scholarly journals Exon definition facilitates reliable control of alternative splicing in the RON proto-oncogene

2019 ◽  
Author(s):  
M. Enculescu ◽  
S. Braun ◽  
S. T. Setty ◽  
K. Zarnack ◽  
J. König ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Epithelial To Mesenchymal Transition ◽  
Intron Retention ◽  
Regulatory Elements ◽  
Protein Isoforms ◽  
Splicing Regulation ◽  
Exon Definition ◽  
Mesenchymal Transition ◽  
Mrna Maturation ◽  
Exon 11

ABSTRACTAlternative splicing is a key step in eukaryotic gene expression that allows the production of multiple protein isoforms from the same gene. Even though splicing is perturbed in many diseases, we currently lack insights into regulatory mechanisms promoting its precision and efficiency. We analyse high-throughput mutagenesis data obtained for an alternatively spliced exon in the proto-oncogene RON and determine the functional units that control this splicing event. Using mathematical modeling of distinct splicing mechanisms, we show that alternative splicing is based in RON on a so-called ‘exon definition’ mechanism. Here, the recognition of the adjacent exons by the spliceosome is required for removal of an intron. We use our model to analyze the differences between the exon and intron definition scenarios and find that exon definition is crucial to prevent the accumulation of deleterious, partially spliced retention products during alternative splicing regulation. Furthermore, it modularizes splicing control, as multiple regulatory inputs are integrated into a common net input, irrespective of the location and nature of the corresponding cis-regulatory elements in the pre-mRNA. Our analysis suggests that exon definition promotes robust and reliable splicing outcomes in RON splicing.SIGNIFICANCEDuring mRNA maturation, pieces of the pre-mRNA (introns) are removed during splicing, and remaining parts (exons) are joined together. In alternative splicing, certain exons are either included or excluded, resulting in different splice products. Inclusion of RON alternative exon 11 leads to a functional receptor tyrosine kinase, while skipping results in a constitutively active receptor that promotes epithelial-to-mesenchymal transition and contributes to tumour invasiveness. Intron retention results in to deleterious isoforms that cannot be translated properly. Using kinetic modeling, we investigate the combinatorial regulation of this important splicing decision, and find that the experimental data supports a so-called exon definition mechanism. We show that this mechanism enhances the precision of alternative splicing regulation and prevents the retention of introns in the mature mRNA.

scholarly journals Widespread separation of the polypyrimidine tract from 3’ AG by G tracts in association with alternative exons in metazoa and plants

2018 ◽  
Author(s):  
Hai Nguyen ◽  
Jiuyong Xie
Keyword(s):  
Alternative Splicing ◽  
Branch Point ◽  
Intron Retention ◽  
Regulatory Elements ◽  
Protein Isoforms ◽  
Model Organisms ◽  
Branch Points ◽  
Splice Sites ◽  
Polypyrimidine Tract ◽  
Whole Genome Analysis

SummaryAt the end of introns, the polypyrimidine tract (Py) is often close to the 3’ AG in a consensus (Y)20NCAGgt in humans. Interestingly, we have found that they could also be separated by purine-rich elements including G tracts in thousands of human genes. These regulatory elements between the Py and 3’AG (REPA) mainly regulate alternative 3’ splice sites (3’SS) and intron retention. Here we show their widespread distribution and special properties across kingdoms. The purine-rich 3’SS are found in up to about 60% of the introns among more than 1000 species/lineages by whole genome analysis, and up to 18% of these introns contain the REPA G tracts in about 2.4 millions of 3’SS in total. In particular, they are significantly enriched over their 3’SS and genome backgrounds in metazoa and plants, and highly associated with alternative splicing of genes in diverse functional clusters. They are also highly enriched (3-6 folds) in the canonical as well as aberrantly used 3’ splice sites in cancer patients carrying mutations of the branch point factor SF3B1 or the 3’AG binding factor U2AF35. Moreover, the REPA G tract-harbouring 3’SS have significantly reduced occurrences of branch point (BP) motifs between the −24 and −4 positions, in particular absent from the −7 - −5 positions in several model organisms examined. The more distant branch points are associated with increased occurrences of alternative splicing in human and zebrafish. The branch points, REPA G tracts and associated 3’SS motifs appear to have emerged differentially in a phylum- or species-specific way during evolution. Thus, there is widespread separation of the Py and 3’AG by REPA G tracts, likely evolved among different species or branches of life. This special 3’SS arrangement contributes to the generation of diverse transcript or protein isoforms in biological functions or diseases through alternative or aberrant splicing.

