Proteomic analyses reveal new features of the box H/ACA RNP biogenesis

The conserved H/ACA RNPs consist of one H/ACA RNA and 4 core proteins: dyskerin, NHP2, NOP10, and GAR1. Its assembly requires several assembly factors. A pre-particle containing the nascent RNAs, dyskerin, NOP10, NHP2 and NAF1 is assembled co-transcriptionally. NAF1 is later replaced by GAR1 to form mature RNPs. In this study, we explore the mechanism leading to the assembly of H/ACA RNPs. We performed the analysis of GAR1, NHP2, SHQ1 and NAF1 proteomes by quantitative SILAC proteomic, and analyzed purified complexes containing these proteins by sedimentation on glycerol gradient. We propose the formation of several distinct intermediate complexes during H/ACA RNP assembly, notably the formation of early protein-only complexes containing at least the core proteins dyskerin, NOP10, and NHP2, and the assembly factors SHQ1 and NAF1. We also identified new proteins associated with GAR1, NHP2, SHQ1 and NAF1, which can be important for box H/ACA assembly or function. Moreover, even though GAR1 is regulated by methylations, the nature, localization, and functions of these methylations are not well known. Our MS analysis of purified GAR1 revealed new sites of arginine methylations. Additionally, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs, even though with less efficiency than methylated ones.