Eukaryotic small ribonucleoproteins. Anti-La human autoantibodies react with U1 RNA-protein complexes

Anti-La sera from patients with autoimmune disorders precipitate a set of nuclear and cytoplasmic small RNA-protein complexes. Up to now, it has been thought that the La antigen is associated only with RNAs transcribed by RNA polymerase III, including precursors of tRNA and 5 S ribosomal RNA. Here we report that anti-La sera also react with ribonucleoprotein particles containing small nuclear RNA U1, which is transcribed by RNA polymerase II. Anti-La sera from 12 out of 12 patients tested were found to precipitate U1 RNA-protein complexes from HeLa cell nuclear extracts, under conditions where nonimmune sera do not. Ribonucleoprotein particles containing a second small nuclear RNA, U2, do not react appreciably with anti-La sera although they are present in HeLa cell nuclei at the same concentration as U1 RNA. Anti-La sera also react with U1 RNA-protein complexes in mouse and frog cells, but not in Drosophila or Chironomus, two organisms which lack the La antigen. Hybridization of cloned U1 DNA with anti-La-reactive RNA from HeLa cell nuclear extracts reveals mature U1 RNA, whereas anti-La-reactive cytoplasmic RNA contains a series of hybridizing bands that represent molecules 1-7 nucleotides longer than U1 and which may include precursors of nuclear U1 RNA (Madore, S. J., Wieben, E. D., and Pederson, T. (1984) J. Cell Biol., 188-192). Pulse-chase experiments suggest that the association of La antigenicity with these cytoplasmic U1 RNA molecules is transient. These results are discussed in relation to the presence of uridylate-rich sequences in the 3' termini of U1 RNA precursors and mature U1 RNA, which are similar to La antigen binding sites in several RNAs transcribed by RNA polymerase III.     

Association of the lupus antigen La with a subset of U6 snRNA molecules.

U6 snRNA is a component of the major class of small RNA-protein complexes, the Sm snRNPs, present in mammalian cell nuclei. Here we report that a substantial fraction (about 10%) of U6 RNA from human and mouse cells is associated with another lupus antigen, the 50 kd La protein. The La-bound U6 subpopulation is characterized by 3' end heterogeneity and partial undermethylation. These U6 molecules have U-rich 3' termini that could be responsible for their selective association with the La protein. The question of whether they are precursors to the major U6 RNAs found in Sm snRNPs is discussed.     

Human SS-B/LA autoantigen contains a covalent protein-RNA linkage

The chemical nature of association of RNA in immunoprecipitates of human SS-B/La ribonucleoprotein, an autoantigen expressed in various autoimmune disorders, was investigated. A fraction of RNA associated with SS-B/La immunoprecipitates was readily dissociated by SDS-polyacrylamide gel electrophoresis, yielding four main subfractions, R1-4, with chain lengths in the range of 90-130 nucleotides (R4), 140-175 nucleotides (R2 and R3) and above 200 nucleotides (R1). Moreover, the immunoreactive protein component, migrating with a molecular mass of 49 kDa, contained a very tightly bound RNA co-migrating with the protein unless the protein was proteolytically degraded. Most of the RNA molecules in this fraction, represented by about 20 components, had a free 3'-terminus but a blocked 5'-terminus and showed chain lengths between 10 and 125 nucleotides. After pretreatment with alkaline phosphatase and a mixture of ribonucleases T1 + T2 + A, adenosine 3',5'-biphosphate (pAp) was liberated by phosphodiesterase (Crotalus durissus) as the blocked 5'-end of the RNA. The chemical nature of the blockage was revealed after alternative treatment of the protein-pAp component with phosphodiesterase or nuclease S7 followed by acid hydrolysis and phosphoamino acid analysis which showed that a threonine residue must be directly involved in the RNA-protein linkage of 49 kDa SS/La antigen, indicating the presence of a covalent threonine-pAp bond.     

Isolation of ribonucleic acid from the unactivated rat liver glucocorticoid receptor

We have reported that the 7-8S form of the rat liver glucocorticoid receptor is associated with RNA. Whether the unactivated 9-10S form of the glucorticoid receptor is also associated with RNA is less clear. Here we provide evidence that the unactivated 9-10S receptor is indeed associated with RNA. Unactivated 9-10S receptor was partially purified by diethylaminoethyl (DEAE)-cellulose chromatography in the presence of molybdate, an activation inhibitor. This preparation was then bound to BuGR-2, a mouse monoclonal antibody of the immunoglobulin G (IgG)-2 class to the rat liver glucocorticoid receptor, or to nonspecific mouse IgG-2. The antibody-antigen complex was then bound to protein A sepharose and washed to remove extraneous RNA. When the receptor was dissociated from the antibody and the RNA extracted and end-labeled, a distinct band of approximately 170 nucleotide (nt) was found that was specific for the BuGR-2 purified receptor. This band could also be found in DEAE-cellulose receptor that had been isolated from sucrose gradients. The DEAE-cellulose receptor was then cross-linked with formaldehyde before mixing with BuGR-2 in order to permit more vigorous washing of the antigen-antibody complex. In addition to the 170 nt RNA band, another distinct band at approximately 400 nt was seen that was specific to the BuGR-2 derived isolate. These results provide evidence that the 9-10S form of the glucocorticoid receptor from rat liver is associated with RNA.     

The Trypanosoma brucei La protein is a candidate poly(U) shield that impacts spliced leader RNA maturation and tRNA intron removal

By virtue of its preferential binding to poly(U) tails on small RNA precursors and nuclear localisation motif, the La protein has been implicated for a role in the stabilisation and nuclear retention of processing intermediates for a variety of small RNAs in eukaryotic cells. As the universal substrate for trans-splicing, the spliced leader RNA is transcribed as a precursor with just such a tail. La protein was targeted for selective knockdown by inducible RNA interference in Trypanosoma brucei. Of three RNA interference strategies employed, a p2T7-177 vector was the most effective in reducing both the La mRNA as well as the protein itself from induced cells. In the relative absence of La protein T. brucei cells were not viable, in contrast to La gene knockouts in yeast. A variety of potential small RNA substrates were examined under induction, including spliced leader RNA, spliced leader associated RNA, the U1, U2, U4, and U6 small nuclear RNAs, 5S ribosomal RNA, U3 small nucleolar RNA, and tRNATyr. None of these molecules showed significant variance in size or abundance in their mature forms, although a discrete subset of intermediates appear for spliced leader RNA and tRNATyr intron splicing under La depletion conditions. 5'-end methylation in the spliced leader RNA and U1 small nuclear RNA was unaffected. The immediate cause of lethality in T. brucei was not apparent, but may represent a cumulative effect of multiple defects including processing of spliced leader RNA, tRNATyr and other unidentified RNA substrates. This study indicates that La protein binding is not essential for maturation of the spliced leader RNA, but does not rule out the presence of an alternative processing pathway that could compensate for the absence of normally-associated La protein.     

Nucleotide sequence of 7 S RNA. Homology to Alu DNA and La 4.5 S RNA

7 S RNA, a component of normal higher eukaryotic cells and several oncornaviruses, was shown to be conserved in evolution (Erikson, E., Erikson, R. L., Henry, B., and Pace, N. R. (1973) Virology 53, 40-46). Recently, 7 S RNA was shown to be partially complementary to Alu family DNA sequences (Weiner, A. (1980) Cell 22, 209-218). In the present study the nucleotide sequence of Novikoff hepatoma 7 S RNA was determined to be: (formula, see text) Comparison of 7 S RNA, Alu and B1 family DNA, and La 4.5 S RNA sequences for homologies showed that 1) one-third of 7 S RNA, mainly the 5'-end, was homologous to Alu and B1 family sequences; 2) one 300-nucleotide long Alu family sequence contained two binding sites for 7 S RNA; and 3) the 5'-ends of 7 S RNA and La 4.5 S RNA also had extensive (60%) homologies. A model for the secondary structure of 7 S RNA based on maximal base pairing and preferential nuclease cleavage sites is also presented.     

