HCP-4, a CENP-C-like protein in Caenorhabditis elegans, is required for resolution of sister centromeres.

The centromere plays a critical role in the segregation of chromosomes during mitosis. In mammals, sister centromeres are resolved from one another in the G2 phase of the cell cycle. During prophase, chromosomes condense with sister centromeres oriented in a back to back configuration enabling only one chromatid to be captured by each half spindle. To study this process, we identified a centromere protein (CENP)-C-like protein, holocentric protein (HCP)-4, in Caenorhabditis elegans based on sequence identity, loss of function phenotype, and centromeric localization. HCP-4 is found in the cytoplasm during interphase, but is nuclear localized in mitosis, where it localizes specifically to the centromere. The localization of HCP-4 to the centromere is dependent on the centromeric histone HCP-3; in addition, HCP-3 and HCP-4 are both required for localization of a CENP-F-like protein, HCP-1, indicating an ordered assembly pathway. Loss of HCP-4 expression by RNA-mediated interference resulted in a failure to generate resolution of sister centromeres on chromosomes, suggesting that HCP-4 is required for sister centromere resolution. These chromosomes also failed to form a functional kinetochore. Thus, the CENP-C-like protein HCP-4 is essential for both resolution sister centromeres and attachment to the mitotic spindle.

Oxygen deprivation causes suspended animation in the zebrafish embryo.

Continuous exposure to oxygen is essential for nearly all vertebrates. We found that embryos of the zebrafish Danio rerio can survive for 24 h in the absence of oxygen (anoxia, 0% O2). In anoxia, zebrafish entered a state of suspended animation where all microscopically observable movement ceased, including cell division, developmental progression, and motility. Animals that had developed a heartbeat before anoxic exposure showed no evidence of a heartbeat until return to terrestrial atmosphere (normoxia, 20.8% O2). In analyzing cell-cycle changes of rapidly dividing blastomeres exposed to anoxia, we found that no cells arrested in mitosis. This is in sharp contrast to similarly staged normoxic embryos that consistently contain more than 15% of cells in mitosis. Flow cytometry analysis revealed that blastomeres arrested during the S and G2 phases of the cell cycle. This work indicates that survival of oxygen deprivation in vertebrates involves the reduction of diverse processes, such as cardiac function and cell-cycle progression, thus allowing energy supply to be matched by energy demands.

SR proteins are autoantigens in patients with systemic lupus erythematosus. Importance of phosphoepitopes.

OBJECTIVE: To determine whether members of the highly phosphorylated SR protein family are autoantigens and, if so, to determine the frequency and molecular basis of antigen recognition. METHODS: Native human SR proteins were purified to homogeneity from HeLa cells, and an enzyme-linked immunosorbent assay (ELISA) was developed. Further studies employed immunoblotting of both phosphorylated and dephosphorylated SR proteins. RESULTS: Anti-SR protein reactivity was frequently detected in the sera of patients with systemic lupus erythematosus (SLE). Sera from 52% of the SLE patients in a group of patients with a variety of autoimmune and other disorders (n = 137) and from 50% of the SLE patients in a separate group (n = 102) were positive in an ELISA. In contrast, sera from patients with other disorders, such as rheumatoid arthritis and primary antiphospholipid syndrome, reacted infrequently. Reactivity with double-stranded DNA (dsDNA), used in the diagnosis of SLE, did not correlate with SR protein reactivity. Anti-SR autoantisera did not bind highly charged unphosphorylated peptides related to the SR domain, which is rich in arginine and phosphoserine residues. Surprisingly, many of the epitopes were influenced by the presence or absence of SR protein phosphorylation. In immunoblots, some patient sera lost reactivity upon SR protein dephosphorylation, while others significantly gained reactivity. CONCLUSION: We have identified a novel set of autoantigens in SLE, the SR protein family of non-small nuclear RNP pre-messenger RNA splicing factors. Anti-SR autoantibodies are distinct from those which bind dsDNA. The identification of this new set of autoantigens and the observation that the auto-epitope(s) involves posttranslational modification offer new possibilities for understanding autoimmunity and its development.

Induction of coiled body-like structures in Xenopus oocytes by U7 snRNA.

