Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct functions. A pool of Smd3 exists in the yeast cytoplasm. This is consistent with the possibility that snRNP assembly in S. cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.     

Expression and native structure of cytosolic class II small heat-shock proteins

Higher plants synthesize small heat-shock proteins (smHSPs) from five related gene families. The class I and II families encode cytosolic smHSPs. We characterized the class II smHSPs of pea (Pisum sativum) and compared them with class I smHSPs. Antibodies against recombinant HSP17.7, a class II smHSP, recognized four heat-inducible 17- to 18-kD polypeptides and did not cross-react with class I smHSPs. On sucrose gradients the class II smHSPs sedimented primarily at 8 Svedberg units, indicating that they are components of large complexes similar in size to class I smHSP complexes. However, the class I and II complexes were readily distinguishable by nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Nondenaturing immune precipitations using anti-HSP17.7 or anti-HSP18.1 (a class I smHSP) antiserum provide further evidence that the class I and II smHSPs exist in different complexes, composed primarily of smHSPs. Recombinant HSP17.7 and HSP18.1 formed complexes of sizes similar to those formed in vivo. When these two smHSPs were mixed, denatured with urea, and then dialyzed, the distinct class I and II complexes again formed, each containing only HSP18.1 or HSP17.7. Thus, cytosolic smHSPs from two related gene families expressed simultaneously form distinct complexes in vivo, suggesting that they have subtly different functions.     

An unusual chemical reactivity of Sm site adenosines strongly correlates with proper assembly of core U snRNP particles

The small nuclear ribonucleoprotein particles (snRNP) U1, U2, U4, and U5 contain a common set of eight Sm proteins that bind to the conserved single-stranded 5'-PuAU3-6GPu-3' (Sm binding site) region of their constituent U snRNA (small nuclear RNA), forming the Sm core RNP. Using native and in vitro reconstituted U1 snRNPs, accessibility of the RNA within the Sm core RNP to chemical structure probes was analyzed. Hydroxyl radical footprinting of in vitro reconstituted U1 snRNP demonstrated that riboses within a large continuous RNA region, including the Sm binding site, were protected. This protection was dependent on the binding of the Sm proteins. In contrast with the riboses, the phosphate groups within the Sm core site were accessible to modifying reagents. The invariant adenosine residue at the 5' end, as well as an adenosine two nucleotides downstream of the Sm binding site, showed an unexpected reactivity with dimethyl sulfate. This novel reactivity could be attributed to N7-methylation of the adenosine and was not observed in naked RNA, indicating that it is an intrinsic property of the RNA- protein interactions within the Sm core RNP. Further, this reactivity was observed concomitantly with formation of the Sm subcore intermediate during Sm core RNP assembly. As the Sm subcore can be viewed as the commitment complex in this assembly pathway, these results suggest that the peculiar reactivity of the Sm site adenosine bases may be diagnostic for proper assembly of the Sm core RNP. Consistent with this idea, a strong correlation was found between the unusual N7-A methylation sensitivity of the Sm core RNP and its ability to be imported into the nucleus of Xenopus laevis oocytes.     

Heterogenous nuclear RNP C1 and C2 core proteins are targets for an autoantibody found in the serum of a patient with systemic sclerosis and psoriatic arthritis

OBJECTIVE: To determine a target recognized by anti-Bh autoantibody, found in the serum of a patient with the unusual coexistence of systemic sclerosis (SSc) and psoriatic arthritis (PsA). METHODS: Antigens recognized by the anti-Bh serum were characterized by indirect immunofluorescence on HeLa cells, by conventional immunoblotting using nuclear extract or partially purified preparation of heterogenous nuclear RNP (hnRNP) proteins, and by 2-dimensional immunoblotting. For the analysis of cross-reactivity and immunofluorescence patterns, autoantibodies were affinity-purified by blot elution and then retested. RESULTS: Comparison of the reactivity of the anti-Bh antibody with the monoclonal antibody 4F4 against both the hnRNP C proteins, together with the determination of biochemical properties of the autoantigens, led to the identification of C1 and C2 core proteins as the targets for the anti-Bh autoantibody. CONCLUSION: Several essential components of the spliceosome are targeted by autoantibodies that are present in the sera of patients with systemic rheumatic diseases. We also found that the hnRNP core proteins C1 and C2 are recognized by the autoantibody present in the serum of a patient with SSc and PsA. C1 and C2 hnRNP proteins should be added to the several intracellular autoantigens recently shown to be cleaved by interleukin-1beta-converting enzyme-like enzymes during apoptosis.     

