12 S small nuclear ribonucleoprotein-associated acidic-and pyrimidine-specific endoribonuclease from calf thymus and L5178y cells

12 S ribonucleoprotein (RNP) particles were separated from a 45 S RNP complex (Bachmann, M., Zahn, R. K. and Müller, W. E. G. (1983) J. Biol. Chem. 258, 7033-7040) isolated from calf thymus and L5178y cells. The particles were determined to be associated with an acidic endoribonuclease (pI 4.1; pH optimum 6.2). the enzyme requires Mg2+ and is sensitively inhibited by higher NaCl concentrations. The nuclease specifically degrades poly(U) and poly(C) in an endonucleolytic manner; the end-products are 3'-UMP (85%) and 2',3'-cyclic UMP (12%). Poly(A) strongly inhibits the pI 4.1 endoribonuclease activity. The Michaelis constant (for poly(U)) was determined as 82 microM and the maximal reaction velocity was 0.54 mumol/microgram per h. The endoribonuclease is distinguished from the known pyrimidine-specific ribonucleases (pancreatic ribonuclease and endoribonuclease VII) by further criteria, e.g., resistance to thiol reagents, inhibition by EDTA, Mg2+ requirement, pI and pH optimum. Using the techniques of counterimmunoelectrophoresis and immunoaffinity column chromatography it was shown that the pI 4.1 endoribonuclease-associated 12 S RNP particles display antigenicity to anti-Sm and anti-(U1)-RNP antibodies. An RNA component, isolated from the 12 S-45 S hypercomplex, was identified as U1-snRNA.     

12 S small nuclear ribonucleoprotein-associated acidic and pyrimidine-specific endoribonuclease from calf thymus and L5178y cells

12 S ribonucleoprotein (RNP) particles were separated from a 45 S RNP complex (Bachmann, M., Zahn, R.K. and Müller, W.E.G. (1983) J. Biol. Chem. 258, 7033–7040) isolated from calf thymus and L5178y cells. The particles were determined to be associated with an acidic endoribonuclease (pI 4.1; pH optimum 6.2). the enzyme requires Mg²⁺ and is sensitively inhibited by higher NaCl concentrations. The nuclease specifically degrades poly(U) and poly(C) in an endonucleolytic manner; the end-products are 3′-UMP (85%) and 2′,3′-cyclic UMP (12%). Poly(A) strongly inhibits the pI 4.1 endoribonuclease activity. The Michaelis constant (for poly(U)) was determined as 82 μM and the maximal reaction velocity was 0.54 μmol/μg per h. The endoribonuclease is distinguished from the known pyrimidine-specific ribonucleases (pancreatic ribonuclease and endoribonuclease VII) by further criteria, e.g., resistance to thiol reagents, inhibition by EDTA, Mg²⁺ requirement, pI and pH optimum. Using the techniques of counterimmunoelectrophoresis and immunoaffinity column chromatography it was shown that the pI 4.1 endoribonuclease-associated 12 S RNP particles display antigenicity to anti-Sm and anti-(U1)-RNP antibodies. An RNA component, isolated from the 12 S-45 S hypercomplex, was identified as U1-snRNA.     

Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally-induced mouse monoclonal antibodies

Small nuclear ribonucleoprotein (snRNP) particles are a class of RNA-containing particles in the nucleus of eukaryotic cells. They consist of uridylate-rich small nuclear RNA complexed with several proteins. snRNP particles U1, U2, U4/U6, and U5 all contain a common protein core consisting of proteins B'/B, D, D', E, F, and G. In addition to this core, U1 snRNP particles contain proteins 70K, A, and C, whereas U2 snRNP particles contain proteins A' and B". Almost any of the small nuclear RNA-associated polypeptides is targeted by autoantibodies in the sera from patients with SLE or related connective tissue diseases. We immunized a genetically non-autoimmune mouse with recombinant human B" protein and obtained three mAb reactive with native U2 snRNP particles. Two of these mAb particles cross-reacted with U1 snRNP, 9A9 and 11A1, via epitopes present on the U2 snRNP B" protein as well as on the U1 snRNP-specific A protein. A third mAb 4g3, reacted exclusively with U2 snRNP via a unique epitope on protein B". Two epitopes mapped at the carboxy-terminal region of the B" protein, whereas binding of the third mAb involved both amino- and carboxy-terminal amino acids of the B" protein. Epitope mapping, employing a DNAse I fragment library of the B" cDNA, revealed that the three mAb-reactive sites were discontinuous. Autoantibodies in sera from patients with SLE and other connective tissue diseases competed for binding with the mAb, implying that the mAb define a major autoantibody-reactive region on protein B".     

