Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions. 182 47

Antibodies to uridylic acid rich small nuclear ribonucleoprotein particles (UsnRNP) are mainly detected in patients with systemic lupus erythematosus (SLE) or mixed connective tissue disease (MCTD). Particularly those directed against epitopes of the 70K protein of U1snRNP serve as important markers for the diagnosis of MCTD. To establish an ELISA for determination of anti-70K protein antibodies in patients' sera a 1239 bp long cDNA insert coding for the epitopes of the 70K protein was ligated into a fusion expression vector. The bacterially expressed fusion protein was purified by chromatography on DEAE cellulose. Microtiter plates were coated with the fusion protein as well as with partially purified calf thymus extract (CTE) containing all natural UsnRNP antigens and RNase digested calf thymus extract (CTERNase) in which the natural 70K antigen was destroyed by the nuclease treatment. 10,888 sera of patients with suspected or overt rheumatic disease were analyzed for antibodies against these antigens simultaneously. Antibodies against CTE or CTERNase were not detected in 9123 sera, none of these showed reactivity with the 70K protein indicating a high degree of specificity of the assay. Positive results in each the 70K protein, CTE as well as the CTERNase ELISAs were obtained with 474 sera. 319 sera were only positive with CTE and 70K protein. Of these 793 anti-70K protein ELISA positive sera, 79% could be confirmed by immunoblot. Of 967 sera reacting with CTE and CTERNase but not with the recombinant 70K protein, 31% contained antibodies against various other UsnRNP proteins as shown by immunoblotting. 2.4% of these sera revealed also antibodies against the 70K protein. The use of the recombinant 70K protein as antigen meets the criterion for a simple and specific assay to detect anti-U1snRNP antibodies. Nevertheless, the sole use of this recombinant protein for anti-U1snRNP antibody screening may not be appropriate, because antibodies against other frequently occurring U1snRNP proteins (A, C) cannot be detected with this test. Therefore it should be used together with a natural UsnRNP antigen until further studies in patients with well established diagnoses will show whether natural antigens may be omitted.
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PMID:A recombinant 70K protein ELISA. Screening for antibodies against U1snRNP proteins in human sera. 183 68

We present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleoprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells.
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PMID:RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection. 213 10

Sera from patients with systemic autoimmune diseases often contain antibodies against small nuclear ribonucleoprotein (snRNP) particles. Anti-Sm antibodies react with the entire set of U1, U2, U4, U5 and U6 (U1-U6) RNP particles whereas anti-(U1)RNP sera specifically recognize particles containing U1 RNA. Here we performed semi-quantitative immunoblotting using 16 human anti-Sm, 15 human anti-(U1)RNP sera and two mouse monoclonal antibodies to establish which snRNA-associated proteins carry antigenic determinants. Almost every (15/16) human anti-Sm sera recognized epitopes present on a 28-kDa (B/B') protein doublet and on a 16-kDa (D) polypeptide. Nine anti-(U1)RNP sera also recognized the B/B' doublet, but in all cases a much stronger reaction was observed with one or more of the specifically U1 RNA-associated 70 kDa, A or C antigens. With affinity-purified antibody fractions eluted from individual antigen bands on nitrocellulose blots it is shown that the anti-Sm-reactive polypeptides B/B' and D contain common epitopes. We also report the finding of one human anti-Sm serum with exclusive specificity for the B/B' doublet and a mouse monoclonal anti-Sm antibody recognizing only the D protein, indicating that these antigens also carry unique epitopes. In immunoprecipitation assays, purified anti-B/B' and -D antibodies react with (U1-U6) RNP while purified anti-70 kDa, anti-A and anti-C antibodies precipitate exclusively U1 RNP particles. Finally, we established the subcellular localization of Sm and U1 RNP antigens using a biochemical cell fractionation procedure. Part of the 70 kDa and B/B' antigens were found in a nuclease and high salt-resistant nuclear substructure, usually referred to as nuclear matrix, while the A and D antigens could be extracted completely from HeLa nuclei by ribonuclease treatment and subsequent high salt extraction.
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PMID:Further characterization and subcellular localization of Sm and U1 ribonucleoprotein antigens. 241 12

