Evolution of AANAT: expansion of the gene family in the cephalochordate amphioxus

<p>Abstract</p> <p>Background</p> <p>The arylalkylamine <it>N</it>-acetyltransferase (AANAT) family is divided into structurally distinct vertebrate and non-vertebrate groups. Expression of vertebrate AANATs is limited primarily to the pineal gland and retina, where it plays a role in controlling the circadian rhythm in melatonin synthesis. Based on the role melatonin plays in biological timing, AANAT has been given the moniker "the Timezyme". Non-vertebrate AANATs, which occur in fungi and protists, are thought to play a role in detoxification and are not known to be associated with a specific tissue.</p> <p>Results</p> <p>We have found that the amphioxus genome contains seven <it>AANAT</it>s, all having non-vertebrate type features. This and the absence of <it>AANATs </it>from the genomes of Hemichordates and Urochordates support the view that a major transition in the evolution of the <it>AANATs </it>may have occurred at the onset of vertebrate evolution. Analysis of the expression pattern of the two most structurally divergent <it>AANAT</it>s in <it>Branchiostoma lanceolatum </it>(<it>bl</it>) revealed that they are expressed early in development and also in the adult at low levels throughout the body, possibly associated with the neural tube. Expression is clearly not exclusively associated with the proposed analogs of the pineal gland and retina. blAANAT activity is influenced by environmental lighting, but light/dark differences do not persist under constant light or constant dark conditions, indicating they are not circadian in nature. bfAANATα and bfAANATδ' have unusually alkaline (> 9.0) optimal pH, more than two pH units higher than that of vertebrate AANATs.</p> <p>Conclusions</p> <p>The substrate selectivity profiles of bfAANATα and δ' are relatively broad, including alkylamines, arylalkylamines and diamines, in contrast to vertebrate forms, which selectively acetylate serotonin and other arylalkylamines. Based on these features, it appears that amphioxus AANATs could play several roles, including detoxification and biogenic amine inactivation. The presence of seven AANATs in amphioxus genome supports the view that arylalkylamine and polyamine acetylation is important to the biology of this organism and that these genes evolved in response to specific pressures related to requirements for amine acetylation.</p

Les récepteurs de la mélatonine dans le cerveau et l'hypophyse de brochet (caractérisations pharmacologique, moléculaire et fonctionnelle)

POITIERS-BU Sciences (861942102) / SudocBANYULS/MER-Observ.Océanol. (660162201) / SudocSudocFranceF

Use of Splicing Reporter Minigene Assay to Evaluate the Effect on Splicing of Unclassified Genetic Variants

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THE BRCA1 c.5434C>G (p.PRO1812ALA) VARIANT INDUCES A DELETERIOUS EXON 23 SKIPPING BY AFFECTING EXONIC SPLICING REGULATORY ELEMENTS

International audienceA large fraction of the sequence variants of unknown significance or unclassified variants (UVs), including exonic variants, could be pathogenic by affecting mRNA splicing. The breast and ovarian cancer susceptibility gene BRCA1 exhibits a large spectrum of sequence variation but only two variants, both located in exon 18, have been shown experimentally to affect splicing regulatory elements. In the present study, we investigate the impact on splicing of the variant BRCA1 c.5434C>G (p.Pro1812Ala), identified in an ovarian cancer patient. This variant has previously been studied at the protein level with inconclusive results concerning its pathogenicity. Here, we show, using patient RNA analyses and hybrid minigene assays, that this variant induces nearly complete skipping of exon 23, resulting in frameshift and predicted protein termination within the second BRCT domain. This argues for its classification as a pathogenic splicing mutation. Moreover, we demonstrate, using an exonic splicing enhancer-dependent minigene assay, that the segment c.5420-5449 of BRCA1, in the centre of exon 23, exhibits splicing enhancer properties. This enhancement is abolished by the c.5434C>G mutation, indicating that the nucleotide change, in this highly conserved region, affects a splicing regulatory element. Bioinformatics analyses predict that the mutation c.5434C>G creates an hnRNPA1-dependent splicing silencer. These results also suggest that UVs in highly conserved nucleotide sequences of short exons may be good candidates for detecting functionally relevant splicing regulatory elements

A Novel Pineal-specific Product of the oligopeptide transporter PepT1 Gene: circadian expression mediated by camp activation of an intronic promoter

The oligopeptide transporter 1, PepT1, is a member of the Slc15 family of 12 membrane-spanning domain transporters; PepT1 has proton/peptide cotransport activity and is selectively expressed in intestinal epithelial cells, where it is responsible for the nutritional absorption of di- and tri-peptides. Here, a novel PepT1 gene product has been identified in the rat pineal gland, termed pgPepT1. It encodes a 150-amino acid protein encompassing the C-terminal 3 membrane-spanning domains of intestinal PepT1 protein, with 3 additional N-terminal residues. Expression of pgPepT1 appears to be restricted to the pineal gland and follows a marked circadian pattern with >100-fold higher levels of mRNA occurring at night; this is accompanied by an accumulation of membrane-associated pgPepT1 protein (∼16 kDa). The daily rhythm in pgPepT1 mRNA is regulated by the well described neural pathway that controls pineal melatonin production. This includes the retina, the circadian clock in the suprachiasmatic nucleus, central structures, and projections from the superior cervical ganglia; activation of this pathway results in the release of norepinephrine. Here it was found that pgPepT1 expression is mediated by a norepinephrine → cyclic AMP mechanism that activates an alternative promoter located in intron 20 of the gene. pgPepT1 protein was found to have transporter-modulator activity; it could contribute to circadian changes in pineal function through this mechanism

Functional Analysis of a Large set of BRCA2 exon 7 Variants Highlights the Predictive Value of Hexamer Scores in Detecting Alterations of Exonic Splicing Regulatory Elements

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Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools

International audienceThe identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient's RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases