Angrisani, Alberto (2010) Novel Transcripts from the human DKC1 gene. [Tesi di dottorato] (Unpublished)

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Dyskeratosis congenita is a rare genetic disorder that causes a variety of symptoms, including mucocutaneous features, stem cell dysfunction, telomere shortening, ribosomal failure and increased susceptibility to cancer. The disease may have either autosomal dominant/recessive or X-linked inheritance. The X-linked form, that is the most common, is caused by mutations of the DKC1 gene. Loss-of-function mutations of this gene, which encodes the nucleolar protein dyskerin, affect telomere stability, ribosome biogenesis, RNA pseudouridylation and production of small nucleolar RNA-derived micro RNAs. Dyskerin participates in fact to at least two essential ribonucleoprotein complexes: the H/ACA small nucleolarRNP complex, involved in rRNA processing and in the pseudouridylation of cellular RNAs, and the telomerase active complex, involved in telomere stability. A single DKC1 ubiquitary transcript has been described to date. However, the wide spectrum of functions in which the gene has been involved raised the possibility that alternative transcripts with different coding properties might also exist. Since a more extensive analysis of the DKC1 may have shed light on the molecular mechanisms underlying the multiple functions accomplished by this gene and suggest new approaches for the therapy, I performed a detailed analysis of the gene transcriptional activity. Here I show that at least 5 alternative transcripts, with different tissue expression profiles and coding potentials, derive from the DKC1 gene. Intriguingly, these transcripts are differentially tuned during in-vitro differentiation of Caco-2 and SK-N-BE(2) cells and, although they present premature stop codons, all escape the mRNA control mediated by Nonsense Mediated Decay, which usually reduces the accumulation of aberrant transcripts. In my thesis I also report the results obtained after constitutive overexpression in HeLa cells of one of the most interesting newly identified DKC1 mRNAs, called Isoform 7. This isoform encodes a variant protein that is identical to canonical dyskerin but lacks the C-terminal NLS signal and is specifically -and unexpectedly- localized in the cytoplasm. Since both types of dyskerin-participated ribonucleoprotein complexes are strictly localized in the nucleus, the biological role of this variant protein is puzzling, but compatible with the hypothesis that it may be directly involved in the translation process or in transport of the small nucleolar RNA derived micro RNAs from the nucleus to the cytoplasm. Intriguingly, Isoform 7 overexpression in stable transformed cells resulted in alteration of cell morphology, increased cell-cell and cell-matrix adhesion, higher proliferation rate and increased survival after X-ray irradiation. Altogether, the results obtained indicated that DKC1 gene expression is more complex than generally accepted, and that previously undetected alternative splice transcripts may be responsible, at least in part, for the plethora of different manifestations of the X-linked DC.

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