Regulation of alternative splicing in the IIICS region of human fibronectin pre-mRNA encoding cell binding sites CS1 and CS5

1992 ◽  
Vol 103 (2) ◽  
pp. 423-433 ◽  
Author(s):  
H.J. Mardon ◽  
G. Sebastio
Keyword(s):  
Alternative Splicing ◽  
Binding Sites ◽  
Donor Site ◽  
Acceptor Site ◽  
Cell Binding ◽  
Adult Liver ◽  
Cellular Factors ◽  
Human Fibronectin ◽  
Iiics Region ◽  
Selection Of

The cell binding sites CS1 and CS5 in the IIICS region of human fibronectin (FN) mediate the adhesion of specific cell types by interacting with the integrin alpha 4 beta 1. IIICS pre-mRNA is alternatively spliced via the use of three alternative splice acceptor sites and one alternative splice donor site. These alternative splicing pathways can potentially give rise to variant FN molecules which are CS1+,CS5+; CS1+,CS5-; CS1-,CS5+ or CS1-,CS5-. Here we show that selection of the acceptor site which incorporates mRNA encoding CS1 and CS5 is more frequent in foetal tissues compared to adult liver, whereas an alternative acceptor site and the alternative donor site, which exclude CS1 and CS5, are used at a higher level in adult liver compared to foetal tissue. All possible splice junctions were accurately processed, and selected at different levels in mRNA expressed from a IIICS minigene transiently transfected into a HeLa cell line which does not express FN, suggesting that all the cellular factors required for alternative processing of IIICS are present in this system. Furthermore, pre-mRNA expressed from a mutant construct lacking IIICS-1 intron sequence, was correctly processed in HeLa cells via selection of all possible splice sites. On the basis of our results we propose that regulation of splice site selection in IIICS and thus expression of CS1 and CS5 is achieved by subtle tuning of splicing systems involving the interaction of local cis elements and cellular factors which are not necessarily restricted developmentally or tissue-specifically, and that expression of CS1 and CS5 is independently regulated.

scholarly journals The rapid evolution of alternative splicing in plants

2017 ◽  
Author(s):  
Zhihao Ling ◽  
Thomas Brockmöller ◽  
Ian T. Baldwin ◽  
Shuqing Xu
Keyword(s):  
Alternative Splicing ◽  
Related Species ◽  
Donor Site ◽  
Rapid Evolution ◽  
Acceptor Site ◽  
Mrna Levels ◽  
Evolutionary Patterns ◽  
Closely Related Species ◽  
Nonsense Mediated Decay ◽  
Splicing Regulators

AbstractAlternative pre-mRNA splicing (AS) is prevalent among all plants and is involved in many interactions with environmental stresses. However, the evolutionary patterns and underlying mechanisms of AS in plants remain unclear. By analyzing the transcriptomes of six plant species, we revealed that AS diverged rapidly among closely related species, largely due to the gains and losses of AS events among orthologous genes. Furthermore, AS that generates transcripts containing premature termination codons (PTC), although only representing a small fraction of the total AS, are more conserved than those that generate non-PTC containing transcripts, suggesting that AS coupled with nonsense-mediated decay (NMD) might play an important role in regulating mRNA levels post-transcriptionally. With a machine learning approach we analyzed the key determinants of AS to understand the mechanisms underlying its rapid divergence. Among the studied species, the presence/absence of alternative splicing site (SS) within the junction, the distance between the authentic SS and the nearest alternative SS, the size of exon-exon junctions were the major determinants for both alternative 5’ donor site and 3’acceptor site, suggesting a relatively conserved AS mechanism. Comparative analysis further demonstrated that variations of the identified AS determinants, mostly are located in introns, significantly contributed to the AS turnover among closely related species in both Solanaceae and Brassicaceae taxa. These new mechanistic insights into the evolution of AS in plants highlight the importance of post-transcriptional regulation in mediating plant-environment interactions.One sentence summaryChanges of intron located splicing regulators contributed to the rapid evolution of alternative splicing in plants.

scholarly journals Prognostic Signatures of Alternative Splicing Events in Esophageal Carcinoma Based on TCGA Splice-Seq Data

2021 ◽  
Vol 11 ◽  
Author(s):  
Ping Ye ◽  
Yan Yang ◽  
Liqiang Zhang ◽  
Guixi Zheng
Keyword(s):  
Alternative Splicing ◽  
Esophageal Carcinoma ◽  
Cox Regression ◽  
Donor Site ◽  
Area Under The Curve ◽  
Clinical Information ◽  
Roc Curves ◽  
The Cancer Genome Atlas ◽  
Acceptor Site ◽  
Cox Regression Analysis