Native small nuclear ribonucleoproteins (snRNPs) purified by several conventional procedures or reconstituted in vitro have no ribonuclease activity. However, when these same snRNPs are centrifuged in cesium chloride gradients at low [Mg2+] and in the presence of sarkosyl, an endoribonuclease is unmasked at the density of core particles (i.e. containing only the set of low molecular weight proteins common to all snRNPs), while an inhibitory component is released in soluble form. The nature of this inhibitor was not further investigated and the molecular events underlying this inhibition/activation process remained only a matter of speculation. On the other hand, evidence was obtained that the nuclease activity is carried by B-B' on the basis of its comigration with B-B' as well as with two of their cleavage products after SDS/polyacrylamide gel electrophoresis of snRNP proteins. One was identified by a B-B'-specific monoclonal antibody. Another one, especially prominent and migrating between D and E core proteins, was identified as the N-terminal half of B-B' by microsequence analysis. Although tightly associated with core snRNPs, the activity is not dependent upon the presence of an snRNA. For the time being, the functional significance of this nuclease remains entirely elusive.
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PMID:B-B' proteins from small nuclear ribonucleoproteins have an endoribonuclease catalytic domain inactive in native particles. 252 74

The complexity of plant U-type small nuclear ribonucleoprotein particles (UsnRNPs) may represent one level at which differences in splicing between animals and plants and between monocotyledonous and dicotyledonous plants could be effected. The maize (monocot.) U2snRNA multigene family consists of some 25 to 40 genes which from RNA blot and RNase protection analyses produce U2snRNAs varying in both size and sequence. The first 77 nucleotides of the maize U2-27 snRNA gene are identical to U2snRNA genes of Arabidopsis (dicot). Despite much lower sequence homology in the remaining 120 nucleotides the secondary structure of the RNA is conserved. The difference in splicing between monocot. and dicot. plants cannot be explained on the basis of sequence differences between monocot, and dicot. U2snRNAs in the region which may interact with intron branch point sequences.
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PMID:Maize U2 snRNAs: gene sequence and expression. 253 69

We identified eight patients whose sera contained autoantibodies to the U2 small nuclear ribonucleoprotein (snRNP), an RNA protein particle involved in the splicing of newly transcribed messenger RNA. Each of these patients had an overlap syndrome that included features of either systemic lupus erythematosus (SLE), scleroderma, and/or polymyositis. We then used these sera to characterize the autoantigenic polypeptides of the U1 and U2 snRNP particles. In immunoblots, all sera contained antibodies to the B" polypeptide of the U2 snRNP. A subset of these sera that more effectively immunoprecipitated the native U2 particle contained an additional antibody system that recognized the A' polypeptide of this snRNP. Antibodies eluted from the B" protein bound the A polypeptide of the U1 snRNP and vice versa. Moreover, antibodies to the B" polypeptide were accompanied by antibodies to the 68K and C polypeptides of the U1 snRNP. Finally, the A' and B" polypeptides remained physically associated after the U2 particle was cleaved with RNase. Thus these sera contain multiple autoantibody systems that, at one level, target two physically associated antigenic polypeptides of the U2 particle and, at another, target two snRNP particles which are associated during the splicing of premessenger RNA. These linked autoantibody sets provide further evidence that intact macromolecular structures are targeted by the immune response in SLE and related diseases.
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PMID:The U2 small nuclear ribonucleoprotein particle as an autoantigen. Analysis with sera from patients with overlap syndromes. 296 64