Crystal structure of the spliceosomal U2B"-U2A' protein complex bound to a fragment of U2 small nuclear RNA

We have determined the crystal structure at 2.4 A resolution of a ternary complex between the spliceosomal U2B"/U2A' protein complex and hairpin-loop IV of U2 small nuclear RNA. Unlike its close homologue the U1A protein, U2B" binds to its cognate RNA only in the presence of U2A', which contains leucine-rich repeats in its sequence. The concave surface of a parallel beta-sheet within the leucine-rich-repeat region of U2A' interacts with the ribonucleoprotein domain of U2B" on the surface opposite its RNA-binding surface. The basic carboxy-terminal region of U2A' interacts with the RNA stem. The crystal structure reveals how protein-protein interaction regulates RNA-binding specificity, and how replacing only a few key residues allows the U2B" and U1A proteins to discriminate between their cognate RNA hairpins by forming alternative networks of interactions.     

Human RanBP3, a group of nuclear RanGTP binding proteins

Willingness to pay (WTP) for a health care program can be estimated in contingent valuation (CV) studies by a nonparametric approach. The nonparametric approach is free from distributional assumptions, which is a strength compared with parametric regression-based approaches. However, using a nonparametric approach it is not clear how to obtain confidence statements for WTP estimates, for example, when testing hypotheses regarding differences in mean WTP for different subsamples. The authors propose a procedure that allows statistical testing and confidence interval estimation by employing bootstrap techniques. The method is easy to implement and has low computational costs with modern personal computers. The method is applied to data from a CV study where the WTP for hormone replacement therapy was investigated. The mean WTP was estimated for the full sample and separately for women with mild and severe menopausal symptoms. Using the proposed method, the mean WTP was significantly higher in the group with severe symptoms.     

Morphological transformation caused by a partial sequence of U5 small nuclear RNA

My colleagues and I have reported that a small nuclear RNA, U5, can cause transformation of and chromosomal aberrations in rat fibroblast 3Y1 cells. In the study described here, I found that a partial U5 sequence, the 3' half of the first stem structure (designated RNA3S), transformed the cells morphologically at a high frequency when expressed with a poly(A) tail (RNA3S+). The transformation critically depended upon a polypurine sequence in RNA3S+. The expressed RNA3S+ was associated with polysomes. The transformed cells did not exhibit significant frequencies of chromosome aberrations when compared with control cells, suggesting that the transformation occurs without significant induction of chromosome damage. Heterogeneous subcolonies emerged in monolayers when the morphologically transformed cells were subcultured and maintained at postconfluence. Cells from subcolonies acquired the ability to form small colonies in soft agar and tumors in nude mice, suggesting that RNA3S+ can drive the cells into the neoplastic stage. RNA3S+ also conferred morphological alterations and a growth advantage and decreased levels of fibronectin protein synthesis in human HeLa cells, confirming the transforming potential of the RNA. RNA3S+ transformation may therefore be a useful model system for studying a transformation process.     

Stoichiometry of reovirus structural proteins in virus, ISVP, and core particles

All eight reovirus structural proteins were resolved in a new tris, glycine, and urea (TGU) electrophoretic gel system. The specific identities of proteins were determined immunologically, biochemically, and genetically. Structural proteins of reovirus type 1 Lang had different mobilities in the TGU gel than did type 3 Dearing proteins. Intertypic reassortant viruses that contained various combinations of parental genes were used to identify each of the viral protein bands. Type 1 Lang virions were metabolically-labelled with either 3H-amino acids or 35S-methionine/cysteine and gradient purified. Aliquots of purified virions were treated to generate infectious subviral particles (ISVPs) and core particles. Radiolabelled virus, ISVP, and core proteins were resolved in the TGU gel and protein band intensities were used to determine copy numbers of each structural protein. These studies confirmed the copy numbers and locations of most reovirus proteins. However, important new findings include the discovery that virions contain approximately 120 copies of major core protein sigma 2 and 20 copies of the polymerase cofactor protein mu 2, and ISVP particles contain about 24 copies of mu 1 C that has not been processed to the delta peptide. These data are used to generate a new model of the arrangement of structural proteins with the reovirus particle.     

