Sm core variation in spliceosomal small nuclear ribonucleoproteins from Trypanosoma brucei

Messenger RNA processing in trypanosomes by cis and trans splicing requires spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5, as well as the spliced leader (SL) RNP. As in other eukaryotes, these RNPs share a core structure of seven Sm polypeptides. Here, we report that the identity of the Sm protein constituents varies between spliceosomal snRNPs: specifically, two of the canonical Sm proteins, SmB and SmD3, are replaced in the U2 snRNP by two novel, U2 snRNP-specific Sm proteins, Sm15K and Sm16.5K. We present a model for the variant Sm core in the U2 snRNP, based on tandem affinity purification-tagging and in vitro protein-protein interaction assays. Using in vitro reconstitutions with canonical and U2-specific Sm cores, we show that the exchange of two Sm subunits determines discrimination between individual Sm sites. In sum, we have demonstrated that the heteroheptameric Sm core structure varies between spliceosomal snRNPs, and that modulation of the Sm core composition mediates the recognition of small nuclear RNA-specific Sm sites.     

Immune responses to small nuclear ribonucleoproteins: antigen-dependent distinct B cell epitope spreading patterns in mice immunized with recombinant polypeptides of small nuclear ribonucleoproteins

Complex patterns of autoantibody reactivities with the small nuclear ribonucleoproteins (snRNPs) are observed in systemic lupus erythematosus. To investigate the role of individual snRNP components in the initiation and diversification of anti-snRNP Ab responses, we immunized A/J mice with recombinant Smith D (SmD), Smith B (SmB), and A ribonucleoprotein (A-RNP) with alum as adjuvant. Sera at different time points after initial immunizations were analyzed by Western blot and immunoprecipitation assays. In SmD-immunized mice, specific Abs to A-RNP and SmB were generated by 2 mo postimmunization, in addition to the detection of cross-reactive Abs between the immunogen and other snRNPs. Whereas Abs reactive with the immunogen decreased by 5 mo, Abs capable of immunoprecipitating A-RNP and SmB increased. In SmB-immunized mice, specific Abs to A-RNP were readily detectable, in addition to cross-reactive Abs. In contrast, A-RNP-immunized mice had only cross-reactive Abs to SmB without detectable Abs to SmD. However, in these mice, specific Abs to the 70-kDa protein were generated. Abs, which precipitated the native snRNP particle, were generated in all three groups of the immunized mice. Our results show that different initiating Ags from the same multiprotein antigenic complex induce distinct patterns of epitope spreading to proteins within that complex. These data have significant implications for the mechanisms of autoantibody diversification in systemic lupus erythematosus.     

Rapid purification of native C protein from nuclear ribonucleoprotein particles

A rapid three step procedure is described for the purification of C protein from HeLa 40 S hnRNP particles. The procedure takes advantage of the salt resistant RNA binding of C protein, the size of the C protein-RNA complex, and the strong binding of C protein to an anion-exchange resin. Typically 120 micrograms of C protein is obtained from 4.0 X 10(9) cells with greater than 95% electrophoretic purity. Proteins C1 and C2 copurify in the ratio of 3.5 Cl to 1 C2. The purified C protein participates in hnRNP particle reconstitution and on this basis is judged to be native. The purified C protein binds to a gel filtration matrix at 0.5 M NaCl but at higher salt concentrations it elutes before the marker protein, apoferritin (Mr = 443,000). An abbreviated two step purification procedure utilizing anion-exchange chromatography is also described. This procedure results in relatively pure C protein, as well as a useful separation of the other hnRNP proteins.     

Nuclear organization of splicing small nuclear ribonucleoproteins in adenovirus-infected cells.