An alternative splicing (AS) event is a highly complex process that plays an essential role in post-transcriptional gene expression. Several studies have suggested that abnormal AS events were the primary element in the pathological process of cancer. However, few works are dedicated to the study of AS events in esophageal carcinoma (EC). In the present study, clinical information and RNA-seq data of EC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The percent spliced in (PSI) values of AS events were acquired from the TCGA Splice-seq. A total of 183 EC patients were enrolled in this study, and 2,212 AS events were found significantly associated with the overall survival of these patients by univariate Cox regression analysis. The prognostic signatures based on AS events were built by multivariate Cox analysis. Receiver operating characteristic (ROC) curves displayed that the area under the curve (AUC) of the following prognostic signatures, including exon skip (ES), alternate terminator (AT), alternate acceptor site (AA), alternate promoter (AP), alternate donor site (AD), retained intron (RI), and total events, was greater than 0.8, suggesting that these seven signatures had valuable prognosis prediction capacity. Finally, the risk score of prognostic signatures was indicated as an independent risk factor of survival. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to explore the function of splicing factors (SFs) that were associated with AS events. Also, the interactive network between AS events and SFs identified several hub genes and AS events which need further study. This was a comprehensive study that explored prognosis-related AS events and established valuable prognosis signatures in EC patients. The network of interactions between AS events and SFs might offer novel insights into the fundamental mechanisms of tumorigenesis and progression of EC.

Mechanisms That Link Promoter Choice with Downstream Alternative Splicing in the Erythroid Protein 4.1R Gene.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1562-1562
Author(s):  
Marilyn Parra ◽  
Jeff Tan ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Rna Processing ◽  
Regulatory Element ◽  
Donor Site ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Exon 2 ◽  
Protein 4.1R

Abstract The protein 4.1R gene is a large transcription unit (240kb) that utilizes complex RNA processing pathways to encode distinct protein isoforms, both during erythropoiesis and also in nonerythroid cells. Proper regulation of these pathways is essential for stage-specific synthesis of the 80-kDa isoforms of 4.1R protein during terminal erythroid differentiation. The 5′ region of the gene contains multiple alternative first exons that map far upstream of the coding exons, and we have shown previously that promoter choice is coupled to alternative splicing decisions 100kb downstream in exon 2′/2. Transcripts that initiate at exon 1A predominate in late stages of erythropoiesis and splice only to a weak internal 3′ splice acceptor site in exon 2, skipping translation start site AUG1 and ensuring proper translation initiation at AUG2 in exon 4 for synthesis of the 80-kDa isoforms. In contrast, 4.1 transcripts initiated at exons 1B or 1C exclusively splice to the strong first 3′ splice acceptor site at exon 2′ to include AUG1 and encode a higher molecular weight 135-kDa isoform known to interact with different affinity to major erythroid membrane proteins in earlier stages of erythropoiesis. Our studies show that this linkage between transcription and splicing is (a) cell type independent; (b) conserved in the 4.1R gene from fish to man; and (c) conserved in the paralogous 4.1B gene. Our recent functional studies suggest that a novel re-splicing mechanism, reminiscent of recursive splicing of large introns previously described in the Drosophila ubx gene, may couple promoter choice with downstream splicing in the 4.1R gene. Using minigenes that reproduce the differential splicing patterns in transfected mammalian cells, we have shown that accurate splicing of exon 1A requires a unique downstream regulatory element. This element maps several kilobases downstream of exon 1A and is conserved among mammals. Analysis of wild type and mutated minigenes suggests a two step splicing model in which this element behaves as a temporary “intra-exon” that is present in a splicing intermediate but eliminated from the mature mRNA. According to this model, the regulatory element behaves as an exon in the first step as its consensus 5′ donor site splices to the strong 3′ splice site of exon 2′, removing this splice site pair and joining the intra-exon directly to exon 2′. In the second step, the juxtaposed region of the intra-exon then behaves as an intron, contributing to the activation of the weak internal splice acceptor at exon 2. This second splicing event joins exon 1A to exon 2, thus deleting the intra-exon, the 2′ region (and AUG1) and generating a mature 5′ end capable of encoding 80-kDa 4.1R. Importantly, pre-mRNA constructs that lack the intra-exon, or have a mutated intra-exon 5′ splice donor site, are uncoupled and exhibit inappropriate splicing of exon 1A to the first acceptor site at exon 2′. In support of the generality of this model, we have identified a candidate intra-exon with similar sequence properties in the long 5′ region of the human 4.1B gene, and have demonstrated that this element successfully rescues proper splicing of 4.1R exon 1A in our minigenes. Detailed molecular analysis is under way to identify the specific cis and trans elements required to effect this unusual, long-distance coupling between RNA processing events which have implications for detailed mechanistic understanding of membrane assembly during erythropoiesis.