Characterization of the SS-B (La) antigen in adenovirus-infected and uninfected HeLa cells.

The molecular composition and subcellular localization of the antigens recognized by anti-SS-B (La or Ha) antibodies was investigated. Ten anti-SS-B sera were selected by indirect immunofluorescence and by their immunological identity in counter-immunoelectrophoresis (CIE) with an anti-SS-B reference serum. All sera precipitated virus-associated (VA) RNA from cellular extracts of adenovirus-infected HeLa cells. Earlier results had shown that in adenovirus-infected HeLa cells a cellular 50 000 mol. wt. protein was tightly associated with VA RNA in situ. Our present results indicate that this 50 000 protein is the only SS-B antigen present in adenovirus-infected as well as in uninfected cells. A major part (greater than 80%) of the SS-B antigen is present in a readily extractable, soluble form. The rest is found in an insoluble form tightly associated with an internal nuclear structure that is mostly referred to as the nuclear matrix. Both forms are very susceptible to proteolytic degradation resulting in at least two distinct breakdown products of mol. wts. 40 000 and 25 000. The cellular 50 000 polypeptide is present in extracts of various types of cells and tissues, indicating that this antigen is very well conserved during evolution. The association of the 50 000 mol. wt. antigen with host- as well as viral-coded RNA polymerase III products also suggests an important function for this protein in the metabolism of these small RNAs.     

A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells

A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (approximately 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.     

Structural determinant of human La protein critical for internal initiation of translation of hepatitis C virus RNA

Human La protein has been implicated in facilitating internal ribosome entry site (IRES)-mediated translation of hepatitis C virus (HCV). Earlier, we demonstrated that the RNA recognition motif (RRM) encompassing residues 112 to 184 of La protein [La (112-184)] interacts with the HCV IRES near the initiator AUG codon. A synthetic peptide, LaR2C (24-mer), derived from La RRM (112-184), retains RNA binding ability, competes with La protein binding to the HCV IRES, and inhibits translation. The peptide interferes with the assembly of 48S complexes, resulting in the accumulation of preinitiation complexes that are incompetent for the 60S ribosomal subunit joining. Here, nuclear magnetic resonance spectroscopy of the HCV IRES-bound peptide complex revealed putative contact points, mutations that showed reduced RNA binding and translation inhibitory activity. The residues responsible for RNA recognition were found to form a turn in the RRM (112-184) structure. A 7-mer peptide comprising this turn showed significant translation inhibitory activity. The bound structure of the peptide inferred from transferred nuclear Overhauser effect experiments suggests that it is a β turn. This conformation is significantly different from that observed in the free RRM (112-184) NMR structure, suggesting paths toward a better-stabilized mimetic peptide. Interestingly, addition of hexa-arginine tag enabled the peptide to enter Huh7 cells and showed inhibition of HCV IRES function. More importantly, the peptide significantly inhibited replication of the HCV monocistronic replicon. Elucidation of the structural determinant of the peptide provides a basis for developing small peptidomimetic structures as potent anti-HCV therapeutics.     

Identification of the Ro and La antigens in the endoribonuclease VII--ribonucleoprotein complex.

45 S RNP (ribonucleoprotein) particles from calf thymus or L5178y mouse lymphoma cells contain the poly(A)-modulated and oligo(U)-binding endoribonuclease VII [Bachmann, Zahn & Müller (1983) J. Biol. Chem. 258, 7033-7040]. From these particles a 4.5 S RNA was isolated that possesses an oligo(U) sequence. By using monospecific and non-cross-reacting antibodies directed against the La or Ro antigen, both proteins were identified in the endoribonuclease VII-RNP complex after phosphorylation in vitro. In a second approach, endoribonuclease VII activity was identified in immunoaffinity-purified Ro RNPs after preparative isoelectric focusing. Therefore we conclude that the 4.5 S RNA belongs to the Ro RNAs. The results indicate a possible function of endoribonuclease VII in activating stored mRNAs.     

