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Identification of alternatively spliced transcripts produced by a gene is a crucial step in deciphering the bulk of its biological roles and the overall processes that regulate its activity. By using a combination of bioinformatic and molecular approaches we identified, cloned, and characterized 3 novel alternative splice isoforms derived from human dyskeratosis congenita 1 (fiDKC1), an essential human gene causative of the X-linked dyskeratosis congenita disease and involved in multiple functions related to cell growth, proliferation, and telomere maintenance. Expression of the new isoforms, all characterized by intron retention, was confirmed by RT-PCR in a panel of diverse cell lines and normal human tissues, and despite the presence of premature termination codons, was not down-regulated by the mechanism of nonsense-mediated decay. Accumulation of these transcripts fluctuated distinctly in the diverse tissues and during in vitro differentiation of Caco2 cells, suggesting that their ratio may contribute to the gene functional diversity across different cell types. Intriguingly, the structure of one isoform leads to exonize an intronically encoded small nucleolar RNA (snoRNA), highlighting an additional layer of complexity that can contribute to overall gene regulation. Key words: alternative splicing, dyskerin, DKC1, pseudouridine synthase, snoRNP. L'identification de transcrits geniques sujets a l'epissage alternatif est une etape cle afin de decrypter l'essentiel de leurs roles biologiques et les processus globaux qui regulent leur activite. En utilisant une combinaison d'approches bioinformatiques et moleculaires, nous avons identifie, clone et caracterise trois nouvelles isoformes epissees de maniere alternative issues du gene hDKC1 (human dyskeratosis congenita 1), un gene essentiel qui cause la dyskeratose congenitale liee au chromosome X et qui implique dans plusieurs fonctions reliees a la croissance cellulaire, a la proliferation et au maintien des telomeres. L'expression des nouvelles isoformes, toutes caracterisees par une retention d'intron, a ete confirmee par RT-PCR dans une serie de lignees cellulaires differentes et de tissus humains normaux et, malgre la presence de codons de terminaison prematures, cette expression n'etait pas regulee a la baisse par un mecanisme de degradation d'ARNm non-sens. L'accumulation de ces transcrits fluctuait distinctement dans les differents tissus et pendant la differenciation in vitro des cellules Caco2, suggerant que leur rapport peut contribuer a la diversite genique fonctionnelle parmi les differents types cellulaires. Fait interessant, la structure d'une isoforme mene a l'exonisation d'un petit ARN nucleolaire (snoARN) code dans un intron, mettant en lumiere un niveau additionnel de complexite qui peut contribuer a la regulation genique generale. [Traduit par la Redaction] Mots-cles: epissage alternatif, dyskerine, DKC1, pseudouridine synthase, snoARN., Introduction In the human transcriptome, current analyses pointed out that 92%-94% of human multi-exon genes undergo alternative splicing (AS) (Pan et al. 2006; Wang et al. 2008). Among diverse types [...]

Abstract Background The "PTEN hamartoma tumor syndrome" (PHTS) includes a group of syndromes caused by germline mutations within the tumor suppressor gene "phosphatase and tensin homolog deleted on chromosome ten" (PTEN), characterized by multiple polyps in the gastrointestinal tract and by a highly increased risk of developing malignant tumours in many tissues. The current work clarifies the molecular basis of PHTS in three unrelated Italian patients, and sheds light on molecular pathway disregulation constitutively associated to PTEN alteration. Methods We performed a combination of RT-PCR, PCR, sequencing of the amplified fragments, Real Time PCR and western blot techniques. Results Our data provide the first evidence of β-catenin accumulation in blood cells of patients with hereditary cancer syndrome caused by germ-line PTEN alteration. In addition, for the first time we show, in all PHTS patients analysed, alterations in the expression of TNFα, its receptors and IL-10. Importantly, the isoform of TNFRI that lacks the DEATH domain (TNFRSF1β) was found to be overexpressed. Conclusion In light of our findings, we suggest that the PTEN pathway disregulation could determine, in non-neoplastic cells of PHTS patients, cell survival and pro-inflammatory stimulation, mediated by the expression of molecules such as β-catenin, TNFα and TNFα receptors, which could predispose these patients to the development of multiple cancers.

Abstract Background Many small vertebrates on islands grow larger, mature later, lay smaller clutches/litters, and are less sexually dimorphic and aggressive than their mainland relatives. This set of observations is referred to as the 'Island Syndrome'. The syndrome is linked to high population density on islands. We predicted that when population density is low and/or fluctuating insular vertebrates may evolve correlated trait shifts running opposite to the Island Syndrome, which we collectively refer to as the 'reversed island syndrome' (RIS) hypothesis. On the proximate level, we hypothesized that RIS is caused by increased activity levels in melanocortin receptors. Melanocortins are postranslational products of the proopiomelanocortin gene, which controls pleiotropically pigmentation, aggressiveness, sexual activity, and food intake in vertebrates. Results We tested the RIS hypothesis performing a number of behavioral, genetic, and ontogenetic tests on a blue colored insular variant of the Italian Wall lizard Podarcis sicula, living on a small island off the Southern Italian coast. The population density of this blue-colored variant was generally low and highly fluctuating from one year to the next. In keeping with our predictions, insular lizards were more aggressive and sexually dimorphic than their mainland relatives. Insular males had wide, peramorphic heads. The growth rate of insular females was slower than growth rates of mainland individuals of both sexes, and of insular males. Consequently, size and shape dimorphism are higher on the Island. As predicted, melanocortin receptors were much more active in individuals of the insular population. Insular lizards have a higher food intake rate than mainland individuals, which is consistent with the increased activity of melanocortin receptors. This may be adaptive in an unpredictable environment such as Licosa Island. Insular lizards of both sexes spent less time basking than their mainland relatives. We suspect this is a by-product (spandrel) of the positive selection for increased activity of melanocortins receptors. Conclusions We contend that when population density is either low or fluctuating annually as a result of environmental unpredictability, it may be advantageous to individuals to behave more aggressively, to raise their rate of food intake, and allocate more energy into reproduction.