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At least 1,171 records · Page 65Linked to original sources
PubMed1996

A unique U5-->A substitution in the Physarum polycephalum U1 snRNA: evidence at the RNA and gene levels.

The 5' terminal sequence of U1 snRNA that base-pairs with the intron 5' splice site in the course of spliceosome assembly was considered to be universally conserved. A study of the P polycephalum U1 snRNA at both RNA and gene levels shows that there are exceptions to this rule: the P polycephalum U1 snRNA has a U to A substitution at position 5, that is partially compensated by a high frequency of T residue at position +4 of introns. In contrast to the yeast genome, the P polycephalum genome contains several U1 snRNA coding sequences (about 20). They either encode the U1A snRNA expressed in microplasmodia or correspond to the previously cloned U1B coding sequence. Both coding sequences show the U5A substitution. The ratio of U1A versus U1B coding sequences is of about 3. A U1A gene was cloned. The 60 nt region upstream of the coding sequence has the same sequence as in the U1B gene. The U1B gene is probably expressed at another stage of the P polycephalum life cycle.

Amino Acid Sequence↗
PubMed1995

The U6 snRNA-encoding gene of the monogenetic trypanosomatid Leptomonas collosoma.

The U6 snRNA (U6) is the most conserved small nuclear RNA (snRNA) and apparently plays a central role in catalysis of the cis-splicing reaction. In trans-splicing, U6 may have an additional function. In the nematode trans-splicing system, a direct interaction between the U6 and spliced leader (SL) RNAs has been demonstrated, suggesting that U6 may serve as a bridge between the SL RNA and the acceptor pre-mRNA. To examine possible phylogenetic conservation of trypanosomatid U6 sequences that may interact with spliceosomal RNAs, we have cloned and sequenced the U6 gene from the monogenetic trypanosomatid Leptomonas collosoma (Lc). The Lc U6 deviates from the Trypanosoma brucei (Tb) RNA only in four positions located in the 5' stem-loop and the central domains. As in Tb, U6 is a single-copy gene and two tRNA genes, tRNAGln and tRNAIle, are found upstream to the gene. The tRNAs are differentially expressed; tRNAGln is transcribed in the opposite direction to U6, whereas tRNAIle is not transcribed. Possible base-pairing between U6 and the U2 and SL RNAs, similar to the interactions that take place in the nematode trans-splicing system, are proposed.

Animals↗
PubMed1991

EM analysis of Drosophila chorion genes: amplification, transcription termination and RNA splicing.

We have used the electron microscope to examine ultrastructurally several events occurring during the biogenesis of two very abundant chorion (eggshell) mRNA molecules in the follicle cells of Drosophila melanogaster--namely, selective gene amplification, transcription initiation and termination, and RNA processing. We find that the highly transcribed s36 and s38 genes are positioned in the central region of large, multi-forked amplified DNA structures. Transcript morphology is consistent with the known presence of a small intron at the 5' end of each gene. Mature transcripts are associated with spliceosomes, demonstrating that splice site selection occurs co-transcriptionally but that splicing is completed after transcript release from the template. We have also mapped the termination sites for the genes. The two genes exhibit efficient termination very near their poly(A) sites--within a 210 bp region for s36 and a 360 bp region for s38.

Animals↗
PubMed1992

RNA processing.

Significant progress has been made over the last year in our understanding of the roles that RNA-binding proteins play in pre-mRNA splicing, the components of the spliceosome and how these components relate to the mechanism of splicing. Of particular importance has been the sequence analysis of the first mammalian splicing factors and structural determination of an RNA-binding domain.

Animals↗
PubMed1994

Small nuclear RNAs and pre-mRNA splicing.

Genetic and biochemical studies have recently shown that the splice sites in nuclear pre-mRNAs are aligned for the transesterification reactions through a collaboration between U5 and U6 small nuclear RNAs, which form the heart of a network of RNA-based interactions in the active spliceosome.

Animals↗
PubMed1994

Introns: evolution and function.