We have studied the effect of adenovirus infection on the nuclear organization of splicing small nuclear ribonucleoproteins (snRNPs) in HeLa cells. In uninfected HeLa cells, snRNPs are widespread throughout the nucleoplasm but also are concentrated in specific nuclear structures, including coiled bodies, interchromatin granules, and perichromatin fibrils. We have used immunofluorescence microscopy to study the localization of splicing snRNPs relative to centers of viral DNA synthesis and accumulation identified with antiserum against the viral 72,000-molecular-weight single-stranded DNA-binding protein (72K protein). Splicing snRNPs were independently detected with both monoclonal and polyclonal antibodies specific for common snRNP antigens, snRNP-specific proteins, and the snRNA-specific 2,2,7-trimethylguanosine 5' cap structure. We have examined infected cells 2 to 24 h after infection, and, in the majority of these cells, we observed no colocalization of the snRNP and 72K-protein staining patterns. In the late phase, snRNPs were found to markedly concentrate in discrete clusters that were distinct from the centers of viral DNA synthesis and accumulation identified with anti-72K protein. We have treated cells with hydroxyurea at various times after infection to inhibit aspects of the virus infectious program. We have found that the accumulation of snRNP clusters is correlated with late gene expression rather than with DNA synthesis or early gene expression. Finally, we show that the late-phase snRNP clusters colocalize with a monoclonal antibody that primarily stains interchromatin granules. These results suggest that the centers of snRNP concentration in late-phase infected cells are likely to correspond to interchromatin granule clusters.     

Dynamic changes in small nuclear ribonucleoproteins of heat-stressed and thermotolerant HeLa cells

Living organisms when subjected to various forms of environmental stress mount a physiological response to survive the long- and short-term ill-effects of the stress. The stress response may involve selective shut down of non-essential metabolic activities and the repair of macromolecular damage resulting from the stress. Messenger RNA splicing in cultured HeLa cells is one of the processes inhibited by heat stress. Splicing is protected from such inhibition in stress-preconditioned cells that have acquired a tolerant state characterised by increased cell survival and resistance to other environmental stresses. Stress tolerant cells have heat shock proteins (HSPs) that had been induced by the preconditioning process. To examine the biochemical changes induced by stress in the splicing apparatus, we analysed the small nuclear ribonucleoprotein (snRNP) particles associated with spliceosomes in normal, stressed, and stress tolerant cells. We show that (a) the spliceosomal component U4/U5/U6 snRNP particle is disassembled by heat stress into intermediates of splicing assembly, (b) prior induction of stress tolerance protects the structural and functional integrity of snRNPs if cells are subsequently exposed to a severe stress and (c) a novel 65 kDa protein is associated with small nuclear ribonucleoprotein particles in stress tolerant cells.     

Nucleic acid binding properties of major proteins from the heterogeneous nuclear ribonucleoproteins of wheat.

Glycine-rich core hnRNP proteins purified from wheat bind tightly to single-stranded but not to double-stranded nucleic acids with a preference for natural RNA over single-stranded DNA. Binding results in i) a progressive disruption of the residual secondary structure of the polynucleotide and the formation of an extended nucleoprotein filament until a protein to polynucleotide weight ratio of about 5:1 is attained. As more protein is added, this is followed by ii) the formation of globular structures along the polynucleotide chain with a concomitant reduction in the contour length of the nucleoprotein complex. These two features of the interaction--unwinding and condensation into beads--are analogous to the previously described behavior of the major glycine-rich core hnRNP protein from Artemia salina (Thomas et al. (1981) Proc. Natl. Acad. Sci. USA 78, 2888) and may represent the basic functional properties of this relatively well conserved group of nuclear proteins.     

A two-tracked approach to analyze RNA-protein crosslinking sites in native,nonlabeled small nuclear ribonucleoprotein particles

Much attention is currently being devoted to questions of protein and RNA tertiary structures and to the quaternary arrangement of the individual macromolecules in ribonucleoprotein (RNP) particles. In this article we describe two complementary strategies that allow the identification of RNA-protein contact sites in assembled, nonlabeled RNP particles after UV crosslinking. The first combines immunoprecipitation of UV-irradiated RNP particles under mildly denaturing conditions followed by primer-extension analysis of the crosslinked (and thus coprecipitated) RNA. The second involves the purification of crosslinked peptide-oligonucleotide from RNP particles and the subsequent analysis of the crosslinked peptide and RNA by Edman degradation and matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS), respectively. Although the first approach provides a rapid method for the exact identification of RNA-protein contact sites in purified nonlabeled RNP particles, the latter adds valuable information about potential RNA binding domains within proteins and, thus, about the arrangement of these proteins within the quaternary structures of complex RNP assemblies. Recently, we applied both these strategies successfully to native purified spliceosomal RNP. These methods may be generally applicable to the analysis of RNP complexes, especially as they avoid labeling and reconstitution, both of which risk introducing artifacts.     

