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J A Steitz

Publications and source records attributed to J A Steitz.

At least 145 records · Page 8Linked to original sources
PubMed1983

The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro.

The ability of purified U1 small nuclear RNA-protein complexes (U1 snRNPs) to bind in vitro to two RNAs transcribed from recombinant DNA clones by bacteriophage T7 RNA polymerase has been studied. A transcript which contains sequences corresponding to the small intron and flanking exons of the major mouse beta-globin gene is bound in marked preference to an RNA devoid of splice site sequences. The site of U1 snRNP binding to the globin RNA has been defined by T1 ribonuclease digestion of the RNA-U1 snRNP complex. A 15-17-nucleotide region, including the 5' splice site, remains undigested and complexed with the snRNP such that it can be co-precipitated by antibodies directed against the U1 snRNP. Partial proteinase K digestion of the U1 snRNP abolishes interaction with the globin RNA, indicating that the snRNP proteins contribute significantly to RNA binding. No RNA cleavage, splicing, or recognition of the 3' splice site by U1 snRNPs has been detected. Our results are discussed in terms of the probable role of U1 snRNPs in the messenger RNA splicing of eucaryotic cell nuclei.

Base Sequence↗
PubMed1982

Precursor molecules of both human 5S ribosomal RNA and transfer RNAs are bound by a cellular protein reactive with anti-La lupus antibodies.

The small ribonucleoproteins recognized by anti-La autoantibodies contain a heterogeneous mixture of small RNAs from uninfected mammalian cells. The identity of many of these has now been established by the discovery of precursor forms of 5S rRNA and of certain tRNAs among La RNAs from HeLa cells. The small fraction of 5S rRNA molecules that exist as La ribonucleoproteins in vivo possess 1 or 2 additional U residues at their 3' ends. Such 5S molecules bound to the La protein have also been identified with in vitro nuclear transcription systems. Pulse-chase experiments performed both in vivo and in vitro support the idea that most newly synthesized 5S rRNA molecules are transiently associated with the La protein. Cell extracts contain a processing activity that converts longer in vitro-synthesized 5S RNA transcripts into molecules of mature size. The presence of in vivo tRNA precursors in the heterogeneous mixture of La RNAs is demonstrated by the identification of precursor forms of five different specific tRNAs (Meti, Asp, Gly, Glu, Asn). After in vitro transcription of a tRNA gene (tRNAiMet), only products the size of precursor molecules are precipitable by anti-La antibodies. The realization that virtually every known RNA polymerase III product associates at least initially with the La antigen suggests that this protein plays an essential role in the synthesis or maturation of all class III transcripts.

Antigens↗
PubMed1982

Antibodies from patients with connective tissue diseases bind specific subsets of cellular RNA-protein particles.

We characterized the RNA-containing antigens precipitated by sera from 260 patients with positive antinuclear antibodies. 49 individuals, most of whom had systemic lupus erythematosus or Sjögren's syndrome, possessed antibodies that precipitated the previously identified RNP, Sm, Ro, and La antigens either singly or in combinations. These antigens, which are located on discrete sets of small nuclear or cytoplasmic RNA-protein particles, exhibited a number of antigenic interrelationships. One patient's serum recognized a new particle containing a small RNA which we have called Th; it also precipitated the Ro complexes. Other patients with systemic lupus erythematosus, hepatitis B virus infection, juvenile rheumatoid arthritis, myositis, and rheumatoid arthritis had antibodies that precipitated specific subsets of ribosomal RNA and transfer RNA. One patient's serum contained a monoclonal immunoglobulin G that precipitated ribosomes. Most of these antibodies identified antigenic determinants constituted at least in part of protein. The specificity of the proteins bound to particular cellular RNA, probably explains the exquisite precision with which antibodies from rheumatic disease patients discriminate among RNA subsets. Such sera should be useful probes for investigating specific roles that different RNA and RNA-protein complexes play in cellular metabolism.

Antibodies, Antinuclear↗
PubMed1982

Structure and function of small ribonucleoproteins from eukaryotic cells.

Autoantibodies from patients with systemic lupus erythematosus and other related diseases have been used to identify and study small RNA-protein complexes from mammalian cells. Properties of three previously described and several new classes of small ribonucleoproteins (RNPs) are reviewed. The sequence of Drosophila U1 RNA reveals that the region proposed to pair with 5' splice junctions is conserved, while that proposed to interact with 3' junctions diverges; this forces some revision of the model for U1 small nuclear (sn)RNP participation in hnRNA splicing. Further characterization of the Ro and La small RNPs has shown that the Ro small cytoplasmic (sc)RNPs are a subclass of La RNPs. Both tRNA and 5S rRNA precursors are at least transiently associated with the La protein. This raises the possibility that the La protein may be an RNA polymerase III transcription factor.