scholarly journals Hypoxia-induced alternative splicing: the 11th Hallmark of Cancer

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Antonietta Rosella Farina ◽  
Lucia Cappabianca ◽  
Michela Sebastiano ◽  
Veronica Zelli ◽  
Stefano Guadagni ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Tumour Cell ◽  
Epithelial To Mesenchymal Transition ◽  
Tumour Progression ◽  
Protein Isoforms ◽  
Metastatic Progression ◽  
Mesenchymal Transition ◽  
Cell Immortalization ◽  
Oncogene Activation ◽  
The Many

Abstract Hypoxia-induced alternative splicing is a potent driving force in tumour pathogenesis and progression. In this review, we update currents concepts of hypoxia-induced alternative splicing and how it influences tumour biology. Following brief descriptions of tumour-associated hypoxia and the pre-mRNA splicing process, we review the many ways hypoxia regulates alternative splicing and how hypoxia-induced alternative splicing impacts each individual hallmark of cancer. Hypoxia-induced alternative splicing integrates chemical and cellular tumour microenvironments, underpins continuous adaptation of the tumour cellular microenvironment responsible for metastatic progression and plays clear roles in oncogene activation and autonomous tumour growth, tumor suppressor inactivation, tumour cell immortalization, angiogenesis, tumour cell evasion of programmed cell death and the anti-tumour immune response, a tumour-promoting inflammatory response, adaptive metabolic re-programming, epithelial to mesenchymal transition, invasion and genetic instability, all of which combine to promote metastatic disease. The impressive number of hypoxia-induced alternative spliced protein isoforms that characterize tumour progression, classifies hypoxia-induced alternative splicing as the 11th hallmark of cancer, and offers a fertile source of potential diagnostic/prognostic markers and therapeutic targets.

scholarly journals miRNA proxy approach reveals hidden functions of glycosylation

2015 ◽  
Vol 112 (23) ◽  
pp. 7327-7332 ◽  
Author(s):  
Tomasz Kurcon ◽  
Zhongyin Liu ◽  
Anika V. Paradkar ◽  
Christopher A. Vaiana ◽  
Sujeethraj Koppolu ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Epithelial To Mesenchymal Transition ◽  
Biological Function ◽  
Regulatory Elements ◽  
Rapid Identification ◽  
Mesenchymal Transition ◽  
Disease States ◽  
Mesenchymal To Epithelial Transition ◽  
Genes Encoding ◽  
Target Network

Glycosylation, the most abundant posttranslational modification, holds an unprecedented capacity for altering biological function. Our ability to harness glycosylation as a means to control biological systems is hampered by our inability to pinpoint the specific glycans and corresponding biosynthetic enzymes underlying a biological process. Herein we identify glycosylation enzymes acting as regulatory elements within a pathway using microRNA (miRNA) as a proxy. Leveraging the target network of the miRNA-200 family (miR-200f), regulators of epithelial-to-mesenchymal transition (EMT), we pinpoint genes encoding multiple promesenchymal glycosylation enzymes (glycogenes). We focus on three enzymes, beta-1,3-glucosyltransferase (B3GLCT), beta-galactoside alpha-2,3-sialyltransferase 5 (ST3GAL5), and (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 (ST6GALNAC5), encoding glycans that are difficult to analyze by traditional methods. Silencing these glycogenes phenocopied the effect of miR-200f, inducing mesenchymal-to-epithelial transition. In addition, all three are up-regulated in TGF-β–induced EMT, suggesting tight integration within the EMT-signaling network. Our work indicates that miRNA can act as a relatively simple proxy to decrypt which glycogenes, including those encoding difficult-to-analyze structures (e.g., proteoglycans, glycolipids), are functionally important in a biological pathway, setting the stage for the rapid identification of glycosylation enzymes driving disease states.

