The RNA-Degrading Exosome Complex Is an Endogenous Suppressor of Erythroid Maturation

Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. We demonstrated that the master regulator of erythropoiesis, GATA-1, induces expression of genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1-mediated transcriptional activation. We conducted studies to assess whether the GATA-1/Foxo3 cooperativity is restricted to the control of autophagy, or if it more broadly impacts the erythroid cell transcriptome. Analysis of the GATA-1/Foxo3-dependent transcriptome in erythroid cells revealed a target gene ensemble extending beyond autophagy, but representing only a small fraction of the complex GATA-1-dependent target gene ensemble. GATA-1/Foxo3 repressed expression of genes encoding two exosome complex components, Exosc5 and Exosc8. The exosome complex functions in one of the major RNA degradation pathways in diverse cell types, mediates splicing and degradation of mRNAs and non-coding RNAs, and functions in epigenetic gene regulation. As the role of the exosome complex in erythropoiesis, and more broadly in hematopoiesis, had not been described previously, we conducted biological and mechanistic studies to determine whether the endogenous exosome complex has important roles in the development and/or function of erythroid cells. Strikingly, downregulating expression of endogenous exosome components, Exosc8, Exosc9 and the catalytic component Dis3 dramatically increased the percentage of primary erythroid precursor cells in the R4 (polychromatic and orthrochromatic orthrochromatic) population from 1% in control cells to 30%, 28% and 16% respectively. We have extended these initial findings to explore key mechanistic and biological questions. Using the exosome complex high-resolution crystal structure as a guide, we are conducting loss-of-function studies to establish whether additional exosome complex components that serve structural roles in the complex (Exosc4 and Exosc7), bind RNA substrates (Exosc1), and degrade RNAs (Dis3L and Exosc10) are also important determinants of erythroid maturation. Initial studies indicate that multiple components suppress maturation, but differ quantitatively in their importance. Studies are underway to test the hypothesis that downregulating Exosc8 or Exosc9 severely disrupts the integrity of the exosome complex, whereas certain other components are less critical for complex integrity and function. We have developed a co-immunoprecipitation assay to measure interactions between endogenous exosome complex components in erythroid cells. This assay is being used to establish the role of the various exosome complex subunits in complex integrity in cultured and primary erythroid cells. Under conditions in which downregulating Exosc8 or Exosc9 induced erythroid maturation, expression of the established regulators of erythropoiesis GATA-1, FOG-1, or KLF1 was unaffected. To test the hypothesis that the exosome complex downregulates a cohort of critical RNAs, including regulatory non-coding RNAs, required for erythroid maturation, we are conducting studies to identify direct exosome complex targets in erythroid cells at distinct stages of maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program. Furthermore, since the exosome complex had not been shown previously to regulate any aspect of hematopoiesis, this work expands the biological repertoire of exosome complex-dependent processes.