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.     

U1 small nuclear ribonucleoprotein studied by in vitro assembly

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody- precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA- protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.     

Purification and mode of action of a microsomal endoribonuclease from rat liver

An endoribonuclease has been purified nearly to homogeneity from rat liver microsomes, and its mode of action and general properties were studied. The enzyme had an apparent molecular weight of 58 000, as estimated by both gel filtration and SDS-polyacrylamide gel electrophoresis and produced oligonucleotides from poly(A), poly(U) and poly(C). No mononucleotide was obtained by the enzymatic hydrolysis of the above substrates. The enzyme made endonucleolytic cleavages which generated 5'-phosphate-terminated oligonucleotides. It was suggested that the existence of at least (Ado5'P)2 residues at both sides of the cleavage bond was necessary for the action of the endoribonuclease. Divalent cations (Mg2+ or Mn2+) were required for the enzymatic activity, while K+ inhibited the enzyme. Spermine stimulated the enzymatic activity in the presence of 1 mM Mg2+.     

Association of a polyuridylate-specific endoribonuclease with small nuclear ribonucleo-proteins which had been isolated by affinity chromatography using antibodies from a patient with systemic lupus erythematosus

Immunoglobulins, containing antibodies against U1-snRNP, have been prepared from a patient with systemic lupus erythematosus. After coupling these antibodies to a Sepharose matrix, U-snRNPs have been isolated and purified from rat liver nuclei by use of immunoaffinity chromatography. The resulting RNPs had the typical protein pattern of U-sn RNPs and a sedimentation coefficient of 12 S. The U-snRNP preparation was associated with an endoribonuclease which required Mg2+ for optimal activity. The enzyme, with an pH optimum of 6.2, degraded only poly(U). Other single-stranded polyribo- and polydeoxyribonucleotides, tRNA, as well as double-stranded RNA and DNA were not digested. The products of a terminal digestion are (U)6-12 with 3'-OH and 5'-P termini. The possible involvement of this endoribonuclease in the splicing of hnRNA is discussed.     

Evidence for an association between U1 RNA and interspersed repeat single-copy RNAs in the cytoplasm of sea urchin eggs

Psoralen crosslinking of RNA-RNA intermolecular duplexes in sea urchin egg extracts reveals that some maternal poly(A)+ RNA molecules are complexed with U1 RNA, a cofactor in somatic nuclear pre-mRNA splicing. Reaction of egg extracts with a monoclonal antibody specific for U1 snRNP selects, in addition to U1, RNAs that contain repeated sequences interspersed with single-copy elements. Antibody-selection experiments with nucleate and anucleate egg halves demonstrate that most of the U1 RNA-interspersed RNA complexes are cytoplasmic, as is the egg's store of total U1 snRNP. These results raise the possibility that maternal interspersed RNAs include unprocessed pre-messenger RNA molecules in arrested complexes with splicing cofactors.     

Three-step purification of retinol-binding protein from rat serum

An endoribonuclease has been purified about 320-fold from the microsomes of rat liver. The enzyme had an apparent molecular weight of 54 000-58 000 and produced oligonucleotides, each consisting of 3-7 nucleotides from poly(A) and poly(U). No mononucleotide was obtained by the enzymatic hydrolysis of poly(A) and poly(U) under standard coditions. The relative rates of breakdown of synthetic polynucleotides by the enzyme under standard conditions were in the order poly(U) = poly(A) > poly(C). Divalent cations (Mg2+ or Mn2+) was required for the enzymatic activity, but monovalent cations (Na+, K+ or NH4+) inhibited the enzyme. The breakdown of poly(C) and poly(U) by the enzyme was inhibited by spermine, but that of poly(A) was not influenced by spermine. The enzyme was inhibited by p-chloromercuribenzoate and poly(G), but not by rat-liver ribonuclease-inhibitor and anti-RNase A serum.     