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.     

Reassembly and protection of small nuclear ribonucleoprotein particles by heat shock proteins in yeast cells

The process of mRNA splicing is sensitive to in vivo thermal inactivation, but can be protected by pretreatment of cells under conditions that induce heat-shock proteins (Hsps). This latter phenomenon is known as "splicing thermotolerance". In this article we demonstrate that the small nuclear ribonucleoprotein particles (snRNPs) are in vivo targets of thermal damage within the splicing apparatus in heat-shocked yeast cells. Following a heat shock, levels of the tri-snRNP (U4/U6.U5), free U6 snRNP, and a pre-U6 snRNP complex are dramatically reduced. In addition, we observe multiple alterations in U1, U2, U5, and U4/U6 snRNP profiles and the accumulation of precursor forms of U4- and U6-containing snRNPs. Reassembly of snRNPs following a heat shock is correlated with the recovery of mRNA splicing and requires both Hsp104 and the Ssa Hsp70 family of proteins. Furthermore, we correlate splicing thermotolerance with the protection of a subset of snRNPs by Ssa proteins but not Hsp104, and show that Hsp70 directly associates with U4- and U6-containing snRNPs in splicing thermotolerant cells. In addition, our results show that Hsp70 plays a role in snRNP assembly under normal physiological conditions.     

Recovery of catalytic activity from an inactive aggregated mutant of l-aspartase.

Two highly conserved lysyl residues have been replaced with an arginine to examine their role in the mechanism of l-aspartase from Escherichia coli. Replacement of an active-site lysine results in a significant loss of catalytic efficiency [A. S. Saribas, J. F. Schindler, and R. E. Viola (1994) J. Biol. Chem. 269, 6313-6319], while replacement of the second lysine leads to a completely inactive and insoluble protein. Fluorescence spectral evidence has suggested that the loss of activity is due to the misfolding of this aspartase mutant. Some catalytic activity is recovered when the mutant is treated with varying levels of denaturants, and extended treatment with high levels of guanidine.HCl results in the recovery of a substantial fraction of the wild-type activity from this inactive mutant. However, upon removal of the denaturant this mutant enzyme slowly reverts to its inactive and insoluble form. Treatment with an artificial chaperone system in which solubilization by detergent is followed by its removal with beta-cyclodextrin leads to a stable enzyme under nondenaturing conditions with about half the catalytic activity of the wild-type enzyme. These results confirm a structural role for lysine-55 in l-aspartase and demonstrate that additional characterization is required before conclusions can be drawn from the production of an inactive mutant.     

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.     

Immunological characterization and intracellular localization of trans-spliceosomal small nuclear ribonucleoproteins in Trypanosoma brucei.

Polyclonal antibodies were raised against purified protein components of the U2 small nuclear ribonucleoprotein (snRNP) from Trypanosoma brucei. Through immunoblot and immunoprecipitation analyses three antisera were characterized that reacted specifically with U2 snRNP proteins of molecular weights 40,000 (anti-40K) and 16,500 (anti-16.5K), and with each of four proteins of molecular weights 14,000, 12,500, 10,000, and 8,500 (anti-CP). Anti-40K antibodies specifically immunoprecipitated the U2 snRNP from trypanosomal extracts, whereas anti-CP antibodies recognized several snRNPs, including the SL RNP and the U2 and U4/U6 snRNPs; in addition, minor RNAs were detected, suggesting that a family of snRNPs with common or related protein components exists in trypanosomes. None of these antibodies cross-reacted significantly with total mammalian snRNP proteins, indicating that the trypanosomal snRNP proteins are immunologically distinct from their mammalian counterparts. Using immunofluorescence microscopy, the snRNP proteins exhibited a differential cellular distribution. Whereas the 40-kDa protein is localized exclusively in the nucleus, with the nucleolus being excluded, a fraction of the common proteins also resides in the cytoplasm.     

