Role and Mechanism of Action of Exonic Splicing Regulatory Sequences

Exonic mutations can result in altered protein function by affecting exon recognition during splicing. To understand this mechanism I have extensively evaluated the exonic regulatory elements affected by the disease G to T mutation at position +6 of BRCA1 exon 18. This substitution induces the exclusion of the exon and it has been suggested that it disrupts an ASF/SF2-dependent enhancer. Using a pulldown assay with an internal standard, I show that WT and T6 sequences bind ASF/SF2 with similar efficiency, which is significantly lower compared to the binding to a typical enhancer derived from the fibronectin EDA exon. Consistent with the absence of an ASF/SF2 enhancing effect, siRNA depletion of ASF/SF2 did not induce exon WT exclusion indicating that ASF/SF2 is not essential for BRCA1 exon 18 splicing. However, depletion or overexpression of ASF/SF2 resulted in a decrease or increase of T6 exon 18 inclusion, respectively. By a series of exonic deletions in the WT and T6 BRCA1 minigenes, an enhancer sequence was identified in position 23-32 which mediates the observed T6-specific, ASF/SF2-dependent splicing activation. Furthermore, extensive mutations analysis indicated that the T6 mutant creates a sequence with a predominantly inhibitory function. Double site-directed mutations showed that the point mutations affecting the “TAG” sequence, between +6 and +8 positions, completely restore normal splicing. Indeed, RNA protein interaction and siRNA experiments showed that the skipping of T6 BRCA1 exon 18 is due to the creation of a silencer element. This sequence specifically binds to the proteins hnRNP A1/A2 and to DAZAP1. By siRNA experiments, DAZAP1 and hnRNP A1/A2 are shown to be involved redundantly in the regulation of the defective BRCA1 exon 18. The present results support a gain-of-function model for the BRCA1 T6 exon 18 and indicate that the binding of the hnRNP A1/A2 and DAZAP1 is the primary determinant of exon skipping

Cold-formed Steel Sections Experimental Data Base

Thin-walled cold-formed member critical load in the coupling point of overall/local buckling modes is defined, together with critical load erosion factor (ψ). A calibration method for (ψ) using an experimental database called DATACOST is presented

Upregulating endogenous genes by an RNA-programmable artificial transactivator

To promote expression of endogenous genes ad libitum, we developed a novel, programmable transcription factor prototype. Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Gene upregulation was small, however sufficient to robustly inhibit neuronal differentiation. The transactivator interacted with target gene chromatin via its RNA cofactor. Its activity was restricted to cells in which the target gene is normally transcribed. Our device might be useful for specific applications. However for this purpose, it will require an improvement of its transactivation power as well as a better characterization of its target specificity and mechanism of action

Quantitative methods for the analysis of CFTR transcripts/splicing variants

AbstractIn cystic fibrosis (CF), transcript analysis and quantification are important for diagnosis, prognosis and also as surrogate markers for some therapies including gene therapy. Classical RNA-based methods require significant expression levels in target samples for appropriate analysis, thus PCR-based methods are evolving towards reliable quantification. Various protocols for the quantitative analysis of CFTR transcripts (including those resulting from splicing variants) are described and discussed here

Interaction of hnRNPA1/A2 and DAZAP1 with an Alu-Derived Intronic Splicing Enhancer Regulates ATM Aberrant Splicing

We have previously identified an Alu-derived Intronic Splicing enhancer (ISE) in the Ataxia Teleangectasia Mutated gene (ATM) that facilitates intron pre-mRNA processing and leads to the inclusion of a cryptic exon in the final mRNA transcript. By using an RNA pull-down assay, we show here that hnRNPA1/A2, HuR and DAZAP1 splicing factors and DHX36 RNA helicase bind to the ISE. By functional studies (overexpression and siRNA experiments), we demonstrate that hnRNPA1 and DAZAP1 are indeed involved in ISE-dependent ATM cryptic exon activation, with hnRNPA1 acting negatively and DAZAP1 positively on splicing selection. On the contrary, HuR and DHX36 have no effect on ATM splicing pattern. These data suggest that splicing factors with both negative and positive effect can assemble on the intronic Alu repeats and regulate pre-mRNA splicing

Antagonistic factors control the unproductive splicing of SC35 terminal intron

Alternative splicing is regulated in part by variations in the relative concentrations of a variety of factors, including serine/arginine-rich (SR) proteins. The SR protein SC35 self-regulates its expression by stimulating unproductive splicing events in the 3′ untranslated region of its own pre-mRNA. Using various minigene constructs containing the terminal retained intron and flanking exons, we identified in the highly conserved last exon a number of exonic splicing enhancer elements responding specifically to SC35, and showed an inverse correlation between affinity of SC35 and enhancer strength. The enhancer region, which is included in a long stem loop, also contains repressor elements, and is recognized by other RNA-binding proteins, notably hnRNP H protein and TAR DNA binding protein (TDP-43). Finally, in vitro and in cellulo experiments indicated that hnRNP H and TDP-43 antagonize the binding of SC35 to the terminal exon and specifically repress the use of SC35 terminal 3′ splice site. Our study provides new information about the molecular mechanisms of SC35-mediated splicing activation. It also highlights the existence of a complex network of self- and cross-regulatory mechanisms between splicing regulators, which controls their homeostasis and offers many ways of modulating their concentration in response to the cellular environment

An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM

We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations