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.

Emerging Role of Eukaryote Ribosomes in Translational Control

“…In yeast abolition of a single base modification, i.e., m1A645 located on the LSU, leads to a change in the quantity of a subset of 18 proteins [ 127 ]. Comparison of the protein abundance with mRNA levels of each protein candidate showed that mRNA content did not correlate with protein content for some of the candidates.…”

Ontogenic mRNA expression of RNA modification writers, erasers, and readers in mouse liver

“…The m 1 A modification in tRNAs was important for tRNA folding, stability, and tRNA-protein interaction [49–52]. The m 1 A modification was also observed at several sites in rRNAs, which affect the tertiary structure of the ribosome and downstream gene translation [53, 54]. In contrast to non-coding RNAs, m 1 A modification has a relatively low abundance in mRNAs, with a potential function in regulating translation process [47].…”

RNA Metabolism Guided by RNA Modifications: The Role of SMUG1 in rRNA Quality Control

“…m1A is found in the large subunit of the ribosome at position A1322 in 28S rRNA, and it is introduced by nucleomethylin (NML). It affects the local structure of the ribosome, promoting proper conformation of the 60S and ultimately translation [ 71 , 72 ]. Two different methods for the detection of m1A have been described and they rely on the ability of m1A to stall transcription [ 71 , 73 , 74 , 75 ].…”