Nonhistone nuclear proteins specific to certain mouse embryonal carcinoma clones having an inactive X chromosome

By means of one-dimensional polyacrylamide slab gel electrophoresis nonhistone nuclear proteins were compared in murine embryonal carcinoma cell clones with two X chromosomes; both are active in some clones and one of them is inactive in others and in a population of cells having only one X chromosome. Under our experimental conditions, we succeeded in finding two extra bands at approximately 46,000 Da in cells having an inactive X chromosome. Furthermore, a band at approximately 71,000 Da was significantly heavier in cells having an inactive X chromosome than in those having two active X or those having only one X chromosome.     

Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs

U₁ snRNP² isolated from HeLa cells and purified by centrifugation in cesium chloride contains a set of proteins that may be resolved into four/five polypeptides by gel electrophoresis. When this particle was submitted to extensive digestion with micrococcal nuclease, RNA fragments of about 25 nucleotides in length were obtained. Sequence analyses showed that these highly protected fragments were derived from the same region of the U₁ molecule, spanning positions 119 to 143. At low concentrations of nuclease, a longer fragment, from nucleotide 119 to the 3′ OH end, was also detected. U₁ core-resistant snRNP, isolated by high performance liquid chromatography, still contains all the protein components of the intact particle.When a less drastically purified U₁ snRNP containing, beside the four/five polypeptides remaining after centrifugation in cesium chloride, a set of at least three polypeptides of larger size, was digested with the nuclease, no other protected RNA fragment was detected.When a mixture of U₁, U₂, U₄, U₅ and U₆ snRNPs, which contains the same four/five polypeptides as U₁ snRNP, was treated with micrococcal nuclease, protected fragments of snRNAs U₂, U₄ and U₅ were found in addition to those derived from U₁. No fragment derived from U₆ was found.In all cases, the region of snRNA shielded from nuclease attack corresponds to a distinctive feature of the molecule. It is a single-stranded region, comprising the sequence A(U)_nG with n ≥ 3, bordered by two double-stranded stems. One of these stems includes the 3′ terminus of the RNA, except in the case of U₂, where there are two stems instead of one on the 3′ side of the single-stranded stretch. Although a comparable structural domain exists also in U₆ snRNA, it does not contain the sequence A(U)_nG which correlates well with the fact that no U₆ snRNA fragment seems to resist micrococcal nuclease digestion.     

YTH: a new domain in nuclear proteins

A novel 100-150-residue domain has been identified in the human splicing factor YT521-B and its Drosophila and yeast homologues. Homology searches show that the domain is typical for the eukaryotes and is particularly abundant in plants. It is predicted to adopt a mixed alpha-helix-beta-sheet fold and to bind to RNA. We propose the name YTH (for YT521-B homology) for the domain.     

Eubacterial components similar to small nuclear ribonucleoproteins: identification of immunoprecipitable proteins and capped RNAs in a cyanobacterium and a gram-positive eubacterium.

Small nuclear ribonucleoprotein (snRNP) particles play an important role in the processing of pre-mRNA. snRNPs have been identified immunologically in a variety of cells, but none have ever been observed in prokaryotic systems. This report provides the first evidence for the presence of snRNP-like components in two types of prokaryotic cells: those of the cyanobacterium Synechococcus leopoliensis and those of the gram-positive eubacterium Bacillus subtilis. These components consist of snRNP-immunoreactive proteins and RNAs, including some with the snRNP-unique 5' m2,2,7G (m3G) cap. Immunoreactivity was determined by immunoprecipitation procedures, with either antinuclear-antibody-positive (RNP- and Sm-monospecific) patient sera or a m3G monoclonal antibody, with radiolabelled cell extracts that were preadsorbed with antinuclear-antibody-negative sera. S. leopoliensis immunoprecipitates showed the presence of high-molecular-mass proteins (14 to 70 kDa) and RNAs (138 to 243 nucleotides) that are analogous in size to proteins and RNAs found in human (HEp-2) cell immunoprecipitates but absent in Escherichia coli immunoprecipitates. Thin-layer chromatography of S. leopoliensis immunoprecipitates confirmed the presence of a capped nucleotide similar to a capped nucleotide in HEp-2 immunoprecipitates; no such nucleotide was observed in E. coli immunoprecipitates. Immunoreactive RNAs (117-170 nucleotides) were identified in a second eubacterium, B. subtilis, as well. This work suggests that snRNPs or their evolutionary predecessors predate the emergence of eukaryotic cells.