The debate continues on the issue of whether nuclear introns were present in eukaryotic protein-coding genes from the beginning (introns-early) or invaded them later in evolution (introns-late). Recent studies concerning the location of introns with respect to gene and protein structure have been interpreted as providing strong support for both positions, but the weight of argument is clearly moving in favour of the latter. Consistent with this, there is now good evidence that introns can function as transposable elements, and that nuclear introns derived from self-splicing group II introns, which then evolved in partnership with the spliceosome. This was only made possible by the separation of transcription and translation. If introns did colonize eukaryotic genes after their divergence from prokaryotes, the original question as to the evolutionary forces that have seen these sequences flourish in the higher organisms, and their significance in eukaryotic biology, is again thrown open. I suggest that introns, once established in eukaryotic genomes, might have explored new genetic space and acquired functions which provided a positive pressure for their expansion. I further suggest that there are now two types of information produced by eukaryotic genes--mRNA and iRNA--and that this was a critical step in the development of multicellular organisms.

Animals↗
PubMed1991

Who's on first? The U1 snRNP-5' splice site interaction and splicing.

U1 small nuclear ribonucleoprotein (snRNP) is important for pre-mRNA splicing both in yeast (Saccharomyces cerevisiae) and mammalian systems. The RNA component of U1 snRNP, U1 snRNA, interacts by base pairing with pre-mRNA 5' splice sites. This article examines recent evidence suggesting that U1 snRNP is important for an early step in spliceosome assembly rather than a late step that contributes to the specificity of 5' splice-site cleavage.

Animals↗
PubMed1995

Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2'-hydroxyl groups.

BACKGROUND: Group II introns are self-splicing RNAs that have mechanistic similarity to the spliceosome complex involved in messenger RNA splicing in eukaryotes. These autocatalytic molecules can be reconfigured into highly specific, multiple-turnover ribozymes that cleave oligonucleotides in trans. We set out to use a simplified system of this kind to study the mechanism of cleavage. RESULTS: Unlike other catalytic RNA molecules, the group II ribozymes cleave DNA linkages almost as readily as RNA linkages. One ribozyme variant cleaves DNA linkages with an efficiency comparable to that of restriction endonuclease EcoRI. Single deoxynucleotide substitutions in the substrate showed that the ribozymes bind substrate without engaging 2'-hydroxyl groups. CONCLUSIONS: The ribose 2'-hydroxyl group at the cleavage site has little role in transition-state stabilization by group II ribozymes. Substrate 2'-hydroxyl groups are not involved in substrate binding, suggesting that only base-pairing is required for substrate recognition.

Animals↗
PubMed2005

Alternative splicing: a new drug target of the post-genome era.

Alternative splicing allows for the creation of multiple distinct mRNA transcripts from a given gene in a multicellular organism. Pre-mRNA splicing is catalyzed by a multi-molecular complex, including serine/arginine-rich (SR) proteins, which are highly phosphorylated in living cells, and thought to play crucial roles in spliceosomal formation and in the regulation of alternative splicing. Recently, reports of low molecular compounds, which alter splicing pattern of genes, have been accumulated. A benzothiazole compound TG003, a kinase inhibitor that targets Clk1 and Clk4, suppressed dissociation of nuclear speckles, altered the splicing patterns, and rescued the embryonic defects induced by excessive Clk activity. The emerging inhibitors of the signal transduction pathways regulating pre-mRNA alternative splicing may open the way to therapies against diseases caused by missplicing.

Alternative Splicing↗
PubMed2004

SKIP modifies gene expression by affecting both transcription and splicing.

SKIP has been described as a transcriptional coregulator as well as a spliceosome component, but the relationship between these functions is not clear. We found that SKIP activated reporter gene expression from the basal promoters of viral origin. SKIP exhibited more prominent effect on the promoters with stronger activities, in an experiment employing a series of reporter constructs carrying different numbers of GC boxes. We also found that SKIP suppressed aberrant splicing at a cryptic splice donor site in the luciferase reporter gene. In addition, SKIP suppressed splicing of an extra intron created by a beta-thalassemia mutation in the human beta-globin gene. In the transfection experiment, an intronless reporter exhibited a higher level of expression, but was less significantly activated by SKIP, than the intron-containing reporter. These results indicate that SKIP affects gene expression by both transcriptional activation and regulation of pre-mRNA splicing.