U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.     

Genetic depletion indicates a late role for U5 snRNP during in vitro spliceosome assembly.

The pre-mRNA splicing pathway is highly conserved from yeast (S. cerevisiae) to mammals. Of the four snRNPs involved in splicing three (U1, U2 and U4/U6) have been shown to be essential for in vitro splicing. To examine the remaining snRNP, we utilized our previously described genetic procedures (Seraphin and Rosbash, 1989) to prepare yeast extracts depleted of U5 snRNP. The results show that U5 snRNP is necessary for both steps of pre- mRNA splicing and for proper spliceosome assembly, i.e., addition of the U4/U5/U6 triple snRNP. The prior steps of U1 and U2 snRNP addition occur normally in the absence of U5 snRNP.     

A new endoribonuclease from Escherichia coli. Ribonuclease N.

A new ribonuclease called RNase N was isolated from Escherichia coli. It is a nonspecific endoribonuclease that can cleave rRNA, poly(U), and poly(C) to small oligonucleotides and 5'-mononucleotides. It requires monovalent cations and is inhibited by divalent cations. It is suggested that this enzyme plays a role in the decay of rRNA,under various starvation conditions and perhaps in the decay of mRNA.     

The low-abundance U11 and U12 small nuclear ribonucleoproteins (snRNPs) interact to form a two-snRNP complex.

A novel small nuclear ribonucleoprotein (snRNP) complex containing both U11 and U12 RNAs has been identified in HeLa cell extracts. This U11/U12 snRNP complex can be visualized on glycerol gradients, on native polyacrylamide gels, and by selection with antisense 2'-O-methyl oligoribonucleotides. RNase H-mediated degradation of the U12 snRNA confirmed a direct interaction between the U11 and U12 snRNPs. This snRNP complex is the first to be identified involving low-abundance snRNPs. Selection of the U11/U12 snRNP complex is sensitive to high salt, suggestive of a protein-mediated interaction. Secondary structure analyses revealed several regions of the U11 snRNP accessible for interaction with other RNAs or proteins but no detectable difference between the accessibility of these regions in the U11 monoparticle compared with the U11/U12 snRNP complex. There are also several accessible single-stranded regions in the U12 snRNP, and oligonucleotide-directed RNase H digestion identified nucleotides 28 to 36 of U12 as containing sequences required for the U11/U12 interaction. Both the U12 snRNP and the U11/U12 snRNP complex can be disrupted without altering the cleavage/polyadenylation activity of a nuclear extract.     

U1 SnRNP association with HnRNP involves an initial non-specific splice-site independent interaction of U1 SnRNP protein with HnRNA

Precursor mRNA is complexed with proteins in the cell nucleus to form heterogeneous nuclear ribonucleoprotein (hnRNP), and these hnRNPs are found associated in vivo with small nuclear RNPs (snRNPs) for the processing of pre-mRNA. In order to better characterize the ATP-independent initial association of U1 snRNP with hnRNP, an important early event in assembly of the spliceosome complex, we have determined some of the components essential to an in vitro reassociation of U1 snRNP with hnRNP. U1 snRNP reassociated in vitro with 40S hnRNP particles from HeLa cells and, similar to the in vivo hnRNP/U1 snRNP association, the in vitro interaction was sensitive to high salt concentrations. U1 snRNP also associated with in vitro reconstituted hnRNP in which bacteriophage MS2 RNA, which lacks introns, was used as the RNA component. Purified snRNA alone would not associate with the MS2 RNA-reconstituted hnRNP, however, intact U1 snRNP did interact with protein-free MS2 RNA. This indicates that the U1 snRNP proteins are required for the hnRNP/U1 snRNP association, but hnRNP proteins are not. Thus, the initial, ATP-independent association of U1 snRNP with hnRNP seems to be mediated by U1 snRNP protein(s) associating with hnRNA without requiring a splice-site sequence. This complex may then be further stabilized by intron-specific interactions and hnRNP proteins, as well as by other snRNPs.