Eukaryotic rRNA Modification by Yeast 5-Methylcytosine-Methyltransferases and Human Proliferation-Associated Antigen p120

<div><p>Modified nucleotide 5-methylcytosine (m⁵C) is frequently present in various eukaryotic RNAs, including tRNAs, rRNAs and in other non-coding RNAs, as well as in mRNAs. RNA:m⁵C-methyltranferases (MTases) Nop2 from <i>S</i>. <i>cerevisiae</i> and human proliferation-associated nucleolar antigen p120 are both members of a protein family called Nop2/NSUN/NOL1. Protein p120 is well-known as a tumor marker which is over-expressed in various cancer tissues. Using a combination of RNA bisulfite sequencing and HPLC-MS/MS analysis, we demonstrated here that p120 displays an RNA:m⁵C- MTase activity, which restores m⁵C formation at position 2870 in domain V of 25S rRNA in a <i>nop2Δ</i> yeast strain. We also confirm that yeast proteins Nop2p and Rcm1p catalyze the formation of m⁵C in domains V and IV, respectively. In addition, we do not find any evidence of m⁵C residues in yeast 18S rRNA. We also performed functional complementation of Nop2-deficient yeasts by human p120 and studied the importance of different sequence and structural domains of Nop2 and p120 for yeast growth and m⁵C-MTase activity. Chimeric protein formed by Nop2 and p120 fragments revealed the importance of Nop2 N-terminal domain for correct protein localization and its cellular function. We also validated that the presence of Nop2, rather than the m⁵C modification in rRNA itself, is required for pre-rRNA processing. Our results corroborate that Nop2 belongs to the large family of pre-ribosomal proteins and possesses two related functions in pre-rRNA processing: as an essential factor for cleavages and m⁵C:RNA:modification. These results support the notion of quality control during ribosome synthesis by such modification enzymes.</p>