Evidence for tissue-specific alternative splicing of the ovine insulin receptor gene

1998 ◽  
Vol 1998 ◽  
pp. 96-96
Author(s):  
P.D. McGrattan ◽  
A.R.G. Wylie ◽  
A.J. Bjourson
Keyword(s):  
Alternative Splicing ◽  
Insulin Receptor ◽  
Receptor Gene ◽  
Protein Isoforms ◽  
Common Mechanism ◽  
Mrna Transcript ◽  
Insulin Receptor Gene ◽  
Tissue Specific ◽  
Specific Regulation ◽  
Exon 11

Alternative splicing of a discrete 36 base pair segment (exon 11) of the human and rat insulin receptor leads to the formation of high and low affinity isoforms differing as much as 3-fold in affinity for insulin. Alternative splicing is a common mechanism for generating protein isoforms and is often regulated in a tissue-specific fashion (Seino & Bell, 1989; Mosthaf et al., 1990). In humans, the lower affinity (B-isoform) mRNA transcript is predominantly expressed in tissues that are important for modulating glucose homeostasis such as the liver and muscle whereas the higher affinity (A-isoform) mRNA transcript is predominantly expressed in haematopoietic tissues such as spleen. Alternative splicing of the region of the ovine insulin receptor gene encoding exon 11 has recently been demonstrated (McGrattan et al., unpublished). The objective of the present study was to establish whether tissue-specific regulation of alternative splicing of the insulin receptor gene occurs in the ruminant animal.

RBM47-regulated alternative splicing of TJP1 promotes actin stress fiber assembly during epithelial-to-mesenchymal transition

Oncogene ◽  
2019 ◽  
Vol 38 (38) ◽  
pp. 6521-6536 ◽  
Author(s):  
Yong-Eun Kim ◽  
Minho Won ◽  
Sung-Gwon Lee ◽  
Chungoo Park ◽  
Chang-Hwa Song ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Epithelial To Mesenchymal Transition ◽  
Stress Fiber ◽  
Actin Stress Fiber ◽  
Mesenchymal Transition ◽  
Fiber Assembly

Evolutionarily Conserved Coupling of Transcription and Alternative Splicing in the Protein 4.1R and 4.1B Genes Regulates N-Terminal Protein Structure.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1664-1664
Author(s):  
Jeff Tan ◽  
Marilyn K. Parra ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Rna Processing ◽  
Regulatory Elements ◽  
Protein Isoforms ◽  
Tissue Type ◽  
Translation Initiation Site ◽  
Acceptor Site ◽  
Exon 2 ◽  
Evolutionarily Conserved ◽  
Protein 4.1R

Abstract The protein 4.1R gene is regulated by complex pre-mRNA processing events that facilitate the synthesis of protein isoforms with different structure, function, and subcellular localization in red cells and various nucleated cell types. One of these events involves the stage-specific activation of exon 16 inclusion in erythroblasts, which mechanically stabilizes the membrane skeleton by increasing the protein’s affinity for spectrin and actin. Some of the splicing factor proteins and RNA regulatory elements responsible for this tissue-specific alternative splicing event have been defined. Here we focus on another RNA processing event, in the 5′ end of the transcript that can affect the structure and function of the membrane binding domain of protein 4.1R. We have shown that 4.1R transcripts originating at three far upstream alternative promoters/first exons splice differentially to alternative acceptor sites in exon 2′/2 in a manner that suggests strict coupling between transcription and alternative splicing events. A precisely analogous gene organization and RNA processing pattern has also been shown to occur in the paralogous 4.1B gene. Now we demonstrate that this coupling is evolutionarily conserved among several vertebrate classes from fish to mammals. The 4.1R and 4.1B genes from fish, bird, amphibian, and mammal genomes exhibit shared features including alternative first exons and differential splice acceptors in exon 2. In all cases, the 5′-most exon (exon 1A) splices exclusively to a weaker internal acceptor site in exon 2, skipping a short sequence designated as exon 2′ (17-33nt). Conversely, alternative first exons 1B and/or 1C always splice to the stronger first acceptor site, retaining exon 2′. These correlations are independent of tissue type or species of origin. Since exon 2′ contains a translation initiation site, this regulated splicing event generate protein isoforms with distinct N-termini. We propose that these 4.1 genes represent a physiologically relevant model system for mechanistic analysis of transcription-coupled alternative splicing. We have recently constructed a 9kb “minigene” that successfully reproduces the differential splicing patterns of exons 1A and 1B to exon 2′/2 in transfected cells. This minigene will facilitate identification of the determinants that guide coupling. Current experiments are testing the importance for proper splicing of the transcriptional promoter, first exon sequences, length and sequence of the intron, and sequence of a conserved element within exon 2′. Ultimately these studies should provide new insights into the mechanisms of coupling between far upstream, transcription-related processes and downstream alternative splicing.

