MS analysis of GAR1 methylation immunopurified from HEK-293 Cells

ABSTRACT: The conserved box H/ACA RNPs consist of one box H/ACA RNA and 4 core proteins: Dyskerin, NHP2, NOP10 and GAR1. The assembly of the H/ACA RNPs is a stepwise process which requires several assembly factors: SHQ1, NAF1, the Survival of Motor Neuron complex SMN, and the R2TP complex. It implicates the co-transcriptional assembly of a pre-particle containing the nascent RNAs, Dyskerin, NOP10, NHP2 and NAF1. The latest keeps the H/ACA RNP inactive, and needs to be replaced by GAR1 to produce mature and functional H/ACA RNPs in the Cajal bodies. In this study, we explore in details the molecular mechanism leading to the assembly of mature box H/ACA RNPs. We performed extensive analysis of GAR1, NHP21 and NAF interactomes by quantitative stable isotope labeling by amino acids in cell culture proteomic analyses (SILAC), and revealed new assembly intermediates: 1) an early protein-only pre-H/ACA RNP complex containing the core proteins DKC1, NOP10 and NHP2, and the assembly factors SHQ1 and NAF1, and 2) a late assembly intermediates containing the RNA, the core proteins and NAF1. We showed that the SMN complex is associated with late forming H/ACA pre-RNP complexes containing GAR1. Moreover, our study identifies new proteins highly and specifically associated with GAR1, NHP2 and NAF1, reflecting their importance in box H/ACA assembly or function, such as the PRMT1 and PRMT5 arginine methylases. MS analysis of purified GAR1 revealed new sites of arginine methylations in the two GAR domains of GAR1, and showed that most of these methylated arginines exist both in mono-methylated and di-methylated forms in cells. By different methods, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs. Therefore, GAR1 methylation should be important for its function.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293t Cell

SUBMITTER: Franck Vandermoere  

LAB HEAD: Severine Massenet

PROVIDER: PXD026752 | Pride | 2023-05-10

REPOSITORIES: Pride