The structures and functions of small nuclear ribonucleoprotein particles have become of interest because of their suggested role in processing heterogeneous nuclear RNA [Lerner, M. R., Boyle, J. A., Mount, S. M., Wolin S. L. & Steitz, J. A. (1980) Nature (London) 283, 220-224]. To determine the conformation of U-1 RNA in U-1 ribonucleoprotein particles and whether proteins of these particles protect segments of U-1 RNA, intact particles and isolated U-1 RNA were digested with T1 RNase. The digested particles were immunoprecipitated with anti-Sm antibodies. A 5'-end fragment containing nucleotides 1-107 and 3'-end fragments containing nucleotides 108-165 and 108-153 were recovered in nearly quantitative yield from digestion of the particles, suggesting that position 107 is the principal cleavage site in them. At the same T1 RNase concentrations, deproteinized U-1 RNA was cleaved into many fragments. At low T1 RNase concentrations, major cleavage site of deproteinized U-1 RNA was at nucleotide 69. Comparison of the cleavage sites of free U-1 RNA and of U-1 RNA in U-1 ribonucleoprotein particles suggested similar secondary structures. The resistance of the 5' end of U-1 RNA to T1 RNase was unexpected inasmuch as this region has been implicated in hydrogen bonding with heterogeneous nuclear RNA splice junctions.
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PMID:Site-specific cleavage by T1 RNase of U-1 RNA in u-1 ribonucleoprotein particles. 616 13

Cell-free translation of human poly(A)+ RNA was carried out to generate and analyze the protein constituents of small nuclear ribonucleoprotein (snRNP) particles. The snRNP proteins were identified by immunoprecipitation with sera from patients with systemic lupus erythematosus. Size fractionation of mRNA prior to translation revealed that these snBNP proteins are all encoded by separate messages. One of the proteins (the A protein, molecular weight 32,000) was seen to lose antigenicity upon RNase treatment either when extracted from cells or when generated in vitro. RNase treatment of immunoprecipitated snRNPs released the A protein in an electrophoretically pure form. Analysis of snRNPs translated in vitro revealed the presence of unassembled and assembled particles as determined by sucrose density gradient sedimentation. Post-translational assembly of snRNPs involving both RNA-protein binding (as revealed by A protein antigenicity) and associations of other snRNP proteins occurred in the in vitro system employed here. In addition, the presence of unassembled snRNP proteins permitted the determination of the precise antigen peptides recognized by Sm and RNP autoimmune sera. It was observed that Sm sera are capable of recognizing each of the eight snRNP proteins, whereas RNP sera recognize only two of the eight.
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PMID:Synthesis and assembly of human small nuclear ribonucleoproteins generated by cell-free translation. 622 47

A clinical laboratory carrying out tests for antinuclear antibodies requires an efficient, reliable preparation method to produce a high yield of nuclear antigens at low cost and a very sensitive, specific assay method for antigen activity. Various tissues were employed for preparation of small nuclear ribonucleoprotein (snRNP) and Sm antigens for these purposes. Fresh calf thymus cells and nuclei, commercially available calf and rabbit thymus acetone powders, fresh rat kidney and liver cells were used as sources of antigens prepared similarly by methods published previously. Preparations of antigens from whole calf thymus cell extracts were prepared with and without inhibitors to protease and RNase. snRNP and Sm antigens were assayed at each preparation step by hemagglutination inhibition (HAI). Using HAI it was possible to routinely assay snRNP and Sm at nanogram/ml quantities which was 10(6) fold more sensitive than Ouchterlony immunodiffusion. Results were expressed as relative specific activity as compared with calf thymus nuclear extract prepared by conventional methods. Protease and RNase inhibitors did not significantly increase yields. Thymus was the best source of snRNP and Sm. Fresh calf thymus extract produced a good, stable, reliable quantity of antigens, whereas calf and rabbit thymus acetone powders provided antigen at higher specific activity with less labor but slightly lower yields. Thus, considering the total cost of preparations, commercial sources may be superior to fresh sources in the clinical laboratory setting. These studies also revealed the utility of the sensitive HAI test not only in the clinical laboratory but also for further research endeavors.
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PMID:Comparison of various preparations of nuclear antigens by hemagglutination inhibition (HAI). 623 76


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