RNA editing and modification are post-transcriptional processes found in all organisms and involved in many biological functions such as splicing, miRNA regulation, control of protein synthesis and mRNA surveillance. More than 100 nucleotide types are modified in rRNA, mRNA and tRNA, suggesting an amazing diversity in the mechanisms of control of RNA function through modification. Defects in RNA modification patters have been shown to cause several diseases in humans. Furthermore, some pathogens are subjected to extensive editing of their mRNA to yield functional protein expression machinery. Despite all this, RNA-editing enzymes are a still unexplored class of drug targets.
During ribosome biogenesis, post-transcriptional modifications of ribonucleotides occur in functionally important regions of pre-rRNA transcripts, such as at intersubunit interfaces, decoding and peptidyltransferase centers. Among the possible modifications, 2’-O-ribose methylation was shown to protect RNA from ribonucleolytic cleavage, stabilize single base pairs, serve as chaperone and impact folding at high temperatures. Defects in the methylation pattern of mRNA have been implied in neurological diseases and scientists suspect the involvement of this modification in many more diseases than currently recognized. For example, RNA methylation has been found to be involved in viral replication and in the host immunologic defense by providing a mechanism to distinguish the viral from the endogenous RNA.
In eukaryotes and archaea, 2’-O-ribose methylation is carried out by the Box C/D small (nucleolar) RNA-protein complex (s(no)RNP). This enzyme is located in the nucleolus and is assembled around snoRNAs containing box C/D and box C’/D’ motives. Archaeal Box C/D sRNPs consist of three core proteins: Fibrillarin, which catalyses the methylation reaction and is equivalent to eukaryotic Nop1; Nop5, which is homologous to eukaryotic Nop56 and Nop58; and L7Ae, which is homologous to eukaryotic Snu13. In addition, the guide sRNA pairs with two different substrate RNAs (10-21 bp) and selects the methylation sites, which are the fifth nucleotides upstream of box D and D’.
In (1) we determined the structure of the 390 kDa Box C/D RNP complex at two stages during catalysis. Despite several years of biochemical investigation and of crystallization experiments, the structure and functional mechanism of this vital cellular enzyme had remained controversial. In our work (1), we followed an interdisciplinary approach consisting of a combination of (i) available high-resolution structures of the complex components, (ii) solution-state NMR data and SANS (Small Angle Neutron Scattering) data (Fig. 1a and b), and (iii) ensemble molecular docking, to solve the structure of the Box C/D enzyme in both its inactive (without substrate RNA) and its active (with substrate RNA) form. A large conformational change is detected upon substrate binding, revealing an unanticipated three-dimensional organization of the catalytic RNP and a mechanism for the regulation of methylation at different rRNA sites. This unexpected regulation mechanism opens a new view on the significance of RNA methylation and suggests that sequentially ordered methylation could serve the purpose of leading the rRNA to productive folding pathways during ribosome biogenesis.
Figure 1. Structures of the apo (a) and holo (b) Box C/D RNP. L7Ae, green; Nop5, gray; Fibrillarin, blue; Box C/D RNA, yellow. A large conformational change occurs upon substrate binding. The complex elongates and the RNA substrates D (firebrick red) and D’ (salmon) occupy different environments. Only two of the four Fibrillarin molecules contact the substrate RNA for methylation. Methylation of the other two substrate RNAs then requires an additional conformational rearrangement that exchanges the position of substrates D and D’. Thus, methylation of substrates D’ and D occurs sequentially.
A failure to supress the transposon in germline cells results in DNA damage leading to defects in gametogenesis and fertility problems. piRNA make up a novel class of 24-32 nucleotide long non coding (nc) RNA molecules that aid in the supression of tranposable elements in the animal germline. The novelty of these molecules is not limited to their length, but also includes their ability to interact with PIWI proteins of the argonaute family in addition to their RNaseIII independent synthesis.
piRNA precursors are synthesised as ssRNA in the nucleus where they are then processed into smaller piRNA fragments, and finally loaded on PIWI proteins. Currently, knowledge of the mechanism behind this process is limited. However, we know that in order to ensure higher levels of piRNA, piRNAs are additionally amplified via a secondary pathway in a ping-pong mechanism. In Drosophila, PIWI protein Aub and AGO3 bring about this secondary biogenesis.
The ping pong cycle occurs in the nuage, which is where the PIWI proteins co-localise. PIWI proteins are known to harbour arginine methylations that Tudor family members recognise, helping in the co-localisation of the PIWI proteins. But these tudor domain containing proteins not only act as a platform for the ping pong cycle, they also actively participate in controlling piRNA levels through regulation of the sense to antisense piRNA ratio in the cell.
This being said, the mechanistic details of the processess still remain elusive. Our lab aims to elucidate the role of the tudor domain containing proteins in the ping-pong cycle by using a variety of biophysical techniques like NMR, SAS and crystallography.
1. Lapinaite, A., Simon, B., Skjaerven, L., Rakwalska-Bange, M., Gabel, F. and Carlomagno, T. (2013) The structure of the box C/D enzyme reveals regulation of RNA methylation. Nature, 502, 519-23.