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J D Keene

Publications and source records attributed to J D Keene.

At least 55 records · Page 3Linked to original sources
PubMed1992

In vitro selection of an RNA epitope immunologically cross-reactive with a peptide.

An antiserum raised against a peptide was used to select a unique RNA species from a degenerate pool of RNAs designed to resemble an autoantibody recognition site in U1 RNA. The peptide and the selected RNA epitope could compete for antibody binding, suggesting that both RNA and peptide epitopes occupy the same or overlapping antigen-combining sites. Thus, the RNA epitope functioned as a specific inhibitor of the antibody-antigen interaction. We demonstrate that the RNA epitope can be used to tag unrelated RNA molecules and also to detect the presence of the antibody. We propose that sequence-specific recognition of RNA by antibodies may involve protein-RNA contacts similar to those occurring in other nucleic acid-binding proteins. In addition, these findings are compatible with the suggestion that nucleic acid-binding autoantibodies may arise through immunological cross-reactivity between proteins and nucleic acids.

Amino Acid Sequence↗
PubMed1992

RNA binding specificity of a Drosophila snRNP protein that shares sequence homology with mammalian U1-A and U2-B" proteins.

We have characterized a recombinant Drosophila melanogaster RNA binding protein, D25, by virtue of its antigenic relationship to mammalian U1 and U2 small nuclear ribonucleoprotein (U snRNP) proteins. Sequence analysis revealed that D25 bears strong similarity to both the human U1 snRNP-A (U1-A) and U2 snRNP-B" (U2-B") proteins. However, at residues known to be critical for the RNA binding specificities of U1-A and U2-B" D25 sequence is more similar to U2-B". Using direct RNA binding assays D25 selected U1 RNA from either HeLa or Drosophila Kc cell total RNA. Furthermore, D25 bound U1 RNA when transfected into mammalian cells. Thus, D25 appears to be a Drosophila homolog of the mammalian U1-A protein, despite its sequence similarity to U2-B".

Amino Acid Sequence↗
PubMed1992

Molecular biology of nuclear autoantigens.

This article provides a historical overview of the application of molecular and immunologic techniques to the analysis of autoantigenic structure and function, as well as to autoantibody recognition of protein and nucleic acid autoantigens. Examples presented here illustrate the role of autoantibodies as tools in the elucidation of the autoimmune components of cellular ribonucleoproteins. In turn, the subsequent molecular dissection of autoantigenic ribonucleoproteins has advanced understanding of autoantibody specificities. The nature of autoantibodies reactive with various proteins and nucleic acids will be the subject of the following articles in this issue. Taken together, these studies of antibody-antigen interactions that arise during the autoimmune response have revealed novel mechanisms of molecular recognition within the RNP autoantigens. These findings are of general importance for understanding basic cellular processes and have contributed to our knowledge of the underlying mechanisms of immunoregulatory abnormalities that arise in autoimmune diseases.

Autoantibodies↗
PubMed1991

U1-snRNP-A protein selects a ten nucleotide consensus sequence from a degenerate RNA pool presented in various structural contexts.

The U1snRNP-A (U1-A) protein was used to select specific RNA sequences from a degenerate pool of transcripts using direct RNA binding and polymerase chain reaction amplification (PCR). Sequences were randomized in loops of 10 or 13 nucleotides or as a linear stretch of 25 nucleotides. From all three structural contexts, an unpaired ten nucleotide consensus sequence was obtained. A selected stem-loop structure that resembled the natural U1-A protein binding site on loop II of U1 RNA demonstrated the highest affinity of binding in comparison with the other structural contexts. A data profile of selected sequences identified U1 RNA upon searching the GenBank database. Thus, this method was useful in determining the sequence specificity of an RNA binding protein and may complement the use of phylogenetic comparisons to predict conserved recognition elements. These findings also suggest that the evolutionary conservation of loop II of U1 RNA results from constraints imposed by protein binding.

Base Sequence↗
PubMed1991

RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins.

An RNA recognition motif (RRM) of approximately 80 amino acids constitutes the core of RNA-binding domains found in a large family of proteins involved in RNA processing. The U1 RNA-binding domain of the A protein component of the human U1 small nuclear ribonucleoprotein (RNP), which encompasses the RRM sequence, was analyzed by using NMR spectroscopy. The domain of the A protein is a highly stable monomer in solution consisting of four antiparallel beta-strands and two alpha-helices. The highly conserved RNP1 and RNP2 consensus sequences, containing residues previously suggested to be involved in nucleic acid binding, are juxtaposed in adjacent beta-strands. Conserved aromatic side chains that are critical for RNA binding are clustered on the surface of the molecule adjacent to a variable loop that influences recognition of specific RNA sequences. The secondary structure and topology of the RRM are similar to those of ribosomal proteins L12 and L30, suggesting a distant evolutionary relationship between these two types of RNA-associated proteins.