The coiled bodies, or "sphere organelles," of amphibian oocytes, first identified by their unique morphology, are large structures of up to 10 microm in diameter. There are 40 to 120 coiled bodies per oocyte nucleus. Most of the coiled bodies are in the nucleoplasm but a few are attached to specific chromosomal loci containing the histone gene repeats. Like the coiled bodies of somatic cells, they contain high concentrations of U7 snRNA, a small nuclear RNA required for 3' end formation of histone transcripts, and of a unique protein called coilin/SPH-1. We show here that increasing the nuclear concentration of U7 snRNA, by injection of either in vitro synthesized U7 snRNA or functional U7 snRNA genes, induces the formation of coiled body-like structures in vivo. In contrast, the formation of these structures is not induced when either a promoterless U7 snRNA gene construct is injected or when functional U7 snRNA genes are co-injected with alpha-amanitin. Increasing the concentration of U1 snRNA or histone mRNA does not induce the formation of these structures, indicating that the formation of these coiled body-like structures is specifically induced by the U7 snRNA. These results suggest that U7 snRNA may be a central nucleating factor of coiled bodies and that the appearance of coiled bodies at histone gene loci results from an increased local concentration of U7 snRNA near the nascent histone pre-mRNAs.

HCP-1, a protein involved in chromosome segregation, is localized to the centromere of mitotic chromosomes in Caenorhabditis elegans.

To learn more about holocentric chromosome structure and function, we generated a monoclonal antibody (mAb), 6C4, that recognizes the poleward face of mitotic chromosomes in Caenorhabditis elegans. Early in mitosis, mAb 6C4 stains dots throughout the nucleoplasm. Later in prophase, mAb 6C4 stains structures on opposing faces of chromosomes which orient towards the centrosomes at metaphase. Colocalization with an antibody against a centromeric histone H3-like protein and the MPM-2 antibody, which identifies a kinetochore-associated phosphoepitope present in a variety of organisms, shows that the mAb 6C4 staining is present adjacent to the centromere. Expression screening using mAb 6C4 identified a protein in C. elegans that we named HCP-1 (for holocentric protein 1). We also identified a second protein from the C. elegans genome sequence database, HCP-2, that is 54% similar to HCP-1. When expression of HCP-1 is reduced by RNA interference (RNAi), staining with mAb 6C4 is eliminated, indicating that hcp-1 encodes the major mAb 6C4 antigen. RNAi with hcp-1 and hcp-2 together results in aberrant anaphases and embryonic arrest at approximately 100 cells with different amounts of DNA in individual nuclei. These results suggest that HCP-1 is a centromere-associated protein that is involved in the fidelity of chromosome segregation.

The relative strengths of SR protein-mediated associations of alternative and constitutive exons can influence alternative splicing.

We have characterized the functional role of SR protein-mediated exon/exon associations in the alternative splicing of exon 5 of chicken cardiac troponin T (cTnT). We have previously shown that SR proteins can promote the association of the alternative exon 5 with the flanking constitutive exon 6 of this pre-mRNA. In this study, we have shown that when exons 2, 3, and 4 of the cTnT pre-mRNA are spliced together, the composite exon 2/3/4 contains an additional SR protein binding site. Furthermore, we have found that SR proteins can also promote interactions between the pairs of exons 2/3/4-5 and 2/3/4-6. We then asked whether the SR protein binding sites in these exons play a role in cTnT alternative splicing in vivo. We found that the SR protein binding sites in exons 2/3/4 and 6 promote exon 5 skipping, and it has previously been shown that the SR protein binding site in exon 5 promotes exon 5 inclusion. Consistent with these results, we find that the SR protein-mediated association of exon 2/3/4 with 6 is preferred over associations involving exon 5, in that exons 2/3/4 and 6 are more efficient than exon 5 in competing an SR protein-mediated exon/exon association. We suggest that the relative strengths of SR protein-mediated associations of alternative and constitutive exons play a role in determining alternative splicing patterns.

SR proteins are sufficient for exon bridging across an intron.

We have developed a defined system to characterize the role of SR proteins and exonic enhancers in directly promoting splice-site interactions across an intron. Using RNA affinity chromatography, we find that SR proteins alone are sufficient to promote the specific association of the enhancer-containing exon 5 with the adjoining exon 6 from avian cardiac troponin-T. Direct visualization of this exon/exon association by electron spectroscopic imaging shows it to be highly specific. Furthermore, using in vivo characterized mutants of exon 5, we also show that this exon/exon association depends on the splicing enhancer within exon 5. These results suggest a model by which SR proteins may function through exonic enhancers to directly promote exon bridging.

ncl-1 is required for the regulation of cell size and ribosomal RNA synthesis in Caenorhabditis elegans.