scholarly journals A broad analysis of splicing regulation in yeast using a large library of synthetic introns

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009805
Author(s):  
Dvir Schirman ◽  
Zohar Yakhini ◽  
Yitzhak Pilpel ◽  
Orna Dahan
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Splice Site ◽  
Donor Site ◽  
Regulation Of Gene Expression ◽  
Combinatorial Design ◽  
Acceptor Site ◽  
Functional Sites ◽  
Splicing Efficiency ◽  
Splicing Machinery

RNA splicing is a key process in eukaryotic gene expression, in which an intron is spliced out of a pre-mRNA molecule to eventually produce a mature mRNA. Most intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient regulation of gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Introns are defined by three functional sites, the 5’ donor site, the branch site, and the 3’ acceptor site. Using a combinatorial design of synthetic introns, we demonstrate how non-consensus splice site sequences in each of these sites affect splicing efficiency. We then show that S. cerevisiae splicing machinery tends to select alternative 3’ splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns’ 3’ ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organism’s splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, demonstrating that merely a synthetic fusion of two introns might be suffice to facilitate alternative splicing in yeast. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome. In addition, it provides a valuable resource to study the regulation of constitutive and alternative splicing in a model organism.

scholarly journals Profiling Novel Alternative Splicing within Multiple Tissues Provides Useful Insights into Porcine Genome Annotation

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1405
Author(s):  
Wen Feng ◽  
Pengju Zhao ◽  
Xianrui Zheng ◽  
Zhengzheng Hu ◽  
Jianfeng Liu
Keyword(s):  
Alternative Splicing ◽  
Genome Annotation ◽  
Intron Retention ◽  
Single Gene ◽  
Donor Site ◽  
Exon Skipping ◽  
Acceptor Site ◽  
Porcine Genome ◽  
Expression Of Genes ◽  
Alternative Donor

Alternative splicing (AS) is a process during gene expression that results in a single gene coding for different protein variants. AS contributes to transcriptome and proteome diversity. In order to characterize AS in pigs, genome-wide transcripts and AS events were detected using RNA sequencing of 34 different tissues in Duroc pigs. In total, 138,403 AS events and 29,270 expressed genes were identified. An alternative donor site was the most common AS form and accounted for 44% of the total AS events. The percentage of the other three AS forms (exon skipping, alternative acceptor site, and intron retention) was approximately 19%. The results showed that the most common AS events involving alternative donor sites could produce different transcripts or proteins that affect the biological processes. The expression of genes with tissue-specific AS events showed that gene functions were consistent with tissue functions. AS increased proteome diversity and resulted in novel proteins that gained or lost important functional domains. In summary, these findings extend porcine genome annotation and highlight roles that AS could play in determining tissue identity.

scholarly journals Profiling novel alternative splicing within multiple tissues puts insight into the porcine genome annotation

2019 ◽  
Author(s):  
Wen Feng ◽  
Pengju Zhao ◽  
Xianrui Zheng ◽  
Jian-Feng Liu
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Genome Annotation ◽  
Intron Retention ◽  
Donor Site ◽  
Exon Skipping ◽  
Acceptor Site ◽  
Novel Proteins ◽  
Genome Wide ◽  
Alternative Donor

Abstract Background Alternative splicing (AS) is a process that mRNA precursor splices intron to form the mature mRNA. AS plays important roles in contributing to transcriptome and proteome divert. However, to date there is no research about pig AS in genome-wide level by RNA sequencing. Results To characterize the AS in pigs, herein we detected genome-wide transcripts and events by RNA sequencing technology (RNA-seq) 34 different tissues in Duroc pigs. In total, we identified 138, 403 AS events and 29, 270 expressed genes. We found alternative donor site was the most common AS form, which is accounted for 44% of the total AS events. The percentage of the other 3 AS forms (Exon skipping, Alternative acceptor site and Intron retention) are all around 19%. The results showed that the most common AS events (alternative donor site) can produce different transcripts or different proteins which affect the biological process. Among these AS events, 109, 483 were novel AS events, and the number of alternative donor splice site has increased the most (Accounting for 44% of the novel AS events). Conclusions The expression of gene with tissue specific AS events showed that the functions of these genes were consistent with the tissue function. AS increased proteome diversity and resulted in novel proteins that gained and lost important functional domains. In summary, these findings extend genome annotation and highlight roles that AS acts in tissue identity in pig.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