Domain dislocation: a change of core structure in periplasmic binding proteins in their evolutionary history

Periplasmic binding proteins (PBPs) serve as receptors for various water-soluble ligands in ATP-binding cassette (ABC) transport systems, and form one of the largest protein families in eubacterial and archaebacterial genomes. They are considered to be derived from a common ancestor, judging from their similarities of three-dimensional structure, their mechanism of ligand binding and the operon structure of their genes. Nevertheless, there are two types of topological arrangements of the central beta-sheets in their core structures. It follows that there must have been differentiation in the core structure, which we call "domain dislocation", in the course of evolution of the PBP family. To find a clue as to when the domain dislocation occurred, we constructed phylogenetic trees for PBPs based on their amino acid sequences and three-dimensional structures, respectively. The trees show that the proteins of each type clearly cluster together, strongly indicating that the change in the core structure occurred only once in the evolution of PBPs. We also constructed a phylogenetic tree for the ABC proteins that are encoded by the same operon of their partner PBP, and obtained the same result. Based on the phylogenetic relationship and comparison of the topological arrangements of PBPs, we obtained a reasonable genealogical chart of structural changes in the PBP family. The present analysis shows that the unidirectional change of protein evolution is clearly deduced at the level of protein three-dimensional structure rather than the level of amino acid sequence.     

Multiple forms of the U2 small nuclear ribonucleoprotein auxiliary factor U2AF subunits expressed in higher plants

Requirements for intron recognition during pre-mRNA splicing in plants differ from those in vertebrates and yeast. Plant introns contain neither conserved branch points nor distinct 3' splice site-proximal polypyrimidine tracts characteristic of the yeast and vertebrate introns, respectively. However, they are strongly enriched in U residues throughout the intron, property essential for splicing. To understand the roles of different sequence elements in splicing, we are characterizing proteins involved in intron recognition in plants. In this work we show that Nicotiana plumbaginifolia, a dicotyledonous plant, contains two genes encoding different homologs of the large 50-65-kDa subunit of the polypyrimidine tract binding factor U2AF, characterized previously in animals and Schizosaccharomyces pombe. Both plant U2AF65 isoforms, referred to as NpU2AF65a and NpU2AF65b, support splicing of an adenovirus pre-mRNA in HeLa cell nuclear extracts depleted of the endogenous U2AF factor. Both proteins interact with RNA fragments containing plant introns and show affinity for poly(U) and, to a lesser extend, poly(C) and poly(G). The branch point or the 3' splice site regions do not contribute significantly to intron recognition by NpU2AF65. The existence of multiple isoforms of U2AF may be quite general in plants because two genes expressing U2AF65 have been identified in Arabidopsis, and different isoforms of the U2AF small subunit are expressed in rice.     

Formation of the mesostructural hierarchic level in a medium composed of randomly packed particles

It is shown that the mesostructural level of the hierarchy in a medium of randomly packed particles is formed as part of the evolution as a complex open system. The conditions for the occurrence of a mesostructure are established and the scenario for the process is discussed. The results are illustrated on a model for random packing of two-dimensional spheres.Materials Science Institute, National Academy of Sciences of the Ukraine, Kiev. Translated from Poroshkovaya Metallurgiya, Nos. 1/2(383), pp. 82–86, January–February, 1996. Original article submitted April 13, 1994.     

Mass transport and phase transformations in a system of small Mo-W-C particles

Translated from Poroshkovaya Metallurgiya, No. 7(319), pp. 17–22, July, 1989.     