Amino Acid Sequence↗
PubMed1991

Anti-La antibody production by MRL-1pr/1pr mice. Analysis of fine specificity.

In evaluating the origin of autoantibodies, patterns of self-Ag recognition have been interpreted to reflect the relative role of Ag in stimulating a response. Few studies, however, have assessed whether human autoantibodies display patterns of autoantigen recognition similar to those of SLE-prone mice. In previous studies, anti-La antibodies from humans have been shown to bind multiple epitopes on recombinant human La Ag, including immunoreactivity with a large fragment, termed La C, representing the middle portion of the La sequence. We report herein for the first time that MRL-1pr mice also spontaneously produce antibodies to recombinant human La protein and resemble human autoantibodies in their reactivity with La C. To further investigate the fine specificity of this response, we tested for antibody binding to six synthetic La peptides representing sequences within La C. Whereas two of the synthetic La peptides reacted with MRL-1pr sera containing anti-La binding, low reactivity was observed with a large panel of human anti-La sera. Our results therefore show that patterns of La antigen recognition displayed by MRL-1pr antibodies differ from those of human autoantibodies, possibly reflecting differences between mouse and man in the induction of these responses.

Animals↗
PubMed1991

Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.

Camptothecin (CPT), a plant alkaloid with antitumor activity, is a specific inhibitor of eukaryotic DNA topoisomerase I. We have previously isolated and characterized a CPT-resistant topoisomerase I isolated from a CPT-resistant human leukemia cell line, CPT-K5. cDNA clones of topoisomerase I were isolated from the CPT-resistant and the parental CPT-sensitive cell lines, respectively. Sequencing of the clones identified two mutations in the cDNA isolated from the resistant cells, which cause amino acid changes from aspartic acid to glycine at residues 533 and 583 of the parental topoisomerase I. When the CPT-K5 topoisomerase I was expressed in E. coli as a fusion protein with Staphylococcal Protein A fragment, the activity was resistant to CPT at a dose level up to 125 microM, whereas the parental fusion protein was sensitive to CPT as low as 1 microM. The resistance index (greater than 125) of the CPT-K5 fusion topoisomerase I is similar to that of the native CPT-K5 topoisomerase I. These results indicate that either or both of the two amino acid changes identified in the mutant enzyme is responsible for the resistance to CPT.

Amino Acid Sequence↗
PubMed1991

RNA recognition: towards identifying determinants of specificity.

Members of a family of proteins containing a conserved approximately 80-amino acid RNA recognition motif (RRM) bind specifically to a wide variety of RNA molecules. Structural studies, in combination with sequence alignments, indicate the structural context of both conserved and non-conserved elements in the motif. These analyses suggest that all RRM proteins share a common fold and a similar protein-RNA interface, and that non-conserved residues contribute additional contacts for sequence-specific RNA recognition.

Amino Acid Sequence↗
PubMed1991

Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions.

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.

Amino Acid Sequence↗
PubMed1991

Recognition of U1 and U2 small nuclear RNAs can be altered by a 5-amino-acid segment in the U2 small nuclear ribonucleoprotein particle (snRNP) B" protein and through interactions with U2 snRNP-A' protein.

We have investigated the sequence elements influencing RNA recognition in two closely related small nuclear ribonucleoprotein particle (snRNP) proteins, U1 snRNP-A and U2 snRNP-B". A 5-amino-acid segment in the RNA-binding domain of the U2 snRNP-B" protein was found to confer U2 RNA recognition when substituted into the corresponding position in the U1 snRNP-A protein. In addition, B", but not A, was found to require the U2 snRNP-A' protein as an accessory factor for high-affinity binding to U2 RNA. The pentamer segment in B" that conferred U2 RNA recognition was not sufficient to allow the A' enhancement of U2 RNA binding by B", thus implicating other sequences in this protein-protein interaction. Sequence elements involved in these interactions have been localized to variable loops of the RNA-binding domain as determined by nuclear magnetic resonance spectroscopy (D. Hoffman, C.C. Query, B. Golden, S.W. White, and J.D. Keene, Proc. Natl. Acad. Sci. USA, in press). These findings suggest a role for accessory proteins in the formation of RNP complexes and pinpoint amino acid sequences that affect the specificity of RNA recognition in two members of a large family of proteins involved in RNA processing.

