1 2 3 4 5 6 Exosome Complex Orchestrates Developmental Signaling to Balance 7 Proliferation and Differentiation During Erythropoiesis 8 9 10 Skye C. McIver1, Koichi R Katsumura1, Elsa Davids1, Peng Liu2, Yoon-A Kang1, 11 David Yang3 and Emery H. Bresnick1 12 13 14 1Department of Cell and Regenerative Biology, UW-Madison Blood Research Program, 15 Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, 16 Madison, WI, 53705, USA; 2Department of Biostatistics and Medical Informatics, Carbone 17 Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, 18 WI, 53705, USA; 3Department of Pathology, University of Wisconsin School of Medicine 19 and Public Health, Madison, WI, 53705, USA 20 21 22 23 24 25 26 To whom correspondence should be addressed: Emery H. Bresnick, 1111 Highland Ave, 27 4009 WIMR, Madison, WI, 53705; email: [email protected]; TEL: 608-265-6446 28 SUMMARY 29 Since the highly conserved exosome complex mediates the degradation and 30 processing of multiple classes of RNAs, it almost certainly controls diverse biological 31 processes. How this post-transcriptional RNA-regulatory machine impacts cell fate 32 decisions and differentiation is poorly understood. Previously, we demonstrated that 33 exosome complex subunits confer an erythroid maturation barricade, and the erythroid 34 transcription factor GATA-1 dismantles the barricade by transcriptionally repressing the 35 cognate genes. While dissecting requirements for the maturation barricade in Mus 36 musculus, we discovered that the exosome complex is a vital determinant of a 37 developmental signaling transition that dictates proliferation and amplification versus 38 differentiation. Exosome complex integrity in erythroid precursor cells ensures Kit receptor 39 tyrosine kinase expression and stem cell factor/Kit signaling, while preventing 40 responsiveness to erythropoietin-instigated signals that promote differentiation. 41 Functioning as a gatekeeper of this developmental signaling transition, the exosome 42 complex controls the massive production of erythroid cells that ensures organismal 43 survival in homeostatic and stress contexts. 44 45 46 47 48 49 50 51 2 52 INTRODUCTION 53 The highly conserved exosome complex, an RNA-degrading and processing 54 machine, is expressed in all eukaryotic cells (Januszyk and Lima, 2014; Kilchert et al., 55 2016). The first subunit was discovered from an analysis of mechanisms controlling yeast 56 rRNA synthesis. Mutations of Exosc2 (Rrp4) disrupt 5.8S rRNA 3’-end processing (Mitchell 57 et al., 1996). Exosc2 assembles into a complex with components homologous to bacterial 58 3’ to 5’ exoribonuclease (PNPase) (Mitchell et al., 1997). Nine exosome complex subunits 59 form a cylindrical core consisting of the RNA binding subunits Exosc1 (Csl4), Exosc2 60 (Rrp4) and Exosc3 (Rrp40), which cap a ring formed by Exosc4 (Rrp41), Exosc5 (Rrp46), 61 Exosc6 (Mtr3), Exosc7 (Rrp42), Exosc8 (Rrp43) and Exosc9 (Rrp45) (Liu et al., 2006; 62 Makino et al., 2013; Makino et al., 2015; Wasmuth et al., 2014) (Figure 1A). Despite 63 homology with bacterial PNPases, the vertebrate core subunits lack RNA-degrading 64 activity (Liu et al., 2006). Whereas Dis3 (Rrp44) (nuclear) and Dis3L (cytoplasmic) catalytic 65 subunits bind the same position of the core complex, adjacent to Exosc4 and Exosc7, the 66 predominantly nuclear catalytic subunit Exosc10 (Rrp6) binds the opposite site 67 (Dziembowski et al., 2007; Makino et al., 2015) (Figure 1A). The catalytic subunits, which 68 may function redundantly in certain contexts, mediate RNA degradation and/or processing 69 (Kilchert et al., 2016). Unlike Dis3L and Exosc10, which are strictly exoribonucleases, Dis3 70 is also an endoribonuclease (Lebreton et al., 2008; Tomecki and Dziembowski, 2010). The 71 core subunits, except Exosc1, are considered to confer structural integrity (Liu et al., 2006). 72 The apparent diversity of exosome complex-regulated RNAs (Schneider et al., 73 2012) suggests the complex controls a plethora of cellular processes. Exosome complex 74 subunits regulate cell differentiation and are implicated in human pathologies. Exosc8, 75 Exosc9 and Dis3 suppress erythroid maturation of primary murine erythroid precursor cells 3 76 (McIver et al., 2014). EXOSC7, EXOSC9 and EXOSC10 maintain human epidermal 77 progenitor function (Mistry et al., 2012). In principle, the exosome complex might control 78 differentiation through complex RNA remodeling mechanisms or by targeting factors 79 mediating the balance between self-renewal and lineage commitment, 80 proliferation/amplification or terminal differentiation. DIS3 has been implicated as a tumor 81 suppressor mutated in multiple myeloma (Chapman et al., 2011) and is overexpressed in 82 colorectal cancer (de Groen et al., 2014). EXOSC8, EXOSC3 or EXOSC2 mutations cause 83 syndromes with complex phenotypes including neurological defects, cerebellar hypoplasia, 84 retinitis pigmentosum, progressive hearing loss and premature aging (Boczonadi et al., 85 2014; Di Donato et al., 2016; Wan et al., 2012). 