by RNA polymerase III (5S) together with proteins translated from mRNAs synthesized by RNA polymerase II. Not only must the appearance of these components be coordinated on the transcriptional level, but those that are not synthesized in the nucleolus (5S and ribosomal proteins) must become specifically concentrated there. In higher eukaryotes the genes for 5S rRNA exist in tandemly repeating units which are not closely linked to the large blocks of rDNA that comprise the nucleolus organizer regions (25). 5S rRNA must therefore travel from the nucleoplasm to its nucleolar site of assembly into ribosomes.In 1967, Knight and Darnell (16) discovered that only about half of the 5S rRNA molecules in mammalian cells are associated with 60S ribosomes. The remainder sediment in a range that could represent either free 5S RNA or 5S bound by one or a few protein molecules. One mammalian protein that is known to bind 5S RNA is the 50-kD La antigen. The La-bound 5S represents only a very small fraction ('M%) of the total and is slightly elongated at its 3' end (called 5S*) (38). Another well-characterized 5S-binding protein is tran- scription factor TF IIIA (10, 33). In the Xenopus oocyte, 5SRNA is stored for later assembly into ribosomes as a 7S RNP (37) containing TF IIIA; TF IIIA is not present in a larger (42S) oocyte particle that contains several other proteins and tRNAs as well as 5S RNA (15,28,36). Mammalian TF IIIA has not yet been identified. A third known 5S-binding protein is ribosomal protein L5 (34 kD) (6). 5S RNA/L5 complexes are released from 60S ribosomes by EI~A (2, 19, 42) or a variety of other treatments (27,34), and the isolated L5/5S particle exchanges 5S rRNA specifically (13, 32). However, it has not been determined whether L5 initially associates with 5S before or only upon ribosome assembly.Here we have used autoantibody probes to characterize a 5S RNP that represents a substantial fraction of the nonribosomal 5S rRNA in mammalian cells. The particle appears to contain a single protein component, which is ribosomal protein L5. Its cellular l...
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...
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