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

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

Development of a novel RNA-programmable artificial transactivator able to upregulate endogenous genes ad libitum: DOI: 10.14800/rd.1142

Here we provide a concise overview of a new platform we recently developed for transactivating endogenous genes ad libitum. It relies on a binary design, including an RNA cofactor in charge of recognizing the target gene, and a polypeptidic apofactor stimulating transcription. Compared to similar CRISPR-based devices, our artificial transactivators are seven-folds smaller and elicit a lower, however robust and biologically effective, expression gain. Remarkably, they only work in cells which already transcribe the gene of interest. These properties make our novel platform an appealing potential tool for restoring normal expression levels of haploinsufficient genes upon generalized delivery

Binding of DAZAP1 and hnRNPA1/A2 to an Exonic Splicing Silencer in a Natural BRCA1 Exon 18 Mutant▿

A disease-causing G-to-T transversion at position +6 of BRCA1 exon 18 induces exclusion of the exon from the mRNA and, as has been suggested by in silico analysis, disrupts an ASF/SF2-dependent splicing enhancer. We show here using a pulldown assay with an internal standard that wild-type (WT) and mutant T6 sequences displayed similar ASF/SF2 binding efficiencies, which were significantly lower than that of a typical exonic splicing enhancer derived from the extra domain A exon of fibronectin. Overexpression or small interfering RNA (siRNA)-mediated depletion of ASF/SF2 did not affect the splicing of a WT BRCA1 minigene but resulted in an increase and decrease of T6 exon 18 inclusion, respectively. Furthermore, extensive mutation analysis using hybrid minigenes indicated that the T6 mutant creates a sequence with a prevalently inhibitory function. Indeed, RNA-protein interaction and siRNA experiments showed that the skipping of T6 BRCA1 exon 18 is due to the creation of a splicing factor-dependent silencer. This sequence specifically binds to the known repressor protein hnRNPA1/A2 and to DAZAP1, the involvement of which in splicing inhibition we have demonstrated. Our results indicate that the binding of the splicing factors hnRNPA1/A2 and DAZAP1 is the primary determinant of T6 BRCA1 exon 18 exclusion

Mis‐splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays

Splicing disruption is a common mechanism of gene inactivation associated with germline variants of susceptibility genes. To study the role of BRCA2 mis-splicing in hereditary breast/ovarian cancer (HBOC), we performed a comprehensive analysis of variants from BRCA2 exons 2-9, as well as the initial characterization of the regulatory mechanisms of such exons. A pSAD-based minigene with exons 2-9 was constructed and validated in MCF-7 cells, producing the expected transcript (1016-nt/V1-BRCA2_exons_2-9-V2). DNA variants from mutational databases were analyzed by NNSplice and Human Splicing Finder softwares. To refine ESE-variant prediction, we mapped the regulatory regions through a functional strategy whereby 26 exonic microdeletions were introduced into the minigene and tested in MCF-7 cells. Thus, we identified nine spliceogenic ESE-rich intervals where ESE-variants may be located. Combining bioinformatics and microdeletion assays, 83 variants were selected and genetically engineered in the minigene. Fifty-three changes impaired splicing: 28 variants disrupted the canonical sites, four created new ones, 10 abrogated enhancers, eight created silencers and three caused a double-effect. Notably, nine spliceogenic-ESE variants were located within ESE-containing intervals. Capillary electrophoresis and sequencing revealed more than 23 aberrant transcripts, where exon skipping was the most common event. Interestingly, variant c.67G>A triggered the usage of a noncanonical GC-donor 4-nt upstream. Thirty-six variants that induced severe anomalies (>60% aberrant transcripts) were analyzed according to the ACMG guidelines. Thus, 28 variants were classified as pathogenic, five as likely pathogenic and three as variants of uncertain significance. Interestingly, 13 VUS were reclassified as pathogenic or likely pathogenic variants. In conclusion, a large fraction of BRCA2 variants (∼64%) provoked splicing anomalies lending further support to the high prevalence of this disease-mechanism. The low accuracy of ESE-prediction algorithms may be circumvented by functional ESE-mapping that represents an optimal strategy to identify spliceogenic ESE-variants. Finally, systematic functional assays by minigenes depict a valuable tool for the initial characterization of splicing anomalies and the clinical interpretation of variants.EAV’s lab was supported by grants from the Spanish Ministry of Economy and Competitivity, Plan Nacional de I+D+I 2013-2016, ISCIII (PI13/01749 and PI17/00227) co-funded by FEDER from Regional Development European Funds (European Union), and grants CSI090U14 (ORDEN EDU/122/2014) and CSI242P18 (actuación cofinanciada P.O. FEDER 2014-2020 de Castilla y León) from the Consejería de Educación, Junta de Castilla y León. EFB is supported by a predoctoral fellowship from the University of Valladolid and Banco de Santander (2015–2019). AVP is supported by a predoctoral fellowship from Consejería de Educación, Junta de Castilla y León (2018–2022).Peer reviewe