86 As downregulating exosome complex subunits can yield compelling phenotypes, 87 and exosome complex subunit mutations yield pathologies, it is instructive to consider 88 whether the phenotypes reflect complex disruption or subunit-specific activities. By 89 conferring exosome complex integrity, all exosome complex activities might require 90 structural subunits, or sub-complexes might have distinct functions (Kiss and Andrulis, 91 2011). The structural subunit requirement for complex stability in vitro (Liu et al., 2006), 92 and lethality due to loss of structural components in yeast (Allmang et al., 1999a; Allmang 93 et al., 1999b) support the importance of the intact complex. However, core subunit 94 downregulation revealed little overlap in the ensembles of regulated RNAs in Drosophila 95 (Kiss and Andrulis, 2010) and differentially influenced RNA processing in humans 96 (Tomecki et al., 2010). Moreover, Arabidopsis Exosc1, Exosc2 and Exosc4 knockouts 97 yielded distinct phenotypes (Chekanova et al., 2007). 98 While investigating these models in the context of erythroid maturation, we 99 discovered that Exosc8 or Exosc9 downregulation disrupted protein/protein interactions 4 100 within the complex and greatly decreased expression of the receptor tyrosine kinase Kit. 101 Loss of Stem Cell Factor (SCF)-induced Kit signaling occurred concomitant with 102 precocious acquisition of erythropoietin signaling, which drives erythroid maturation. As Kit 103 stimulates erythroid precursor cell proliferation, our results establish a paradigm in which 104 the exosome complex regulates a receptor tyrosine kinase to orchestrate a vital 105 developmental signaling transition dictating proliferation/amplification versus differentiation. 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 5 124 RESULTS 125 Dismantling protein-protein interactions within the exosome complex 126 Previously, we demonstrated that downregulating exosome complex subunits 127 (Exosc8, Exosc9 or Dis3) in murine fetal liver erythroid precursor cells induced erythroid 128 maturation (McIver et al., 2014). Analogous to Exosc8 and Exosc9 (McIver et al., 2014), 129 experiments in which Exosc3 expression is impaired suggest that this protein also 130 suppresses maturation of primary murine fetal liver lineage-negative hematopoietic 131 precursor cells. In particular, Exosc3 downregulation using two distinct shRNAs increased 132 the R4 (late basophilic/orthochromatic erythroblasts) cell population 9 fold (p = 0.006 and p 133 = 0.01 for the two shRNAs, respectively) (Figure 1 - figure supplement 1). However, it 134 remains unclear whether the single subunit perturbations impact exosome complex 135 integrity. To address this, we developed a co-immunoprecipitation assay to test whether 136 individual components mediate complex integrity (Figure 1A). Using the X-ray crystal 137 structure of the exosome complex as a guide (Liu et al., 2006), the strategy involved 138 testing whether downregulating endogenous Exosc8 or Exosc9 alter interactions between 139 endogenous Exosc2 and Exosc3, subunits that do not interact directly in the complex 140 (Figure 1A). As Exosc2 and Exosc3 are only expected to co-immunoprecipitate when 141 residing in the complex or a sub-complex, the extent of co-immunoprecipitation constitutes 142 a metric of complex integrity. 143 We conducted the protein/protein interaction analysis in G1E cells stably expressing 144 a conditionally active GATA-1 allele (G1E-ER-GATA-1), which mimic a normal erythroid 145 precursor cell, the proerythroblast (Weiss et al., 1997). Estradiol activation of ER-GATA-1 146 induces an erythroid transcriptional program and recapitulates a physiological window of 147 erythroid maturation (Welch et al., 2004). G1E-ER-GATA-1 cells were infected with 6 148 retroviruses expressing control (luciferase) shRNA or shRNAs targeting Exosc8 or Exosc9. 149 Whole cell lysates prepared 48 hours post-infection were immunoprecipitated with anti- 150 Exosc3 or isotype-matched control antibody, and Western blotting was conducted with 151 anti-Exosc2 antibody. Whereas knocking down Exosc8 or Exosc9 by ~75% (Figure 1B) did 152 not alter Exosc2 levels in the input, the knockdowns reduced the amount of Exosc2 153 recovered with the anti-Exosc3 antibody (Figure 1C). Densitometric analysis indicated that 154 Exosc8 and Exosc9 knockdowns reduced the amount of Exosc2 co-immunoprecipitated 155