A single N¹- methyladenosine on the large ribosomal subunit rRNA impacts locally its structure and the translation of key metabolic enzymes

Abstract

The entire chemical modification repertoire of yeast ribosomal RNAs and the enzymes responsible for it have recently been identified. Nonetheless, in most cases the precise roles played by these chemical modifications in ribosome structure, function and regulation remain totally unclear. Previously, we demonstrated that yeast Rrp8 methylates m1A645 of 25S rRNA in yeast. Here, using mung bean nuclease protection assays in combination with quantitative RP-HPLC and primer extension, we report that 25S/28S rRNA of S. pombe, C. albicans and humans also contain a single m1A methylation in the helix 25.1. We characterized nucleomethylin (NML) as a human homolog of yeast Rrp8 and demonstrate that NML catalyzes the m1A1322 methylation of 28S rRNA in humans. Our in vivo structural probing of 25S rRNA, using both DMS and SHAPE, revealed that the loss of the Rrp8-catalyzed m1A modification alters the conformation of domain I of yeast 25S rRNA causing translation initiation defects detectable as halfmers formation, likely because of incompetent loading of 60S on the 43S-preinitiation complex. Quantitative proteomic analysis of the yeast Δrrp8 mutant strain using 2D-DIGE, revealed that loss of m1A645 impacts production of specific set of proteins involved in carbohydrate metabolism, translation and ribosome synthesis. In mouse, NML has been characterized as a metabolic disease-associated gene linked to obesity. Our findings in yeast also point to a role of Rrp8 in primary metabolism. In conclusion, the m1A modification is crucial for maintaining an optimal 60S conformation, which in turn is important for regulating the production of key metabolic enzymes.

Programmable System of Cas13-Mediated RNA Modification and Its Biological and Biomedical Applications

“…TRMT10C-SDR5C1 also introduces m 1 A, in which TRMT10C requires SDR5C1 to be active as a methyltransferase ( Vilardo et al, 2012 ). Rrp8 (NML1 in humans) and BMT2 regulate the formation of m 1 A in rRNA, thereby controlling ribosome assembly ( Sharma et al, 2013 , 2018 ; Waku et al, 2016 ). Many demethylases of m 1 A have been characterized successively.…”

Variations in transfer and ribosomal RNA epitranscriptomic status can adapt eukaryote translation to changing physiological and environmental conditions

“…Surprisingly, despite having a reduced translation initiation efficiency, most proteins are produced in similar amounts in wt and rrp8 mutant lines. Exceptions to this rule concern several enzymes involved in carbohydrate metabolism that are either up or down regulated in rrp8 mutant compared to wt, suggesting that ribosomes lacking m 1 A 645 translate corresponding mRNAs more or less efficiently [169]. This suggests that, under some growth conditions, ribosomes lacking m 1 A 645 could be synthesized to specifically regulate the translation of mRNAs involved in producing key carbohydrate metabolism enzymes.…”

“…Another key rRNA modifying enzyme is the methylase RRP8 that generates m 1 A in position 645 of yeast 25S rRNA [169]. The loss of m 1 A 645 results in the production of ribosomes altered in their general ability to initiate translation, possibly linked to a reduced competence for the 60S subunit lacking m 1 A 645 to bind to the 40S subunit [169]. Surprisingly, despite having a reduced translation initiation efficiency, most proteins are produced in similar amounts in wt and rrp8 mutant lines.…”

The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

“…Both of these enzymes are non-essential and, like Tsr3, do not demonstrate any phenotype upon their deletion (at 30 ºC). In contrast, non-functional mutants of Rrp8 and Bmt2 both lead to the accumulation of half-mer ribosomes, indicative of defects in 60S maturation (Peifer et al, 2012;Sharma et al, 2018). Like Tsr3, Rrp8 functions after snoRNA mediated rRNA modification at A649/650.…”