Tackling rRNA Methylation: Molecular Bases and Ribosome Translational Impact – MetRibo

Ribosome assembly and regulation of translation activity is crucial for cell growth and response to external cues or environmental conditions in all eukaryotic organisms.

Ribosomal RNA (rRNA) are the linchpins of ribosome assembly and function. The rRNAs 25S, 5.8S, 5S and 18S form, respectively, the large (LSU) and the small (SSU) subunits of the ribosome. The LSU acts as a ribozyme, catalyzing peptide bond formation, while SSU harbors the decoding center which monitors the complementarity of tRNA anticodon with mRNA codon during translation. In all eukaryotic cells, rRNA are subjects of two major types of nucleotide modifications: methylation of sugars (2’-O-ribose) and conversion of uridine to pseudouridine. Over 90% of the rRNA methylations are 2’-O-ribose methylations which are guided by antisense small nucleolar RNA (snoRNA) referred as C/D-box snoRNA. The C/D-box snoRNA associates to four nucleolar proteins called the C/D core proteins (Nop56p/p62, Nop58p, snu13/15.5K and the methyltransferase Nop1p/Fibrillarin) to form the C/D-box snoRNP.

The extent and positions of rRNA 2’-O-Me fluctuate between species, however the majority of modifications occurs in functionally conserved regions. Remarkably, modulation of rRNA 2'-O-methylation affects ribosome translation fidelity and is linked to human diseases. In plants, only limited information is available on rRNA 2'-O-methylation profile, mechanism of modification and role(s) of modified nucleotides in ribosome activity.

In the plant model A. thaliana, ~100 C/D-box snoRNA have been identified by computational screening of genomic sequences. Later, analysis of small RNA-seq data and targeted sequencing of nucleolar RNAs expanded the list of known C/D-box snoRNA to ~200 species. Notably, studies in Arabidopsis demonstrated that the knockout of a single C/D-box snoRNA triggers strong developmental and growth defects, and gene disruption of NUFIP, a C/D-box snoRNP assembly factor, also leads to severe developmental phenotypes and inhibited 2’-O-methylation at specific rRNA sites.

We have recently performed RiboMethSeq analysis of Arabidopsis thaliana rRNA and identified both conserved and plant-specific rRNA 2’-O-Me sites. Remarkably; our data suggest that some rRNA 2’-O-Me sites do not have matching snoRNAs, suggesting 2’-O-Me through a non-canonical snoRNA and/or another guiding mechanism. Furthermore, we also observed alterations of rRNA 2’-O-Me level in both conserved and plant-specific rRNA positions in response to specific developmental conditions. Furthermore, RNA-seq analysis of Arabidopsis C/D-box snoRNPs identified a subset of non-coding RNA, other than known C/D-box snoRNA, that might be implicated in 2’-O-methylation of rRNA and /or other RNAs.

In this project, we will perform deep investigation of the mechanisms controlling 2’-O-methylation of rRNA and determine connections with other rRNA modifications and translation activity of plant ribosomes. We will determine how 2’-O-ribose methylation of rRNA contributes to the production of heterogeneous ‘specialized’ ribosomes in plants and impact plant resistance and/or adaptation to heat stress conditions.
The MetRibo project is intended to contribute substantially into the intense efforts currently devoted to decipher how ribosome modifications contributes to regulatory pathways and how is tuned during adaptive responses of multicellular organisms to external cues, a field to which plants have much to offer.