Amino Acid Sequence↗
PubMed1990

The fine specificity of anti-La antibodies induced in mice by immunization with recombinant human La autoantigen.

Because of increasing evidence suggesting that anti-La autoantibodies are induced in humans by an Ag-specific mechanism, we investigated the antibody response of animals immunized with the human La Ag and studied its relationship to the anti-La response of autoimmune patients. Anti-La antibodies were raised in 6- to 8-wk-old male MRL(-)+/+, C57BL/6J, BALB/c, and A/J mice by immunizing with authentic human La protein obtained by recombinant expression in Escherichia coli. As we have shown previously for human autoantibodies, induced mouse anti-La antibodies reacted with recombinant fusion proteins containing nonoverlapping sequences from different portions of the La molecule. The epitope specificity of antibodies to the middle region of the La Ag was further evaluated using six synthetic La peptides predicted to be antigenic based on their hydrophilic properties. Although the induced mouse anti-La antibodies bound to five of the six synthetic La peptides, human anti-La autoantibodies failed to recognize any of the peptide homologs. These results suggest that mice respond to immunization with human La protein differently than humans who develop autoimmunity to this self Ag.

Amino Acid Sequence↗
PubMed1990

Antigenicity of a recombinant Ro (SS-A) fusion protein.

The antigenicity of the 60-kd human Ro (SS-A) synthesized in vitro from its complementary DNA as a beta-galactosidase fusion protein (beta-gal-Ro) was evaluated by Western blotting. In this analysis, almost all the anti-Ro (SS-A)-positive sera that bound beta-gal-Ro also bound affinity-purified 60-kd human Ro (SS-A) (P less than 0.005). Three of the 27 anti-Ro (SS-A) precipitin-positive sera, however, did not show reactivity on Western blot analysis, which suggests that in some sera, antigenicity to Ro (SS-A) is destroyed by denaturation. Of the 22 sera that were reactive with beta-gal-Ro, 2 were not reactive with affinity-purified human Ro (SS-A). Two serum samples that did not react with beta-gal-Ro were also reactive with affinity-purified human Ro (SS-A). Nevertheless, except for a small percentage of Ro (SS-A) precipitin-positive sera, the frequency of antibody binding to the fusion protein was similar to the frequency of binding to the purified antigen in Western blots. Recombinant Ro (SS-A) antigen may therefore be valuable in the serologic evaluation of anti-Ro (SS-A) autoantibodies.

Autoantigens↗
PubMed1990

Expression of autoantibodies to recombinant (U1) RNP-associated 70K antigen in systemic lupus erythematosus.

To determine the specificity of antibodies to the (U1) ribonucleoprotein antigen in systemic lupus erythematosus (SLE), patient sera were tested for binding to a recombinant human 70K antigen. By solid-phase immunoassay, we detected anti-70K reactivity in sera from 31 of 96 patients with systemic lupus erythematosus (SLE), demonstrating that anti-70K antibodies may occur in patients with SLE as well as other clinical diagnoses. In sequential sera from 2 of these patients, we found that anti-70K binding varied dramatically over the course of disease. The changes in anti-70K antibody levels did not correlate with clinical events nor evolving antibody reactivity with the Sm-specific antigens.

Autoantibodies↗
PubMed1990

Quantitative determination that one of two potential RNA-binding domains of the A protein component of the U1 small nuclear ribonucleoprotein complex binds with high affinity to stem-loop II of U1 RNA.

Many RNA-associated proteins contain a ribonucleoprotein (RNP) consensus octamer encompassed by a conserved 80 amino acid sequence, which we have termed an RNA recognition motif (RRM). RRM family members contain either one (class I) or multiple (class II) copies of this motif. We report here that a class II component of the U1 small nuclear RNP (snRNP), the A protein of U1 snRNP (U1snRNP-A), contains two RRMs (RRM1 and -2), yet has only one binding domain (RRM1) that interacts specifically with stem-loop II of U1 RNA. Quantitative analysis of binding affinities of fragments of U1snRNP-A demonstrated that an 86-amino acid polypeptide was competent to bind to U1 RNA with an affinity comparable to that of the full-length protein (Kd approximately 80 nM). The carboxyl-terminal RRM2 of U1snRNP-A did not bind to U1 RNA and may recognize an unidentified heterologous RNA. We propose that class II proteins may function as bridges between RNA components of RNP complexes such as the spliceosome.

Binding Sites↗