Regulation of ribosome synthesis is an essential aspect of growth control. Thus far, little is known about the factors that control and coordinate these processes. We show here that the Caenorhabditis elegans gene ncl-1 encodes a zinc finger protein and may be a repressor of RNA polymerase I and III transcription and an inhibitor of cell growth. Loss of function mutations in ncl-1, previously shown to result in enlarged nucleoli, result in increased rates of rRNA and 5S RNA transcription and enlarged cells. Furthermore, ncl-1 adult worms are larger, have more protein, and have twice as much rRNA as wild-type worms. Localization studies show that the level of NCL-1 protein is independently regulated in different cells of the embryo. In wild-type embryos, cells with the largest nucleoli have the lowest level of NCL-1 protein. Based on these results we propose that ncl-1 is a repressor of ribosome synthesis and cell growth.

smg mutants affect the expression of alternatively spliced SR protein mRNAs in Caenorhabditis elegans.

The expression of alternatively spliced mRNAs from genes is an ubiquitous phenomenon in metazoa. A screen for trans-acting factors that alter the expression of alternatively spliced mRNAs reveals that the smg genes of Caenorhabditis elegans participate in this process. smg genes have been proposed to function in degradation of nonsense mutant mRNAs. Here we show that smg genes affect normal gene expression by modulating the levels of alternatively spliced SRp20 and SRp30b mRNAs. These SR genes contain alternatively spliced exons that introduce upstream stop codons. The effect of smg genes on SR transcripts is specific, because the gene encoding the catalytic subunit of the cAMP-dependent protein kinase, which also contains an alternatively spliced exon that introduces upstream stop codon, is not effected in a smg background. These results suggest that the levels of alternatively spliced mRNAs may, in part, be regulated by alternative mRNA stability.

Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription.

If pre-mRNA splicing begins during RNA synthesis, then transcriptionally active genes may be expected to contain high concentrations of pre-mRNA splicing factors. However, previous studies have localized splicing factors to a network of "speckles," which is distinct from individual sites of gene transcription where pre-mRNA is spliced. Speckles have been detected with antibodies specific for splicing snRNPs and members of the SR family of splicing factors. Here we report that dilution of these probes results in the visualization of hundreds of sites throughout the HeLa cell nucleus, the size and distribution of which are consistent with transcription units viewed with light microscopy. Importantly, these sites of highest SR protein concentration frequently coincide in three-dimensional space with active sites of RNA polymerase II transcription. A newly developed reagent specific for a single member of the SR family, SRp20, detects a subset (approximately 20%) of these sites, suggesting the gene-specific accumulation of these splicing regulators, which have distinct functions in pre-mRNA splicing. These observations question the view that the nucleus and its functions are highly compartmentalized; instead, they support a model in which the localization of these and possibly other gene regulators is determined primarily by their function.

Canine parvovirus vaccine elicits protection from the inflammatory and clinical consequences of the disease.

Inflammatory changes following infection are central to the clinical manifestation of disease. However, information regarding such changes in animal disease is limited. In canine parvovirus infected puppies we measured the levels of acute phase proteins and changes in leukocyte phenotypes and cell trafficking by flow cytometry. These parameters correlated with conventional assessment of clinical disease in a vaccine efficacy study. Seropositive (CPV-2) 6-week-old puppies given three doses of a CPV-2 containing vaccine developed significant antibody titers and remained healthy after experimental infection with CPV-2b. Unvaccinated controls developed clinical signs and shed virus. Importantly, acute phase proteins became elevated, and lymphopenia, neutropenia and modulation of neutrophil-CD4 were detected in controls but not in vaccinates.

A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer.

The cardiac troponin T pre-mRNA contains an exonic splicing enhancer that is required for inclusion of the alternative exon 5. Here we show that enhancer activity is exquisitely sensitive to changes in the sequence of a 9-nucleotide motif (GAGGAAGAA) even when its purine content is preserved. A series of mutations that increased or decreased the level of exon inclusion in vivo were used to correlate enhancer strength with RNA-protein interactions in vitro. Analyses involving UV cross-linking and immunoprecipitation indicated that only four (SRp30a, SRp40, SRp55, and SRp75) of six essential splicing factors known as SR proteins bind to the active enhancer RNA. Moreover, purified SRp40 and SRp55 activate splicing of exon 5 when added to a splicing-deficient S100 extract. Purified SRp30b did not stimulate splicing in S100 extracts, which is consistent with its failure to bind the enhancer RNA. In vitro competition of SR protein splicing activity and UV cross-linking demonstrated that the sequence determinants for SR protein binding were precisely coincident with the sequence determinants of enhancer strength. Thus, a subset of SR proteins interacts directly with the exonic enhancer to promote inclusion of a poorly defined alternative exon. Independent regulation of the levels of SR proteins may, therefore, contribute to the developmental regulation of exon inclusion.

Spheres, coiled bodies and nuclear bodies.