BRCA 2 mis-splicing: regulación de los exones 17 y 18

Resumen del trabajo presentado al I Congreso Interdisciplinar en Genética Humana, celebrado en Madrid del 25 al 28 de abril de 2017.[Objetivos]: El rastreo de mutaciones en BRCA2 ha identificado un gran número de variantes de significado clínico desconocido (VUS). Nuestro objetivo es investigar el impacto de este tipo de variantes en el splicing de los exones 17 y 18 de BRCA2, así como caracterizar elementos reguladores en los mismos que constituyan hotspots para variantes de splicing. [Material y Método]: Se construyó un minigen con los exones 14-20 de BRCA2 en el vector pSAD®. En él, se evaluaron 52 variantes seleccionadas a través de programas de predicción de splicing. Cada una de ellas, fue introducida en el minigen mediante mutagénesis dirigida y ensayada en células MCF7. El estudio de regulación se llevó a cabo mediante microdeleciones, ensayos de pulldown y siRNAs. [Resultados]: El minigen wild type produjo un transcrito estable de tamaño (1.802nt) y estructura (V1-[BRCA2_exons_14-20]-V2) esperada. El mapeo mediante microdeleciones reveló regiones esenciales en el extremo 3' del exón 17 (c.7944-7973) y en el 5' del exón 18 (c.7979-7988, c.7999-8013). Estas contienen secuencias de unión a proteínas SR, importantes en el reconocimiento de los exones. Por otro lado, 30/52 variantes, provocaron alteraciones en el splicing. Se encontraron más de 16 tipos de transcritos diferentes, siendo el skipping el evento más común. Encontramos alteraciones en un amplio número de elementos de splicing, incluidos los sitios canónicos (15), sitios alternativos de novo (3), tracto de polipirimidinas (3) y enhancer/silenciadores (9). Cabe destacar que 18 de las variantes estudiadas podrían ser reclasificadas como patogénicas, lo cual supone un 28.6% de las variantes patogénicas reportadas en los exones 17 y 18 de BRCA2. [Conclusiones]: Las mutaciones de splicing son especialmente prevalentes en los exones 17 y 18 debido a la presencia de ESEs activos. Los minigenes son una herramienta valiosa para discriminar entre variantes patogénicas y neutras, así como para estudiar los mecanismos reguladores del procesamiento del RNA mensajero.Peer Reviewe

BRCA2 mis-splicing: exons 17 and 18 regulation

Póster presentado a la European Human Genetics Conference, celebrada en Copenague (Dinamarca) del 27 al 30 de mayo de 2017.Mutation screening of BRCA2 identifies a large fraction of variants of uncertain clinical significance (VUS). Our goal was to investigate the impact of reported variants of BRCA2 exons 17 and 18 on splicing to assess their role in hereditary breast cancer and to identify critical regulatory elements that may constitute hotspots for spliceogenic variants. A splicing reporter minigene with BRCA2 exons 14 to-20 (MGBR2_ex14-20) was constructed in the pSAD vector. Fifty-two candidate variants were selected with splicing prediction programs, introduced in MGBR2_ex14-20 by site-directed mutagenesis and assayed in MCF-7 cells. Functional mapping by microdeletions revealed essential sequences for exon recognition on the 3' end of exon 17 and the 5' end of exon 18. Thirty out of the 52 selected variants induced anomalous splicing in minigene assays with >16 different aberrant transcripts, where exon skipping was the most common event. According to the biological indicators of pathogenicity, 18 variants could be classified as disease-causing, accounting for 28.6% of all pathogenic variants of exons 17-18 at the BRCA Share database. Aberrant splicing is especially prevalent in BRCA2 exons 17 and 18 due to the presence of active ESEs, which are recognized by splicing factor as SC35 and Tra2ß that play an important role in exon recognition. Splicing functional assays with minigenes are a valuable strategy for the interpretation of VUS of any disease-gene.Projects FIS PI13/01749 (ISCIII,Spanish Ministry of Economy and Competitivity), BIO/VA34/15 (Junta Castilla-León); EF-B is supported by a predoctoral fellowship (University of Valladolid/Banco Santander) and by a Short-Fellowship (EMBO).Peer Reviewe