Circular Box C/D RNA in P. abyssi : An atypical circularization mechanism mediated by Pab1020 RNA ligase and their role in rRNA Methylation. - CBI Toulouse - Salle De Conférence Nicole Le Douarin (4R4)

Circular RNAs (circRNAs) result from the covalent ligation of the 5' and 3' free ends of linear RNA, making them more resistant to exonucleases and therefore more stable. CircRNAs are conserved across all three domains of life with a specific mechanism of formation in archaea involving the RNA ligase family (Rnl3). We identified Pab1020 an RNA ligase belonging to the Rnl3 family, as a homodimeric ATP-dependent RNA ligase responsible for RNA circularization in the hyperthermophilic archaeon Pyrococcus abyssi. RIP-seq experiments revealed 133 individual circRNA loci in P. abyssi, among which circular C/D Box RNAs are particularly abundant. Box C/D RNAs are guide RNAs involved in the 2’-O-methylation of ribosomal RNA, but how their circular form modulates their own biological function remains unknown. using molecular biology, biochemical and structural biology approaches, we have investigated i) the atypical mechanism underlying the intramolecular ligation of singlestranded RNA molecules and ii) the biological role of circular RNA in archaeal cells. Using catalytic and structural variants of Pab1020, we showed that both the homodimeric state and the C-terminal domain are essential for RNA binding and circularization. The cryo-EM structure of Pab1020 bound to a box C/D RNA reveals an RNA-induced rearrangement of the C-terminal domain and an asymmetric mode of substrate recognition (Col. C. Madru & E. Schmitt, Ecole Polytechnique). We also sought to determine the cellular context of Pab1020-dependent RNA circularization by identifying its protein partners and cellular localization. Pull-down and mass spectrometry analyses revealed an association of Pab1020 with the RNA degradation and maturation machinery, including RNase W and the RNA exosome. Immunofluorescence analysis further showed that Pab1020 and its interacting proteins are enriched at the cell periphery. We have also began to elucidate the functional importance of box C/D RNA circularization in rRNA 2′- O-methylation in P. abyssi. First, we reconstitute the archaeal RNP box C/D methylation complex in vitro, to monitor rRNA nucleotide methylation activity in the presence of either linear or circular box C/D RNA. Second, we initiated the mapping 2′-O-methylated sites of 16S and 23S rRNA from P. abyssi using RiboMethSeq (Epitranscriptomics platform, Y. Motorin, Nancy). Our results identified modified nucleotides consistent with bioinformatic predictions and structural data, including sites guided by circular box C/D RNAs. Together, our work links the atypical homodimeric architecture of Pab1020 to its circularization mechanism, and provides a framework for determining the functional importance of circular box C/D RNAs in rRNA modification in hyperthermophilic archaea.