RESUMO
Heterozygous de novo missense variants of SRP54 were recently identified in patients with congenital neutropenia (CN) who display symptoms that overlap with Shwachman-Diamond syndrome (SDS). Here, we investigate srp54 knockout zebrafish as the first in vivo model of SRP54 deficiency. srp54-/- zebrafish experience embryonic lethality and display multisystemic developmental defects along with severe neutropenia. In contrast, srp54+/- zebrafish are viable, fertile, and show only mild neutropenia. Interestingly, injection of human SRP54 messenger RNAs (mRNAs) that carry mutations observed in patients (T115A, T117Δ, and G226E) aggravated neutropenia and induced pancreatic defects in srp54+/- fish, mimicking the corresponding human clinical phenotypes. These data suggest that the various phenotypes observed in patients may be a result of mutation-specific dominant-negative effects on the functionality of the residual wild-type SRP54 protein. Overexpression of mutated SRP54 also consistently induced neutropenia in wild-type fish and impaired the granulocytic maturation of human promyelocytic HL-60 cells and healthy cord blood-derived CD34+ hematopoietic stem and progenitor cells. Mechanistically, srp54-mutant fish and human cells show impaired unconventional splicing of the transcription factor X-box binding protein 1 (Xbp1). Moreover, xbp1 morphants recapitulate phenotypes observed in srp54 deficiency and, importantly, injection of spliced, but not unspliced, xbp1 mRNA rescues neutropenia in srp54+/- zebrafish. Together, these data indicate that SRP54 is critical for the development of various tissues, with neutrophils reacting most sensitively to the loss of SRP54. The heterogenic phenotypes observed in patients that range from mild CN to SDS-like disease may be the result of different dominant-negative effects of mutated SRP54 proteins on downstream XBP1 splicing, which represents a potential therapeutic target.
Assuntos
Síndrome Congênita de Insuficiência da Medula Óssea/genética , Neutropenia/congênito , Partícula de Reconhecimento de Sinal/genética , Proteína 1 de Ligação a X-Box/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Modelos Animais de Doenças , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Células HL-60 , Humanos , Modelos Moleculares , Mutação , Neutropenia/genética , Splicing de RNA , RNA Mensageiro/genéticaRESUMO
Stemness and pluripotency are mediated by transcriptional master regulators that promote self-renewal and repress cell differentiation, among which is the high-mobility group (HMG) box transcription factor SOX2. Dysregulated SOX2 expression, by contrast, leads to transcriptional aberrations relevant to oncogenic transformation, cancer progression, metastasis, therapy resistance, and relapse. Here, we report a post-transcriptional mechanism by which the cytosolic pool of SOX2 contributes to these events in an unsuspected manner. Specifically, a low-complexity region within SOX2's C-terminal segment connects to the ribosome to modulate the expression of cognate downstream factors. Independent of nuclear structures or DNA, this C-terminal functionality alone changes metabolic properties and induces non-adhesive growth when expressed in the cytosol of SOX2 knockout cells. We thus propose a revised model of SOX2 action where nuclear and cytosolic fractions cooperate to impose cell fate decisions via both transcriptional and translational mechanisms.