Incomplete splicing of neutrophil-specific genes affects neutrophil development in a zebrafish model of poikiloderma with neutropenia

Abstract

Keywords: poikiloderma with neutropenia, RNA disease, splicing, U6 biogenesis, zebrafish Abbreviations: CHT, caudal haematopoietic tissue; CRISPR, clustered regularly interspaced short palindromic repeats; GFP, green fluorescent protein; hpf, hours post fertilization; MO, morpholino antisense oligo; MPN1, mutated in poikiloderma with neutropenia; PN; poikiloderma with neutropenia; snRNPs; small nuclear ribonucleoproteins; sqRT-PCR; semi-quantitative reverse transcription and polymerase chain reaction; USB1, U Six Biogenesis 1Poikiloderma with neutropenia (PN) is a rare inherited disorder characterized by poikiloderma, facial dysmorphism, pachyonychia, short stature and neutropenia. The molecular testing of PN patients has identified mutations in the C16orf57 gene, which encodes a protein referred to as USB1 (U Six Biogenesis 1). In this study, we developed a zebrafish model of PN by the microinjection of morpholino antisense oligos to suppress usb1 gene function. Severe morphological defects, including a bent tail, thin yolk extension and reduced body length, were predominant in the Usb1-suppressed embryos (morphants). We also observed significantly decreased number of neutrophils in the morphants by Sudan Black staining. Interestingly, the splicing of genes involved in neutrophil differentiation and development, such as mpx, ncf1, ela3l and npsn, was aberrant in the morphants. However, the splicing of haematopoietic precursors and erythroid-specific genes was unaltered. Importantly, the neutrophil defects were almost completely rescued by co-injection of ela3l mRNA, the most markedly affected gene in the morphants. Our study demonstrated a possible role of USB1 in modulating the tissue-specific gene splicing that eventually leads to the impaired development of neutrophils. This zebrafish model could serve as a valuable tool to investigate the causative role of USB1 in PN pathogenesis.

Human Mpn1 promotes post‐transcriptional processing and stability of U6atac

“…It is therefore likely that Mpn1 deficiency might compromise pre‐mRNA splicing only in certain tissues or during specific developmental stages. It is also possible that only a restricted fraction of U2 and/or U12 introns might be mispliced in cells lacking Mpn1, a possibility supported by the observation of splicing defects of only some pre‐mRNAs involved in neutrophil differentiation and development, in Mpn1‐depleted zebrafish embryos [15]. Moreover, a large number of human U12 introns are spliced specifically upon stress through stabilization of cellular U6atac, a process that might involve Mpn1 [21].…”

“…Intriguingly, human lymphoblastoid cells from PN patients do not display any obvious defects in canonical pre‐mRNA splicing, suggesting that the impact of U6 misprocessing varies among different organisms or, possibly, cell types [5,7]. Indeed, morpholino‐mediated Mpn1 depletion in zebrafish larvae induced aberrant splicing of a subset of pre‐mRNAs encoding polypeptides required for neutrophil differentiation and development [15]. Injection of spliced mRNA in Mpn1 depleted animals rescued neutrophil developmental defects, indicating that splicing defects of a small subset of transcripts is responsible for tissue‐specific abnormalities [15].…”

“…Indeed, morpholino‐mediated Mpn1 depletion in zebrafish larvae induced aberrant splicing of a subset of pre‐mRNAs encoding polypeptides required for neutrophil differentiation and development [15]. Injection of spliced mRNA in Mpn1 depleted animals rescued neutrophil developmental defects, indicating that splicing defects of a small subset of transcripts is responsible for tissue‐specific abnormalities [15]. Besides U6, no other cellular substrate of Mpn1 has been identified so far, raising the question of whether all defects associated to Mpn1 deficiency arise from inappropriate U6 processing.…”

A zebrafish model of Poikiloderma with Neutropenia recapitulates the human syndrome hallmarks and traces back neutropenia to the myeloid progenitor

“…In line with the substantial evidence that the zebrafish is an excellent system for the study of haematopoiesis during development 23 , our model, together with that recently generated by Patil et al . 24 , demonstrate that the usb1 gene has a key role in normal haematopoiesis and, when dysfunctional, leads to significant reduction of mature mpx -neutrophils, making it a candidate for a leukaemia-causing gene.…”

“…Finally we want to comment our data taking into account the work of Patil et al . 24 who exploited as we did the morpholino knockdown technology for successful modelling Poikiloderma with Neutropenia in zebrafish. Despite differing in the main focus and the prioritised experimental tools, a few merging issues are highlighted by both studies, in particular the impact of usb1 deficit on the neutrophil commitment and differentiation.…”