STUDIES ON ACTH-BINDING ANTIBODIES: CHARACTERIZATION OF IMMUNOLOGICAL SPECIFICITIES

1969 ◽  
Vol 62 (3) ◽  
pp. 521-536 ◽  
Author(s):  
M. L. Aubert ◽  
J.-P. Felber
Keyword(s):  
Binding Sites ◽  
Competitive Inhibition ◽  
Biologically Active ◽  
Active Part ◽  
Terminal Portion ◽  
Plasma Acth ◽  
Acth Fragments ◽  
Selection Of

ABSTRACT In investigations on the production and the specificity of anti-ACTH antibodies used for radioimmunoassay, differences have been observed between the various antibodies obtained. It was shown by means of competitive inhibition with different ACTH fragments that the binding of the ACTH molecule to its antibody can occur at different sites along the ACTH peptide. By varying the concentrations of the fragments and the conditions of the assays, it was possible to study the properties of each antibody. Thus antibodies which bind the N-terminal portion, or which exclusively bind the biologically active part of the ACTH chain (1–20), are the most suitable for radioimmunoassay. It was found, however, that the production of antiserum was generally more frequent when binding occurred to the C-terminal portion of the ACTH peptide. Should the presence of such fragments in plasma be confirmed, then the use of these antisera could lead to erroneous measurement of biologically inactive ACTH fragments. Thus, this study reveals that a selection of the antibody for specificity might be necessary for its application to the radioimmunoassay of plasma ACTH, and that this selection could be performed with the use of ACTH fragments. An approach to the problem of binding sites between antigen and antibody has been described and the possibility of introducing a radioimmunoassay for plasma ACTH fragments discussed.

scholarly journals Nova Regulates GABAA Receptor γ2 Alternative Splicing via a Distal Downstream UCAU-Rich Intronic Splicing Enhancer

2003 ◽  
Vol 23 (13) ◽  
pp. 4687-4700 ◽  
Author(s):  
B. Kate Dredge ◽  
Robert B. Darnell
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Splicing Regulation ◽  
Globin Genes ◽  
Enhancer Element ◽  
Intronic Splicing Enhancer ◽  
Splicing Enhancer

ABSTRACT Nova is a neuron-specific RNA binding protein targeted in patients with the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia, which is characterized by failure of inhibition of brainstem and spinal motor systems. Here, we have biochemically confirmed the observation that splicing regulation of the inhibitory GABAA receptor γ2 (GABAARγ2) subunit pre-mRNA exon E9 is disrupted in mice lacking Nova-1. To elucidate the mechanism by which Nova-1 regulates GABAARγ2 alternative splicing, we systematically screened minigenes derived from the GABAARγ2 and human β-globin genes for their ability to support Nova-dependent splicing in transient transfection assays. These studies demonstrate that Nova-1 acts directly on GABAARγ2 pre-mRNA to regulate E9 splicing and identify an intronic region that is necessary and sufficient for Nova-dependent enhancement of exon inclusion, which we term the NISE (Nova-dependent intronic splicing enhancer) element. The NISE element (located 80 nucleotides upstream of the splice acceptor site of the downstream exon E10) is composed of repeats of the sequence YCAY, consistent with previous studies of the mechanism by which Nova binds RNA. Mutation of these repeats abolishes binding of Nova-1 to the RNA in vitro and Nova-dependent splicing regulation in vivo. These data provide a molecular basis for understanding Nova regulation of GABAARγ2 alternative splicing and suggest that general dysregulation of Nova's splicing enhancer function may underlie the neurologic defects seen in Nova's absence.

Two-dimensional gel selection of protein binding sites on DNA

1993 ◽  
Vol 14 (1) ◽  
pp. 259-265 ◽  
Author(s):  
Andrea Boffini ◽  
Pierre Prentki
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Two Dimensional ◽  
Protein Binding Sites ◽  
Selection Of
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