Of several distinct aggregations or nuclear bodies in the nucleus, two of the most prominent are the sphere organelle and the coiled body. Recent molecular studies indicate that the coiled body found in interphase nuclei may be identical to the sphere organelle found in the amphibian oocyte nucleus. This is not the first time an interphase nuclear body has been suspected of being the same as a structure in the oocyte nucleus; the first such structure was the nucleolus. Both the sphere organelle and the nucleolus are attached to site-specific loci on oocyte lampbrush chromosomes. Because of their unique morphologies and utility in reproducibly identifying specific genetic elements, they have been referred to as lampbrush chromosomal landmarks. The discovery of the way nucleoli arise has led to an understanding of their function, and for this reason I discuss current models for the genesis of spheres.

A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors.

The removal of introns from eukaryotic pre-mRNA occurs in a large ribonucleoprotein complex called the spliceosome. We have generated a monoclonal antibody (mAb 16H3) against four of the family of six SR proteins, known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins, SRp20, SRp40, SRp55, and SRp75, mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription which suggests their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope was mapped to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, we propose that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors.

Distinct functions of SR proteins in recruitment of U1 small nuclear ribonucleoprotein to alternative 5' splice sites.

Alternative splicing of precursor messenger RNAs (pre-mRNAs) is an important mechanism for the regulation of gene expression. The members of the SR protein family of pre-mRNA splicing factors have distinct functions in promoting alternative splice site usage. Here we show that SR proteins are required for the first step of spliceosome assembly, interaction of the U1 small nuclear ribonucleoprotein complex (U1 snRNP) with the 5' splice site of the pre-mRNA. Further, we find that individual SR proteins have distinct abilities to promote interaction of U1 snRNP with alternative 5' splice junctions. These results suggest that SR proteins direct 5' splice site selection by regulation of U1 snRNP assembly onto the pre-mRNA.

Identification and characterization of a sphere organelle protein.

Sphere organelles are nuclear structures in amphibian oocytes that are easily visible by light microscopy. These structures are up to 10 microns in diameter and have been described morphologically for decades, yet their function remains obscure. The present study defines a protein component of the sphere organelle, named SPH-1, which is recognized by a mAb raised against purified Xenopus laevis oocyte nucleoplasm. SPH-1 is an 80-kD protein which is localized specifically to spheres and is undetectable elsewhere on lampbrush chromosomes or in nucleoli. We show using confocal microscopy that SPH-1 is localized to the cortex of sphere organelles. Furthermore, we have isolated a cDNA that can encode SPH-1. When epitope-tagged forms of SPH-1 are expressed in X. laevis oocytes the protein specifically localizes to spheres, demonstrating that the cloned cDNA encodes the sphere antigen. Comparison of the predicted amino acid sequence with sequence databases shows SPH-1 is related to p80-coilin, a protein associated with coiled bodies; coiled bodies are nuclear structures found in plant and animal cells. The sphere-specific mAb stains X. laevis tissue culture cells in a punctate nuclear pattern, showing that spheres or sphere antigens are present in somatic cells as well as germ cells and suggesting a general and essential function for spheres in all nuclei.

Human SR proteins and isolation of a cDNA encoding SRp75.

SR proteins are a family of proteins that have a common epitope recognized by a monoclonal antibody (MAb104) that binds active sites of polymerase II transcription. Four of the SR family members have been shown to restore activity to an otherwise splicing-deficient extract (S100 extract). Here we show that two untested SR proteins, SRp20 and SRp75, can also complement the splicing-deficient extract. We isolated a cDNA encoding SRp75 and found that this protein, like other SR proteins, contains an N-terminal RNA recognition motif (RRM), a glycine-rich region, an internal region homologous to the RRM, and a long (315-amino-acid) C-terminal domain composed predominantly of alternating serine and arginine residues. The apparent molecular mass of dephosphorylated SRp75 is 57 kDa, the size predicted from the cDNA clone. We also detected mobility shifts after dephosphorylating SRp55, SRp40, SRp30a, and SRp30b; the sizes of the shifts are proportional to the length of the SR domain, suggesting that serines in this domain are phosphorylated.

Distinct functions of SR proteins in alternative pre-mRNA splicing.

Alternative splicing of precursor messenger RNAs (pre-mRNAs) is a common mechanism of regulating gene expression. SR proteins are a family of pre-mRNA splicing factors that are structurally related and evolutionarily conserved. Any member of the SR family can complement a splicing-deficient extract that lacks the entire family of SR proteins. Here it is demonstrated that particular SR proteins have distinct functions in alternative pre-mRNA splicing in vitro. In addition, SR proteins are differentially expressed in a variety of tissues. These results suggest a fundamental role for SR proteins in the regulation of alternative splicing.