Animals↗
PubMed2004

The human Ski-interacting protein functionally substitutes for the yeast PRP45 gene.

The PRP45 gene has been identified as encoding a protein involved in mRNA splicing that is essential in Saccharomyces cerevisiae. PRP45's human homolog SKIP has been identified as a mediator of transcriptional programming in a variety of signal transduction pathways including TGFbeta, nuclear hormones, Notch, and retinoblastoma signaling. However, Skip has been also identified in purified spliceosomal complexes but an explicit role in splicing has not been identified in mammalian cells. To determine if the Skip protein could function as a splicing factor we investigated if the SKIP gene could functionally complement the yeast PRP45 gene. We show that SKIP complements the PRP45 deletion and rescues the lethal phenotype. These results show that the human SKIP gene can functionally substitute for the mRNA splicing gene PRP45 of S. cerevisiae.

Amino Acid Sequence↗
PubMed2005

Potentiation of estrogen receptor transcriptional activity by breast cancer amplified sequence 2.

Breast cancer amplified sequence 2 (BCAS2) was initially identified as a gene that was overexpressed and amplified in some breast cancer cell lines. It was later found to be a component of the spliceosome. Here, we identified BCAS2 as an estrogen receptor (ER) alpha interacting protein by yeast two-hybrid screening. In addition to ER alpha, BCAS2 also interacted with ER beta, TR beta, PR, and PPAR gamma in a ligand-independent way. Transient transfection assays revealed that overexpression of BCAS2 enhanced while inhibition of BCAS2 expression attenuated the estrogen receptor-mediated transcription. BCAS2 potentiated the activation function-2 (AF-2) activity of ER alpha but had no effect on the AF-1 activity. This study suggested that BCAS2 might play an important role in breast cancer development by increasing the estrogen receptor's function.

Animals↗
PubMed2005

Human genetic evidence that OX40 is implicated in myocardial infarction.

We recently showed that genetic variants in OX40L are associated with myocardial infarction (MI) and severity of coronary artery disease in human. A number of studies also suggest a possible role for OX40 (the OX40L receptor) as a factor contributing to atherosclerosis. In the present study, the OX40 gene was screened for variants associated with precocious MI, using individuals with MI before the age of 60 and controls. Despite the fact that the OX40 gene is highly conserved between species and that relatively few common genetic variants were encountered, an association with MI was seen for a polymorphism in intron 5 (rs2298212). In silico investigation suggested that genetic variation (rs2298211), linked to this intronic variant, is possibly affecting spliceosome function. Our results provide evidence that variants in human OX40 might influence susceptibility to MI. The relevance of these findings is supported by the vital functions fulfilled by OX40 in mammals as reflected by the high level of evolutionary conservation.

Adolescent↗
PubMed2006

Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo.

SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function.

Alternative Splicing↗
PubMed2006

Emp is a component of the nuclear matrix of mammalian cells and undergoes dynamic rearrangements during cell division.

Emp, originally detected in erythroblastic islands, is expressed in numerous cell types and tissues suggesting a functionality not limited to hematopoiesis. To study the function of Emp in non-hematopoietic cells, an epitope-tagged recombinant human Emp was expressed in HEK cells. Preliminary studies revealed that Emp partitioned into both the nuclear and Triton X-100-insoluble cytoskeletal fractions in approximately a 4:1 ratio. In this study, we report investigations of Emp in the nucleus. Sequential extractions of interphase nuclei showed that recombinant Emp was present predominantly in the nuclear matrix. Immunofluorescence microscopy showed that Emp was present in typical nuclear speckles enriched with the spliceosome assembly factor SC35 and partially co-localized with actin staining. Coimmunoprecipitation and GST-pull-down assays confirmed the apparent close association of Emp with nuclear actin. During mitosis, Emp was detected at the mitotic spindle/spindle poles, as well as in the contractile ring during cytokinesis. These results suggest that Emp undergoes dynamic rearrangements within the nuclear architecture that are correlated with cell division.

Actins↗
PubMed2006

A new RNA branching activity: the GIR1 ribozyme.