scholarly journals Bleomycin-enhanced alternative splicing of fibroblast growth factor receptor 2 induces epithelial to mesenchymal transition in lung fibrosis

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Kui-Jun Chen ◽  
Qing Li ◽  
Chang-Mei Weng ◽  
Zhao-Xia Duan ◽  
Dong-Dong Zhang ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Lung Fibrosis ◽  
Epithelial To Mesenchymal Transition ◽  
Fibroblast Growth Factor Receptor ◽  
Growth Factor Receptor ◽  
Fibroblast Growth ◽  
Transgenic Mouse Models ◽  
Mesenchymal Transition

Idiopathic pulmonary fibrosis (IPF) is an important public health problem, and it has few treatment options given its poorly understood etiology; however, epithelial to mesenchymal transition (EMT) of pneumocytes has been implicated as a factor. Herein, we aimed to explore the underlying mechanisms of lung fibrosis mediated by EMT, with a focus on the alternative splicing of fibroblast growth factor receptor 2 (FGFR2), using bleomycin (BLM)-induced lung fibrotic and transgenic mouse models. We employed BLM-induced and surfactant protein C (SPC)-Cre and LacZ double transgenic mouse models. The results showed that EMT occurred during lung fibrosis. BLM inhibited the expression of epithelial splicing regulatory protein 1 (ESRP1), resulting in enhanced alternative splicing of FGFR2 to the mesenchymal isoform IIIc. BLM-induced lung fibrosis was also associated with the activation of TGF-β/Smad signaling. These findings have implications for rationally targetted strategies to therapeutically address IPF.

scholarly journals PRPF6 Induces the Alternative Splicing of lncRNA SNHG16 To Promote Progression and Paclitaxel Resistance of Ovarian Cancer via Regulating GATA3 Transcription

2021 ◽  
Author(s):  
Han Wang ◽  
Yingying Zhou ◽  
Siyang Zhang ◽  
Ya Qi ◽  
Min Wang
Keyword(s):  
Ovarian Cancer ◽  
Alternative Splicing ◽  
Epithelial To Mesenchymal Transition ◽  
Binding Protein ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mesenchymal Transition ◽  
Gene Assay

Abstract Background Small nucleolar RNA host gene 16 (SNHG16) and pre-mRNA processing factor 6(PRPF6) play vital roles in regulatory mechanisms of multiple cancers, but the mechanisms in ovarian cancer (OC) remains poorly understood. Methods The expression of SNHG16 transcripts-SNHG16-L/S in OC tissues were analyzed by real-time PCR (RT-PCR). The expression of PRPF6 in OC tissues were detected by Immunohistochemistry (IHC). Tumorigenesis, epithelial-to-mesenchymal transition (EMT) and PTX-resistance were detected by western blot, transwell, CCK-8 assays, colony formation assays and flow cytometry analyses. Molecular interactions were examined by dual-luciferase reporter gene assay, RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP). Results The results indicated the expression of SNHG16-L/S was opposite in chemo-resistance and chemo-sensitivity tissues of OC. And SNHG16-L/S had different effects on the progression and PTX-resistance of OC cells. SNHG16-L inhibited GATA binding protein 3 (GATA3) transcription through CCAAT/enhancer-binding protein b (CEBPB) to further promote tumorigenesis, EMT and PTX-resistance of OC. Moreover, PRPF6 was upregulated in chemo-resistance tissues of OC. PRPF6 promoted tumorigenesis and PTX-resistance in vitro and in vivo. Mechanistically, PRPF6 induced the alternative splicing of SNHG16 to downregulate SNHG16-L, which further mediated progression and PTX-resistance through upregulating GATA3 in OC. Conclusions Totally, the results demonstrated that PRPF6 promoted progression and PTX-resistance in OC through SNHG16-L/CEBPB/GATA3 axis. Thus, PRPF6 may become a valuable target for OC therapy.