Antigen-Antibody Complex↗
PubMed1981

Sequence of U1 RNA from Drosophila melanogaster: implications for U1 secondary structure and possible involvement in splicing.

U1 RNA from cultured Drosophila melanogaster cells (Kc) was identified by its ability to be recognized, as an RNP, by anti-(U1)RNP antibodies from human lupus patients. Its sequence was deduced largely from direct analysis of the RNA molecule and then confirmed by DNA sequence determinations on a genomic clone isolated by hybridization to Drosophila U1 RNA. The Drosophila U1 RNA sequence exhibits 72% agreement with human U1 RNA. Nucleotides 3-11, which are complementary to the entire consensus sequence for donor (5') splice junctions in hnRNA, and to part of the acceptor (3') consensus, are exactly conserved. However, nucleotides 14-21, postulated to interact only with acceptor junctions, differ. Comparison of the Drosophila U1 sequence with vertebrate U1 sequences allows a particular secondary structure model to be preferred over others. These results are consistent with the hypothesis that U1 snRNPs are involved in splicing, but suggest specific modifications of the model detailing molecular interactions between U1 RNA and hnRNA during the splicing reaction.

Animals↗
PubMed1981

Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus.

The RNP and Sm antigens recognized by lupus erythematosus antibodies are located on discrete particles containing single small nuclear RNA's complexed with proteins. The antigens Ro and La are also on ribonucleoproteins. The small RNA's in ribonucleoproteins with Ro are discrete, like those associated with RNP and Sm; in contrast, ribonucleoproteins with La contain a striking highly banded spectrum of small RNA's from uninfected cells as well as virus-associated RNA from adenovirus-infected cells.

Animals↗
PubMed1981

Two small RNAs encoded by Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus.

Primate cells harboring the Epstein-Barr virus (EBV) genome synthesize large amounts of two small RNAs:EBER 1 and EBER 2 (EBV-encoded RNA). These RNAs are approximately 180 nucleotides long, possess 5' pppA termini, and lack poly(A). They have different T1 and pancreatic RNase digestion fingerprints. They are not found in normal B lymphocytes, in transformed B lymphocytes that lack EBV DNA, in T lymphocytes transformed by Herpesvirus ateles, or in a variety of other nonlymphoid mammalian cells. Hybridization analyses indicate that EBER 1 and EBER 2 are encoded by the EcoRI-J fragment of EBV (B95-8) DNA. In vivo both RNAs are associated with protein(s), allowing their specific precipitation by the systemic lupus erythematosus-associated antibody anti-La. The La antigen in uninfected mammalian cells consists of a heterogeneous class of small ribonucleoprotein particles, some of whose RNA components exhibit sequence homology with a highly repetitive, interspersed class of human DNA designated the Alu family. Possible functions for EBER 1 and EBER 2 in infection and cell transformation by EBV and their potential relationship to the pathogenesis of systemic lupus erythematosus are discussed.

Antibodies↗
PubMed1981

A small nuclear ribonucleoprotein is required for splicing of adenoviral early RNA sequences.

The size and structure of viral RNA species synthesized in nuclei isolated during the early phase of productive infection by adenovirus type 2 have been examined by electrophoresis in denaturing polyacrylamide cells and the nuclease S1 assay. The major products of transcription in vitro of early regions 1 and 2 in the adenoviral genome are processed RNA molecules that appear to be correctly spliced in isolated nuclei. Splicing of adenoviral RNA molecules is inhibited when nuclei are preincubated with antibodies from systemic lupus erythematosus patients that immunoprecipitate small nuclear ribonucleoprotein particles. The specificity of these antibodies suggests that ribonucleoprotein particles containing U1 RNA are required for splicing of the adenoviral RNA sequences we have examined.

Adenoviruses, Human↗
PubMed1981

Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease.

