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Intron retention: a human DKC1 gene common splicing event.
No full textIdentification 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 (hDKC1), 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
A new role for human dyskerin in vesicular trafficking
No full textDyskerin is an essential, conserved, multifunctional protein found in the nucleolus, whose loss of function causes the rare genetic diseases X-linked dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome. To further investigate the wide range of dyskerin's biological roles, we set up stable cell lines able to trigger inducible protein knockdown and allow a detailed analysis of the cascade of events occurring within a short time frame. We report that dyskerin depletion quickly induces cytoskeleton remodeling and significant alterations in endocytic Ras-related protein Rab-5A/Rab11 trafficking. These effects arise in different cell lines well before the onset of telomere shortening, which is widely considered the main cause of dyskerin-related diseases. Given that vesicular trafficking affects many homeostatic and differentiative processes, these findings add novel insights into the molecular mechanisms underlining the pleiotropic manifestation of the dyskerin loss-of-function phenotype
Impiego di un bioreattore ad alta pressione per lo studio di fattori di trascrizione implicati nella meccanotrasduzione di cellule cartilaginee
No full textLAUREA MAGISTRAL
Molecular mechanisms underlying the functional multiplicity of human DKC1 gene
Full text linkHypomorphic mutations of the DKC1 gene cause the Xlinked
dyskeratosis congenita, an inherited multisystemic disorder characterized by a variety of symptoms, including mucocutaneous features, stem cell dysfunction, bone marrow failure, and increased susceptibility to cancer. DKC1 encodes an evolutionarily conserved protein, called dyskerin, whose activity is essential for cellular vitality. Dyskerin participates in at least two functional ribonucleoprotein (RNP) complexes that play essential roles in
the cell. The protein is in fact an essential component of H/ACA snoRNPs,
which are involved in the processing and pseudouridylation of rRNAs. In
addition, through its binding to the telomerase RNA component (TERC), that
contains an H/ACA RNA motif, dyskerin participates in the active complex of
telomerase, ensuring telomeres stability and maintenance.
In order to investigate and characterize in more detail the effects triggered by
DKC1 loss of function on human cells, we generated a stable cellular model
able to reduce gene expression, upon inducible RNA interference, without
affecting the expression of the alternatively spliced cytoplasmic isofom 3.
I focussed my analyses on the effects triggered by dyskerin depletion well
before telomere erosion, in order to define the spectrum of telomere independent outcomes. Intriguingly, I found that dyskerin depletion affected
cell adhesion. In fact, dyskerin dowregulation perturbed both cell-cell and cell substrate adhesion, causing reduced expression of some proteins involved in the tight and adherens junctions, and in the focal adhesions. Moreover, coimmunoprecipitations analyses revealed interactions between dyskerin and some component of focal adhesions, while confocal microscopy indicates that this interaction can occur at level of the plasma membrane. Finally, I observed that dyskerin depletion alter cell morphology and increases cell motility.
Altogether, these results could be of valuable help to understanding the
puzzling relationship that links pseudouridine synthases loss-of-function to
cancer predisposition
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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