Characterization of the intron-encoded U19 RNA, a new mammalian small nucleolar RNA that is not associated with fibrillarin.

We have characterized a new member (U19) of a group of mammalian small nuclear RNAs that are not precipitable with antibodies against fibrillarin, a conserved nucleolar protein associated with most of the small nucleolar RNAs characterized to date. Human U19 RNA is 200 nucleotides long and possesses 5'-monophosphate and 3'-hydroxyl termini. It lacks functional boxes C and D, sequence motifs required for fibrillarin binding in many other snoRNAs. Human and mouse RNA are 86% homologous and can be folded into similar secondary structures, a finding supported by the results of nuclease probing of the RNA. In the human genome, U19 RNA is encoded in the intron of an as yet not fully characterized gene and could be faithfully processed from a longer precursor RNA in HeLa cell extracts. During fractionation of HeLa cell nucleolar extracts on glycerol gradients, U19 RNA was associated with higher-order structures of approximately 65S, cosedimenting with complexes containing 7-2/MRP RNA, a conserved nucleolar RNA shown to be involved in 5.8S rRNA processing in yeast cells.     

Binding of the rat liver 7-8 S dexamethasone receptor to deoxyribonucleic acid

The 7-8 S form of the [3H]dexamethasone (9 alpha-fluoro-11 beta,17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3, 20-dione) receptor from rat liver cytosol can be converted to the 3-4 S form by RNase treatment or high salt, suggesting a salt-sensitive association between the receptor protein and RNA. In DNA-cellulose column assays, the gradient-purified 3-4 S form bound DNA more efficiently than the 7-8 S form, though the 7-8 S form was also capable of binding to DNA-cellulose to a significant extent. Activated 7-8 S dexamethasone receptor could be released from its association with soluble DNA by treatment with DNase I. Sucrose gradient analysis showed that the released receptor sedimented as the 7-8 S form and was sensitive to RNase treatment, which induced a conversion to the 3-4 S form. Activated RNase-generated 3-4 S receptor again displayed a higher degree of binding to soluble DNA and was recovered in the 3-4 S form following DNase extraction. The fact that the 3-4 S form bound immobilized or soluble DNA more efficiently suggests that the associated RNA of the 7-8 S form interferes directly or indirectly with the receptor association with DNA. The observation that the receptor binds to DNA in its 7-8 S form suggests that the receptor complex is capable of binding RNA and DNA concurrently.     

Cellular La protein shields nonsegmented negative-strand RNA viral leader RNA from RIG-I and enhances virus growth by diverse mechanisms

The La antigen (SS-B) associates with a wide variety of cellular and viral RNAs to affect gene expression in multiple systems. We show that La is the major cellular protein found to be associated with the abundant 44-nucleotide viral leader RNA (leRNA) early after infection with respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus. Consistent with this, La redistributes from the nucleus to the cytoplasm in RSV-infected cells. Upon RNA interference knockdown of La, leRNA is redirected to associate with the RNA-binding protein RIG-I, a known activator of interferon (IFN) gene expression, and this is accompanied by the early induction of IFN mRNA. These results suggest that La shields leRNA from RIG-I, abrogating the early viral activation of type I IFN. We mapped the leRNA binding function to RNA recognition motif 1 of La and showed that while wild-type La greatly enhanced RSV growth, a La mutant defective in RSV leRNA binding also did not support RSV growth. Comparative studies of RSV and Sendai virus and the use of IFN-negative Vero cells indicated that La supports the growth of nonsegmented negative-strand RNA viruses by both IFN suppression and a potentially novel IFN-independent mechanism.