Mice of the strain MRL/Mp-lpr/lpr develop a lupus erythematosus-like syndrome that includes the production of autoantibodies specific for nucleic acid-containing cellular components. We have fused spleen cells from such a mouse with the myeloma SP 2/0 and examined the antibodies produced by the resultant cloned hybrid cell lines by using immunoprecipitation and immunofluorescence techniques. Three types of monoclonal antibodies, specific for Sm, DNA, or rRNA, all antigens to which patients who have lupus make antibodies, have been identified. Patient anti-Sm antibody had previously been reported to precipitate five small nuclear ribonucleoproteins that contain U-1, U-2, U-4, U-5, and U-6 RNAs. The monoclonal anti-Sm antibody gives the same immunoprecipitation pattern, providing direct evidence that the Sm antigen resides on all these RNA-protein complexes. Monoclonal anti-Sm antibody will be valuable in deciphering the biological function of these ubiquitous small nuclear RNPs. A simple competition radioimmunoassay using the monoclonal anti-Sm antibody to titer patient sera is also presented. Uses of monoclonal antibodies for the study of autoimmune disease are discussed.

Animals↗
PubMed1981

Arrangement of 30S heterogeneous nuclear ribonucleoprotein on polyoma virus late nuclear transcripts.

Heterogeneous nuclear ribonucleic acid (hnRNA) molecules in eucaryotic cell nuclei associate with a well-defined group of abundant, highly conserved proteins to form heterogeneous nuclear ribonucleoproteins (hnRNP). The exact manner in which these 30S complexes assemble on nuclear transcripts, however, has not been well documented. To determine whether any site selectivity in the formation of hnRNP can be detected (e.g., preferential recognition of intervening sequences or of premessage regions), we investigated the distribution of 30S hnRNP on a particular nuclear RNA, the polyoma virus late transcript. Hybridization studies showed not only that the majority of polyoma late nuclear RNA sequences can be isolated in the form of 30S complexes, but that the RNP were located equally on intervening sequences and premessage portions of the transcript. The latter conclusion was confirmed by ribonuclease T1 oligonucleotide fingerprint analysis of polyoma virus-specific RNA recovered from native 30S complexes. However, fingerprint analysis of the small segments of viral RNA in the 30S fraction that survived extensive ribonuclease treatment revealed that oligonucleotides corresponding to intervening sequences were preferentially lost. We discuss these findings in relation to the structure of 30S hnRNP and their function in RNA biogenesis.

Heterogeneous-Nuclear Ribonucleoproteins↗
PubMed1981

Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells.

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) classes, the numbers of different RNA molecules and their sequences vary between mouse and human cells. The complex mixtures of La RNAs include two previously sequenced 4.5S RNAs from mouse cells and 5S ribosomal RNA-like molecules from both mouse and human cells. All Ro and La RNAs possess 5-triphosphates. Some La RNAs have internal modifications typical of transfer RNAs. The Ro RNPs are quite stable and are localized by immunofluorescence in the cell cytoplasm, whereas the majority of the La RNPs turn over rapidly and reside in the nucleus. Despite these differences, reconstitution experiments show that the Ro particles carry the La as well as the Ro determinant. Studies using a nuclear transcription system demonstrate that most of the La RNAs are synthesized by RNA polymerase III. The possibility that the La protein(s) functions in the transcription or maturation of all RNA polymerase III transcripts is discussed.

Animals↗
PubMed1981

Striking similarities are exhibited by two small Epstein-Barr virus-encoded ribonucleic acids and the adenovirus-associated ribonucleic acids VAI and VAII.

The nucleotide sequence of the region of the Epstein-Barr virus genome that specifies two small ribonucleic acids (RNAs), EBER 1 and EBER 2, has been determined. Both of these RNAs are encoded by the right-hand 1,000 base pairs of the EcoRI J fragment of EBV deoxyribonucleic acid. EBER 1 is 166 (167) nucleotides long and EBER 2 is 172 +/- 1 nucleotides long; the heterogeneity resides at the 3' termini. The EBER genes are separated by 161 base pairs and are transcribed from the same deoxyribonucleic acid strand. In vitro, both EBER genes can be transcribed by RNA polymerase III; sequences homologous to previously identified RNA polymerase III intragenic transcription control regions are present. Striking similarities are therefore apparent both between the EBERs and the two adenovirus-associated RNAs, VAI and VAII, and between the regions of the two viral genomes that specify these small RNAs. We have shown that VAII RNA as well as VAI RNA and the EBERs exist in ribonucleoprotein complexes which are precipitable by anti-La antibodies associated with systemic lupus erythematosus. Finally, we have demonstrated that the binding of protein(s) from uninfected cells confers antigenicity on each of the four virus-encoded small RNAs.

Adenoviruses, Human↗