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

Thomas Preiss,Gabrielle Bourgeois,Matthias Schaefer,Mark Helm,Michel Ney,Imre Gaspar,Christelle Aigueperse,Stefanie Kellner,Yuri Motorin

doi:10.1371/journal.pone.0133321

Abstract

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 S. cerevisiae 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 nop2Δ 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.

Highlights

Post-transcriptional RNA modification is an integral part of global RNA maturation in all cell types
The human proliferation-associated nucleolar antigen p120 belongs to a large family of putative RNA:m5C-MTases and its MTase domain displays approximately 60% sequence identity with Nop2 at the amino acid sequence level
The slow growth phenotype observed for haploid yeast strains expressing human p120 was not related to the potential toxicity of the heterologous protein, as normal growth was restored upon co-expression of WT Nop2

Summary

Introduction

Post-transcriptional RNA modification is an integral part of global RNA maturation in all cell types. During this step, single enzymes or enzymatic systems form numerous chemically distinct modified residues. The great majority of RNA modifications are methylations, consisting of the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to various positions in the base or 2'-OH of ribose [1]. Among these modifications, methylation of cytosine at position 5 is one of the most widespread. There is no evidence for the presence of m5C modifications in human or yeast 5S and 18S rRNA [11]

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Results

Conclusion