Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing

Hernández-Imaz, Elisabete; Martín, Yolanda; Conti, Laura De; Melean, Germán; Valero, Ana; Baralle, Marco; Hernández-Chico, Concepción

doi:10.1371/journal.pone.0141735

Cited by 9 publications

(6 citation statements)

References 40 publications

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“…Overexpression of hnRNPA1 decreases expression of an HMGCR splice variant lacking exon 13 that is important in response to statins, a class of cholesterol-lowering drugs (42). A single base change (c.1007G.A) within the neurofibromatosis type 1 (NF1) gene generates a pathogenic mutation that creates a binding site for hnRNPA1 and exon 9 exclusion (43). hnRNPA1 also plays an important role in generation of androgen receptor splice variants (AR-V7).…”

Section: Discussionmentioning

confidence: 99%

The Splicing Factor hnRNPA1 Regulates Alternate Splicing of the MYLK Gene

Mascarenhas¹,

Tchourbanov

Danilov

et al. 2018

Am J Respir Cell Mol Biol

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Profound lung vascular permeability is a cardinal feature of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI), two syndromes known to centrally involve the nonmuscle isoform of myosin light chain kinase (nmMLCK) in vascular barrier dysregulation. Two main splice variants, nmMLCK1 and nmMLCK2, are well represented in human lung endothelial cells and encoded by MYLK, and they differ only in the presence of exon 11 in nmMLCK1, which contains critical phosphorylation sites (Y and Y) that influence nmMLCK enzymatic activity, cellular translocation, and localization in response to vascular agonists. We recently demonstrated the functional role of SNPs in altering MYLK splicing, and in the present study we sought to identify the role of splicing factors in the generation of nmMLCK1 and nmMLCK2 spliced variants. Using bioinformatic in silico approaches, we identified a putative binding site for heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a recognized splicing factor. We verified hnRNPA1 binding to MYLK by gel shift analyses and that hnRNPA1 gene and protein expression is upregulated in mouse lungs obtained from preclinical models of ARDS and VILI and in human endothelial cells exposed to 18% cyclic stretch, a model that reproduces the excessive mechanical stress observed in VILI. Using an MYLK minigene approach, we established a direct role of hnRNPA1 in MYLK splicing and in the context of 18% cyclic stretch. In summary, these data indicate an important regulatory role for hnRNPA1 in MYLK splicing, and they increase understanding of MYLK splicing in the regulation of lung vascular integrity during acute lung inflammation and excessive mechanical stress, such as that observed in ARDS and VILI.

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Section: Discussionmentioning

confidence: 99%

The Splicing Factor hnRNPA1 Regulates Alternate Splicing of the MYLK Gene

Mascarenhas¹,

Tchourbanov

Danilov

et al. 2018

Am J Respir Cell Mol Biol

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“…Therefore, it remains to be determined if OCRL mutation c.741G>T has the same effect in the context of its natural gene using patient-derived RNA. Mutations in other genes associated with different diseases have been shown to have similar effects [ 58 , 59 , 60 , 61 , 62 ].…”

Section: Discussionmentioning

confidence: 99%

Splicing Analysis of Exonic OCRL Mutations Causing Lowe Syndrome or Dent-2 Disease

Suarez-Artiles

Perdomo‐Ramirez

Ramos-Trujillo

et al. 2018

Genes

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Mutations in the OCRL gene are associated with both Lowe syndrome and Dent-2 disease. Patients with Lowe syndrome present congenital cataracts, mental disabilities and a renal proximal tubulopathy, whereas patients with Dent-2 disease exhibit similar proximal tubule dysfunction but only mild, or no additional clinical defects. It is not yet understood why some OCRL mutations cause the phenotype of Lowe syndrome, while others develop the milder phenotype of Dent-2 disease. Our goal was to gain new insights into the consequences of OCRL exonic mutations on pre-mRNA splicing. Using predictive bioinformatics tools, we selected thirteen missense mutations and one synonymous mutation based on their potential effects on splicing regulatory elements or splice sites. These mutations were analyzed in a minigene splicing assay. Results of the RNA analysis showed that three presumed missense mutations caused alterations in pre-mRNA splicing. Mutation c.741G>T; p.(Trp247Cys) generated splicing silencer sequences and disrupted splicing enhancer motifs that resulted in skipping of exon 9, while mutations c.2581G>A; p.(Ala861Thr) and c.2581G>C; p.(Ala861Pro) abolished a 5′ splice site leading to skipping of exon 23. Mutation c.741G>T represents the first OCRL exonic variant outside the conserved splice site dinucleotides that results in alteration of pre-mRNA splicing. Our results highlight the importance of evaluating the effects of OCRL exonic mutations at the mRNA level.

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“…Aberrant splicing resulting from mutation in the constitutive splice site has been described in patients with hereditary diffuse gastric cancer [ 6 ]. Mutations that disrupt cis splicing regulatory elements have been studied in patients with neurofibromatosis type 1 (NF1) [ 7 ]. trans -acting mutations include those that affect the splicing machinery or factors that regulate alternative splicing.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Bioinformatic Programmes for the Analysis of Variants within Splice Site Consensus Regions

Tang

Prosser

Love

2016

Advances in Bioinformatics

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The increasing diagnostic use of gene sequencing has led to an expanding dataset of novel variants that lie within consensus splice junctions. The challenge for diagnostic laboratories is the evaluation of these variants in order to determine if they affect splicing or are merely benign. A common evaluation strategy is to use in silico analysis, and it is here that a number of programmes are available online; however, currently, there are no consensus guidelines on the selection of programmes or protocols to interpret the prediction results. Using a collection of 222 pathogenic mutations and 50 benign polymorphisms, we evaluated the sensitivity and specificity of four in silico programmes in predicting the effect of each variant on splicing. The programmes comprised Human Splice Finder (HSF), Max Entropy Scan (MES), NNSplice, and ASSP. The MES and ASSP programmes gave the highest performance based on Receiver Operator Curve analysis, with an optimal cut-off of score reduction of 10%. The study also showed that the sensitivity of prediction is affected by the level of conservation of individual positions, with in silico predictions for variants at positions −4 and +7 within consensus splice sites being largely uninformative.

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Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing

Cited by 9 publications

References 40 publications

The Splicing Factor hnRNPA1 Regulates Alternate Splicing of the MYLK Gene

The Splicing Factor hnRNPA1 Regulates Alternate Splicing of the MYLK Gene

Splicing Analysis of Exonic OCRL Mutations Causing Lowe Syndrome or Dent-2 Disease

Evaluation of Bioinformatic Programmes for the Analysis of Variants within Splice Site Consensus Regions

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