scholarly journals Alternative splicing takes charge of interleukin-22 and interferon lambda 4 signaling

2018 ◽  
Author(s):  
Chrissie Lim
Keyword(s):  
Alternative Splicing ◽  
Intron Retention ◽  
Protein Isoforms ◽  
Functional Domain ◽  
Type I ◽  
Protein Coding ◽  
Interleukin 22 ◽  
Interferon Lambda ◽  
Type Iii Ifn ◽  
Overexpression System

Immune responses require the tight control of dose, location, strength and duration through genetic, epigenetic or biochemical regulation. Of these, the generation of alternatively-spliced constructs increases transcriptional and proteomic diversity in post-transcriptional modification, localization and functional domain integrity. Specifically, this thesis explores how splice variation engenders profound differences in the biological functions of interleukin-22 (IL-22) binding protein (IL-22BP) and interferon lambda 4 (IFNλ4), which are both central components of distinct cytokine pathways in mucosal immunity and inflammation. IL-22BP is a soluble receptor for IL-22 that is expressed as three isoforms in humans, though the physiological relevance of the three human isoforms has remained a mystery due to the absence of this variation in mice. We present novel findings that IL-22BPi1 is inactive due to intracellular retention by its unique exon, while IL-22BPi3 is also an antagonist but with differential activity from IL-22BPi2. Importantly, while IL-22BPi3 has widespread expression in steady-state homeostatic conditions, IL-22BPi2 is the only isoform induced by inflammatory TLR2/retinoic acid stimulation, highlighting important spatiotemporal control of the two isoforms that exploit their differential activities. IFNλ4 presents a different mystery in which the protein-coding variant is genetically associated with poorer clearance, but the mechanism for this association remains unclear. We investigated several non-canonical functions proposed by the field, including intrinsic differences in activity of the three protein isoforms and their interference with antiviral activites of other type I or III interferons. Establishing an overexpression system and purifying recombinant proteins, we found that only the full-length isoform is active and exhibits similar effects to canonical type III IFN IFNλ3, without any blockade of other IFN signaling. Simultaneously, functional IFNλ4 expression is suppressed in hepatocytes and dendritic cells through preferential splicing to increase intron retention and expression of inactive isoforms. Therefore, alternative splicing in IFNλ4 is an important mechanism to control IFNλ4 bioactivity. The divergent manners in which alternative splice forms impact the activity of both IL-22BP and IFNλ4 highlight the important contributions of this process to cytokine biology and bigger implications that escape detection by genomic analyses.

scholarly journals Splice-Junction Elements and Intronic Sequences Regulate Alternative Splicing of the Drosophila Myosin Heavy Chain Gene Transcript

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 725-741 ◽  
Author(s):  
David M Standiford ◽  
Mary Beth Davis ◽  
Weitao Sun ◽  
Charles P Emerson
Keyword(s):  
Alternative Splicing ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Alternative Exon ◽  
Chain Gene ◽  
Gene Transcript ◽  
Specific Alternative ◽  
Exon 11

The Drosophila muscle myosin heavy chain (Mhc) gene primary transcript contains five alternatively spliced exon groups (exons 3, 7, 9, 11 and 15), each of which contains two to five mutually exclusive members. Individual muscles typically select a specific alternative exon from each group for incorporation into the processed message. We report here on the cis-regulatory mechanisms that direct the processing of alternative exons in Mhc exon 11 in individual muscles using transgenic reporter constructs, RT-PCR and directed mutagenesis. The 6.0-kilobase exon 11 domain is sufficient to direct the correct processing of exon 11 alternatives, demonstrating that the alternative splicing cis-regulatory elements are local to Mhc exon 11. Mutational analysis of Mhc exon 11 reveals that the alternative exon nonconsensus 5′-splice donors are essential for alternative splicing regulation in general, but do not specify alternative exons for inclusion in individual muscles. Rather, we show, through exon substitutions and deletion analyses, that a 360-nucleotide intronic domain precisely directs the normal processing of one exon, Mhc exon 11e, in the indirect flight muscle. These and other data indicate that alternative exons are regulated in appropriate muscles through interactions between intronic alternative splice-specificity elements, nonconsensus exon 11 splice donors and, likely, novel exon-specific alternative splicing factors.

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