In situ stress-strain response of small metal particles embedded in a ceramic matrix

The in situ stress-strain response of metal particles embedded in a ceramic matrix was obtained by combining the measurement and the modeling of the crack opening displacement field of a crack in a brittle material bridged by metal particles. The experiments were done on a composite made from platinum particles with a volume fraction of 10% in a magnesium aluminate spinel matrix. The size of the platinum particles was varied from 1 to 12 μm to study the influence of scale on the deformation behavior. Large strain to failure (85%) and ultimate tensile strength of 550 MPa were obtained for the 1 μm particles. But the larger particles failed at a strain of less than 25%; the ultimate tensile strength was also lower. This difference in ductility is explained in terms of debonding at the metal ceramic interface. It is argued that the debonding depends on the length of the dislocation pile up at the interface, and, therefore, on the particle size. The results and the metallographic observations are consistent with a model presented here; in this model the failure condition is given by a combination of the intrinsic yield stress of platinum, and the hydrostatic constraining stress in the metal particle.     

Specific cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic cell death

The U1 small nuclear ribonucleoprotein particle is essential for splicing of precursor mRNA, an activity that depends upon both the RNA and protein components of the U1 particle. One of the U1-specific proteins that is functionally important in this splicing reaction is the 70-kDa protein (U1-70kDa). We report here that U1-70kDa is specifically cleaved in apoptotic cells, resulting in the generation of a 40-kDa fragment. The kinetics of this cleavage coincided with the appearance of cells with apoptotic morphology in the population, and the proportion of 40-kDa fragment observed was markedly increased in apoptotic cells that had become detached from the substratum. Although the inhibitor characteristics of the activity cleaving U1-70kDa suggest that interleukin 1 beta-converting enzyme (ICE) might be responsible, the specific ICE inhibitor N-(N-acetyl-tyrosinyl-valinyl-alaninyl)-3-amino-4-oxob utanoic acid (YVAD-CHO) did not prevent cleavage, and U1-70kDa was not cleaved by purified ICE in vitro. Further study of this novel cleavage and the enzyme responsible will yield information about proteolytic events that might be central in the mechanism and control of apoptosis.     

Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8-homologous gene from Trypanosoma brucei provides evidence for a U5-analogous RNP

In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6. In the absence of conventional cis mRNA splicing, the structure and function of a U5-analogous snRNP in trypanosomes has remained an open question. In cis splicing, a U5 snRNP-specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice-site recognition and selection. We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220-homologous gene (p277) coding for a 277 kDa protein. Using an antibody against a C-terminal region of the trypanosomal p277 protein, a small RNA of approximately 65 nucleotides could be specifically co-immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al. (1996). Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans-spliceosomal snRNPs. Together these results demonstrate that a U5-analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing.     

Characterization of a Drosophila proximal-sequence-element-binding protein involved in transcription of small nuclear RNA genes

The semisynthesis of eel[L-alpha-aminosuberic acid]calcitonin (elcatonin) was accomplished by alpha-chymotrypsin-catalyzed coupling of two peptide segments in a single reaction without the protection of any functional group. The eel calcitonin-(10-32)-peptide was prepared by a gene manipulation. The esters of cyclic desamino nonapeptide (segment 1-9) were synthesized by the conventional solution method including a thermolysin-mediated resolution of DL-alpha-aminosuberic acid via one-step tripeptide synthesis leading to the 7-9 sequence. The main aim of this work was to determine the conditions for protease-catalyzed segment condensation while avoiding a concurrent cleavage of other proteolytically labile peptide bonds in the hormone. The alpha-chymotrypsin condensation strategy under usual conditions led to a complicated mixture of split products with an insignificant amount of the required peptide. When the coupling reaction was carried out at 0 degrees C, the reaction resulted in a satisfactory yield of elcatonin with the complete conversion of the acyl donor (1-9 segment) accompanied by negligible concurrent peptide bond digestion. The same strategy was employed for the preparation of analogous dicarba salmon calcitonin using a synthetic elcatonin-(10-32)-peptide. Both calcitonin analogs exhibited hypocalcemic activity corresponding to the international standard of elcatonin. We demonstrate in this work a peptide synthesis based on the combination of genetic engineering, chemical synthesis and proteinase-catalyzed segment condensation. This approach enables effective incorporation of an unnatural amino acid into calcitonins without the side-chain protection.