The formation of lariat intermediates during the first step of splicing of group II introns and spliceosomal introns is a well-studied fundamental reaction in molecular biology. Apart from this prominent example, there are surprisingly few occurrences of branched nucleotides or even 2',5'-phosphodiester bonds in biology. We recently described a new ribozyme, the GIR1 branching ribozyme, which catalyzes the formation of a tiny lariat that caps an mRNA. This new example together with work on artificial branching ribozymes and deoxyribozymes shows that branching is facile and points to the possibility that branching reactions could be more prevalent than previously recognized.

Catalysis↗
PubMed2026

Transcriptomic characterization of the intestine in Stichopus monotuberculatus under gradient temperature stress and HSP gene family-mediated molecular adaptation.

The increasing frequency of extreme temperature events under climate change poses a growing threat to the stability of tropical sea cucumber aquaculture. To characterize the molecular responses of the tropical sea cucumber Stichopus monotuberculatus to acute temperature stress, juveniles were exposed for 96 h to 15 °C, 20 °C, 25 °C, 30 °C, and 35 °C, followed by transcriptomic profiling of the intestine. By transcriptomic analysis, 2258, 634, 1618, and 2980 differentially expressed genes (DEGs) were identified at 15, 20, 30, and 35 °C compared to control, respectively. More DEGs were generally detected at temperatures further from 25 °C, with the 35 °C group showing the largest transcriptional response. Although cold and heat stress both affected metabolism and protein homeostasis, their enrichment profiles differed. At 15 °C, DEGs were mainly enriched in the spliceosome and p53 signaling pathways, highlighting RNA processing and p53 signaling as prominent features of the cold-stress response. At 35 °C, DEGs were mainly enriched in the PI3K-Akt signaling pathway, ubiquitin-mediated proteolysis, and mitophagy, indicating enhanced regulation of cell survival, protein turnover, and mitochondrial quality control. HSP genes also responded differently to cold and heat stress. Most HSP70 and HSP90 family members were downregulated at low temperatures, whereas HSP70 genes and small heat shock proteins were markedly upregulated at high temperatures. Overall, the intestinal transcriptome showed distinct responses to cold and heat stress. These results identify pathways and HSP genes potentially involved in the temperature response of S. monotuberculatus and provide useful information for evaluating temperature tolerance and defining suitable temperatures for its aquaculture.

Heat shock protein↗
PubMed2004

The clinical value of intracellular autoantigens B-cell epitopes in systemic rheumatic diseases.

A hallmark of autoimmune diseases is the production of autoantibodies against intracellular autoantigens. Although their pathogenetic and their etiologic relationship are not fully understood, these autoantibodies are important tools for establishing the diagnosis, classification and prognosis of autoimmune diseases. Systemic rheumatic diseases are among the most complex disorders because their clinical presentation and constellation of findings are in part reflected by the wide spectrum of autoantibodies found in the sera of patients suffering from these disorders. These autoantibodies usually target large complexes consisting of protein antigens noncovalently associated with (ribo)-nucleic acid(s), like the spliceosome or Ro/La-RNPs. In this review, we first address the main characteristics and the clinical value of several autoantibodies, with respect to their diagnostic sensitivity and specificity. Subsequently, we provide a brief overview of the antigenic determinant types that have been identified on the corresponding autoantigens. The antibody targets of autontigens include primary, secondary, tertiary and quarternary structure epitopes, as well as cryptotopes, neoepitopes and mimotopes. We next focus on antigenic structures corresponding to B-cell epitopes with high disease specificity and sensitivity for all the major autoantigens in systemic autoimmunity including the Ro/La and U1 ribonucleoprotein complexes and the Ku70/80, ribosomal P, DNA topoisomerase I, filaggrin, Jo-1 and PM/SCl-100 autoantigens. These epitopes, defined at the peptide level, can be chemically synthesized and engineered for the development of new inexpensive and easier to perform assays and the improvement of the methods for autoantibody detection. Specific examples of newly developed assays that incorporate (i) epitopes with high disease specificity and sensitivity, (ii) modified epitopes, (iii) conformational epitopes and (iv) complementary epitopes are discussed in detail. Finally, we examine the potential of combining these synthetic epitopes for future development of multiplex diagnostic tests based on miniaturized autoantigen arrays.

Autoantigens↗