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Functional properties and evolutionary splicing constraints on a composite exonic regulatory element of splicing in CFTR exon 12
- Nucleic Acids Res
, 2010
"... ABSTRACT In general, splicing regulatory elements are defined as Enhancers or Silencers depending on their positive or negative effect upon exon inclusion. Often, these sequences are usually present separate from each other in exonic/ intronic sequences. The Composite Exonic Splicing Regulatory Ele ..."
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ABSTRACT In general, splicing regulatory elements are defined as Enhancers or Silencers depending on their positive or negative effect upon exon inclusion. Often, these sequences are usually present separate from each other in exonic/ intronic sequences. The Composite Exonic Splicing Regulatory Elements (CERES) represent an extreme physical overlap of enhancer/silencer activity. As a result, when CERES elements are mutated the consequences on the splicing process are difficult to predict. Here, we show that the functional activity of the CERES2 sequence in CFTR exon 12 is regulated by the binding, in very close proximity to each other, of several SR and hnRNP proteins. Moreover, our results show that practically the entire exon 12 sequence context participate in its definition. The consequences of this situation can be observed at the evolutionary level by comparing changes in conservation of different splicing elements in different species. In conclusion, our study highlights how it is increasingly difficult to define many exonic sequences by simply breaking them down in isolated enhancer/silencer or even neutral elements. The real picture is close to one of continuous competition between positive and negative factors where affinity for the target sequences and other dynamic factors decide the inclusion or exclusion of the exon.
Co-regulation of alternative splicing by diverse splicing factors in
, 2010
"... Caenorhabditis elegans ..."
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Dual Role of G-runs and hnRNP F in the Regulation of a Mutation-Activated Pseudoexon in the Fibrinogen Gamma-Chain Transcript
"... Most pathological pseudoexon inclusion events originate from single activating mutations, suggesting that many intronic sequences are on the verge of becoming exons. However, the precise mechanisms controlling pseudoexon definition are still largely unexplored. Here, we investigated the cis-acting e ..."
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Most pathological pseudoexon inclusion events originate from single activating mutations, suggesting that many intronic sequences are on the verge of becoming exons. However, the precise mechanisms controlling pseudoexon definition are still largely unexplored. Here, we investigated the cis-acting elements and trans-acting regulatory factors contributing to the regulation of a previously described fibrinogen gamma-chain (FGG) pseudoexon, which is activated by a deep-intronic mutation (IVS6-320A.T). This pseudoexon contains several G-run elements, which may be bound by heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H. To explore the effect of these proteins on FGG pseudoexon inclusion, both silencing and overexpression experiments were performed in eukaryotic cells. While hnRNP H did not significantly affect pseudoexon splicing, hnRNP F promoted pseudoexon inclusion, indicating that these two proteins have only partially redundant functions. To verify the binding of hnRNP F and the possible involvement of other trans-acting splicing modulators, pulldown experiments were performed on the region of the pseudoexon characterized by both a G-run and enrichment for exonic splicing enhancers. This 25-bp-long region strongly binds hnRNP F/H and weakly interacts with Serine/Arginine-rich protein 40, which however was demonstrated to be dispensable for FGG pseudoexon inclusion in overexpression experiments. Deletion analysis, besides confirming the splicing-promoting role of the G-run within this 25-bp region, demonstrated that two additional hnRNP F binding sites might instead function as silencer elements. Taken
Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage. Nucleic Acids Res
"... Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utiliz-ing a novel minigene that mimics endogenous MDM ..."
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Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utiliz-ing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily con-served sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted ex-clusion of exon 11, while its siRNA-mediated knock-down prevented the stress-induced alternative splic-ing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. No-tably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation.
Deregulation of splicing factors and breast cancer development
, 2015
"... D ow nloaded from 2 It is well known that many genes implicated in the development and progression of breast cancer undergo aberrant alternative splicing events to produce proteins with pro-cancer properties. These changes in alternative splicing can arise from mutations or single nucleotide polymor ..."
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D ow nloaded from 2 It is well known that many genes implicated in the development and progression of breast cancer undergo aberrant alternative splicing events to produce proteins with pro-cancer properties. These changes in alternative splicing can arise from mutations or single nucleotide polymorphisms (SNPs) within the DNA sequences of cancer-related genes, which can strongly affect the activity of splicing factors and influence the splice site choice. However, it is important to note that absence of mutations is not sufficient to prevent misleading choices in splice site selection. There is now increasing evidence to demonstrate that the expression profile of ten splicing factors (including SRs and hnRNPs) and eight RNA-binding proteins changes in breast cancer cells compared with normal cells. These modifications strongly influence the alternative splicing pattern of many cancer-related genes despite the absence of any detrimental mutations within their DNA sequences. Thus, a comprehensive assessment of the splicing factor status in breast cancer is important to provide insights into the mechanisms that lead to breast cancer development and metastasis. Whilst most studies focus on mutations that affect alternative splicing in cancer-related genes, this review focuses on splicing factors and RNA-binding proteins that are themselves deregulated in breast cancer and implicated in cancer-related alternative splicing events.
unknown title
, 2010
"... Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving ..."
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Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving
unknown title
, 2010
"... Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving ..."
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Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving
unknown title
, 2009
"... Functional properties and evolutionary splicing constraints on a composite exonic regulatory element of splicing in CFTR exon 12 ..."
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Functional properties and evolutionary splicing constraints on a composite exonic regulatory element of splicing in CFTR exon 12
unknown title
, 2010
"... Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving ..."
Abstract
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Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving
