Stéphane Labialle

The Prader–Willi/Angelman syndrome (PWS/AS) locus is regulated by the epigenetic mechanism of parental genomic imprinting. This region holds two eutherian-specific, large tandem repeats of box C/D small nucleolar RNA (Snord) genes called SNORD115 and SNORD116, whose loss of paternal expression is key in the development of the PWS. Snords represent an ancient class of noncoding RNAs that typically direct the 2′-O-methylation of specific nucleotides of ribosomal RNAs. However, Snord115 and Snord116 belong to the large class of orphan Snords whose functions remain unclear. The constraints that generated and maintained their unusual genetic organization for mammalian genomes have been poorly addressed to date. Here, a comparative analysis of the evolutionary history of both tandem repeats reveals that several genetic events affected them concomitantly, including copy gains and losses between species, emergence of gene subfamilies in catarrhines or partial tandem duplication in rats. Several indications suggest that parental genomic imprinting orchestrated this coordination of events, adding a new effect on mammalian genome structure and evolution to its roles in gene dosage, meiotic recombination and replication timing. Finally, our work provides a functional rationale for the existence of closely located tandem repeats of small RNA genes in mammalian genomes.

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.