Abstract
Poikiloderma with Neutropenia (PN) is an autosomal recessive genodermatosis characterized by early-onset poikiloderma, pachyonychia, hyperkeratosis, bone anomalies and neutropenia, predisposing to myelodysplasia. The causative C16orf57/USB1 gene encodes a conserved phosphodiesterase that regulates the stability of spliceosomal U6-RNA. The involvement of USB1 in splicing has not yet allowed to unveil the pathogenesis of PN and how the gene defects impact on skin and bone tissues besides than on the haematological compartment. We established a zebrafish model of PN using a morpholino-knockdown approach with two different splicing morpholinos. Both usb1-depleted embryos displayed developmental abnormalities recapitulating the signs of the human syndrome. Besides the pigmentation and osteochondral defects, usb1-knockdown caused defects in circulation, manifested by a reduced number of circulating cells. The overall morphant phenotype was also obtained by co-injecting sub-phenotypic dosages of the two morpholinos and could be rescued by human USB1 RNA. Integrated in situ and real-time expression analyses of stage-specific markers highlighted defects of primitive haematopoiesis and traced back the dramatic reduction in neutrophil myeloperoxidase to the myeloid progenitors showing down-regulated pu.1 expression. Our vertebrate model of PN demonstrates the intrinsic requirement of usb1 in haematopoiesis and highlights PN as a disorder of myeloid progenitors associated with bone marrow dysfunction.
Highlights
The introduction of the genetic test to validate diagnosis and provide patients with the appropriate oncological surveillance[7,8,9,10,11]
It has been demonstrated that in Saccharomyces cerevisiae and human cells the USB1 gene and its yeast ortholog encode a phosphodiesterase that is essential for the biogenesis of the RNA splicing apparatus as it regulates the stability of U6 small nuclear RNA16,17
X-ray crystallography of the human USB1 protein has definitely confirmed that USB1 is an RNase that trims the 3′ end of the U6 transcript implicating aberrant oligoadenylation of U6 small nuclear RNA (snRNA) in the pathogenesis of PN18
Summary
The introduction of the genetic test to validate diagnosis and provide patients with the appropriate oncological surveillance[7,8,9,10,11]. X-ray crystallography of the human USB1 protein has definitely confirmed that USB1 is an RNase that trims the 3′ end of the U6 transcript implicating aberrant oligoadenylation of U6 snRNA in the pathogenesis of PN18. While these insights advance our understanding of the PN pathomechanism, how USB1 mutations impact on the morphogenesis and differentiation of tissues affected in PN remains unknown. To address this issue, we have exploited the zebrafish as a model organism. Using a morpholino (MO) knockdown approach, we show that the morphants display developmental abnormalities that reproduce the signs of the human syndrome, namely defective pigmentation, osteochondral alterations and severe haematopoietic defects, mainly affecting the myeloid lineage
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