Abstract
Proteomic analyses reveal new features of the box H/ACA RNP biogenesisFlorence Schlotter1, Salim Mérouani1, Valentyne Kogey1, Amani Issa1, Justine Flayac1, Edouard Bertrand2, Stéphane Labialle1, Franck Vandermoere3, Céline Verheggen2 and Séverine Massenet1*1 Université de Lorraine, CNRS, IMoPA, Nancy, France 2 IGH, CNRS, Univ Montpellier, Montpellier, France3 IGF, CNRS, INSERM, Univ Montpellier, Montpellier, France.The conserved box H/ACA RNPs consist of one box H/ACA RNA and 4 core proteins: DKC1, NHP2, NOP10 and GAR1. Most of them catalyze the formation of pseudouridine residues on various RNAs, or participate in the nucleolytic processing of pre-ribosomal RNAs. The assembly of the H/ACA RNPs is a stepwise process that requires several assembly factors: SHQ1, NAF1, and the R2TP complex. SHQ1 interacts with DKC1 in the cytoplasm as soon as it is synthesized. It is released with the help of the R2TP complex in the nucleus. NAF1 interacts with the RNA polymerase II, and allows the co-transcriptional assembly of a pre-particle containing the nascent RNAs, DKC1, NOP10, NHP2, and NAF1. NAF1 needs to be replaced by GAR1 to produce mature and functional H/ACA RNPs in the CBs. Here, we explore the mechanism for assembling box H/ACA RNPs. MS analysis of purified GAR1 revealed new sites of arginine methylations in GAR1. Most of them exist both in mono-methylated and di-methylated forms. We showed that unmethylated GAR1 is correctly incorporated in the RNPs. Moreover, we performed the analysis of GAR1, NHP2, SHQ1, and NAF1 proteomes by quantitative SILAC proteomic, and analyzed the complexes containing these proteins. Based on all the data obtained, we propose the formation of additional assembly intermediates during the biogenesis of box H/ACA RNA. Moreover, our study identifies new proteins associated with GAR1, NHP2, SHQ1, and NAF1, which could be important for box H/ACA assembly or function.