RESUMO
Cellular differentiation requires vast remodeling of transcriptomes, and therefore machinery mediating remodeling controls differentiation. Relative to transcriptional mechanisms governing differentiation, post-transcriptional processes are less well understood. As an important post-transcriptional determinant of transcriptomes, the RNA exosome complex (EC) mediates processing and/or degradation of select RNAs. During erythropoiesis, the erythroid transcription factor GATA1 represses EC subunit genes. Depleting EC structural subunits prior to GATA1-mediated repression is deleterious to erythroid progenitor cells. To assess the importance of the EC catalytic subunits Dis3 and Exosc10 in this dynamic process, we asked if these subunits function non-redundantly to control erythropoiesis. Dis3 or Exosc10 depletion in primary murine hematopoietic progenitor cells reduced erythroid progenitors and their progeny, while sparing myeloid cells. Dis3 loss severely compromised erythroid progenitor and erythroblast survival, rendered erythroblasts hypersensitive to apoptosis-inducing stimuli and induced γ-H2AX, indicative of DNA double-stranded breaks. Dis3 loss-of-function phenotypes were more severe than those caused by Exosc10 depletion. We innovated a genetic rescue system to compare human Dis3 with multiple myeloma-associated Dis3 mutants S447R and R750K, and only wild type Dis3 was competent to rescue progenitors. Thus, Dis3 establishes a disease mutation-sensitive, cell type-specific survival mechanism to enable a differentiation program.
Assuntos
Eritropoese , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/metabolismo , Processamento Pós-Transcricional do RNA , Animais , Apoptose , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Eritroblastos/citologia , Eritroblastos/metabolismo , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Exossomos/genética , Fator de Transcrição GATA1/metabolismo , Humanos , Mutação com Perda de Função , Camundongos , Camundongos Endogâmicos C57BL , TranscriptomaRESUMO
Given the complexity of intracellular RNA ensembles and vast phenotypic remodeling intrinsic to cellular differentiation, it is instructive to consider the role of RNA regulatory machinery in controlling differentiation. Dynamic post-transcriptional regulation of protein-coding and non-coding transcripts is vital for establishing and maintaining proteomes that enable or oppose differentiation. By contrast to extensively studied transcriptional mechanisms governing differentiation, many questions remain unanswered regarding the involvement of post-transcriptional mechanisms. Through its catalytic activity to selectively process or degrade RNAs, the RNA exosome complex dictates the levels of RNAs comprising multiple RNA classes, thereby regulating chromatin structure, gene expression and differentiation. Although the RNA exosome would be expected to control diverse biological processes, studies to elucidate its biological functions and how it integrates into, or functions in parallel with, cell type-specific transcriptional mechanisms are in their infancy. Mechanistic analyses have demonstrated that the RNA exosome confers expression of a differentiation regulatory receptor tyrosine kinase, downregulates the telomerase RNA component TERC, confers genomic stability and promotes DNA repair, which have considerable physiological and pathological implications. In this review, we address how a broadly operational RNA regulatory complex interfaces with cell type-specific machinery to control cellular differentiation.
Assuntos
Cromatina/química , Eritrócitos/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Animais , Diferenciação Celular , Cromatina/metabolismo , Cromatina/ultraestrutura , Reparo do DNA , Eritrócitos/ultraestrutura , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/ultraestrutura , Instabilidade Genômica , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/ultraestrutura , Humanos , Camundongos , Especificidade de Órgãos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
By inducing the generation and function of hematopoietic stem and progenitor cells, the master regulator of hematopoiesis GATA-2 controls the production of all blood cell types. Heterozygous GATA2 mutations cause immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. GATA2 disease mutations commonly disrupt amino acid residues that mediate DNA binding or cis-elements within a vital GATA2 intronic enhancer, suggesting a haploinsufficiency mechanism of pathogenesis. Mutations also occur in GATA2 coding regions distinct from the DNA-binding carboxyl-terminal zinc finger (C-finger), including the amino-terminal zinc finger (N-finger), and N-finger function is not established. Whether distinct mutations differentially impact GATA-2 mechanisms is unknown. Here, we demonstrate that N-finger mutations decreased GATA-2 chromatin occupancy and attenuated target gene regulation. We developed a genetic complementation assay to quantify GATA-2 function in myeloid progenitor cells from Gata2 -77 enhancer-mutant mice. GATA-2 complementation increased erythroid and myeloid differentiation. While GATA-2 disease mutants were not competent to induce erythroid differentiation of Lin-Kit+ myeloid progenitors, unexpectedly, they promoted myeloid differentiation and proliferation. As the myelopoiesis-promoting activity of GATA-2 mutants exceeded that of GATA-2, GATA2 disease mutations are not strictly inhibitory. Thus, we propose that the haploinsufficiency paradigm does not fully explain GATA-2-linked pathogenesis, and an amalgamation of qualitative and quantitative defects instigated by GATA2 mutations underlies the complex phenotypes of GATA-2-dependent pathologies.
Assuntos
Fator de Transcrição GATA2/genética , Leucemia Mieloide Aguda/genética , Mutação/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação da Expressão Gênica/genética , Haploinsuficiência/genética , Hematopoese/genética , Humanos , Camundongos , Síndromes Mielodisplásicas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Células-Tronco/metabolismo , Dedos de Zinco/genéticaRESUMO
The RNA-regulatory exosome complex (EC) posttranscriptionally and cotranscriptionally processes and degrades RNAs in a context-dependent manner. Although the EC functions in diverse cell types, its contributions to stem and progenitor cell development are not well understood. Previously, we demonstrated that the transcriptional regulator of erythrocyte development, GATA1, represses EC subunit genes, and the EC maintains erythroid progenitors in vitro. To determine if this mechanism operates in vivo, we used the hematopoietic-specific Vav1-Cre and "conditional by inversion" mouse system to ablate Exosc3, encoding an EC structural subunit. Although Exosc3C/C Cre+ embryos developed normally until embryonic day 14.5, Exosc3 ablation was embryonic lethal and severely reduced erythromyeloid progenitor activity. RNA sequencing analysis of Exosc3-ablated burst-forming unit-erythroid revealed elevated transcripts encoding multiple proapoptotic factors, and the mutant erythroid progenitors exhibited increased apoptosis. We propose that the EC controls an ensemble of apoptosis-regulatory RNAs, thereby promoting erythroid progenitor survival and developmental erythropoiesis in vivo.