Project Details
Projekt
Functional and Structural Analysis of Eukaryotic snoRNP Complexes Catalysing rRNA Ribose Methylation
Subject Area
Structural Biology
Biochemistry
Cell Biology
Biochemistry
Cell Biology
Term
from 2015 to 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 277251038
During the biosynthesis and processing of the pre-rRNA transcripts post-transcriptional modifications of ribonucleotides occur in functional regions, including intersubunit interfaces, decoding and peptidyltransferase centers. Among the possible modifications, 2(prime)-O-ribose methylation was shown to protect RNA from ribonucleolytic cleavage, stabilize single base pairs, serve as chaperone and impact folding at high temperatures. rRNA methylation is essential for both pre-rRNA processing and ribosome assembly, with complete suppression of methylation leading to cell death. In eukaryotes, ribose methylation is carried out by the Box C/D small nucleolar RNA-protein complex (snoRNP), where the guide snoRNAs target the modification machinery to the sites of modification, due to their complementarity to the RNA sequences to be modified. In addition to rRNA, snoRNAs can guide ribose-methylation of a few mRNAs with intriguing consequences. Failure to methylate the ribose of a specific adenosine residue within the serotonin 2C receptor mRNA has been recently linked to human neurogenetic Prader-Willi syndrome (PWS). This suggests that RNA editing, and in particular ribose methylation, might be involved in many more diseases than currently recognized. In addition, RNA methylation has been found to play a role in viral replication and in the host immunologic defense, by providing a mechanism to distinguish the viral from the endogenous RNA. The enzymology, the particular function of rRNA modifications, and the molecular structure of eukaryotic snoRNP complexes remain a scientific challenge. Most recent information on snoRNPs has been obtained from studies with reconstituted archaeal sRNPs. However, eukaryotic snoRNPs differ from archaeal sRNPs in many aspects and display a higher level of complexity and regulation. Thus, much of the knowledge achieved for archaeal sRNPs cannot be directly transferred to the eukaryotic system. Here we propose a competitive work plan that aims at providing a molecular understanding of ribose-methylation in eukaryotes. Within SSP1784 we aim at determining the structure and the enzymatic mechanism of eukaryotic snoRNPs, as well as the principles of regulation. We will answer the question about whether and how the levels of methylation are modulated at the different sites and which functional purpose this regulation serves. From the methodological point of view, we will use an interdisciplinary approach consisting of molecular engineering, HPLC/MS-based quantification of ribose methylation in vivo as well as structural, biophysical and biochemical analysis of reconstituted and natively isolated yeast snoRNPs. Besides the impact on revealing basic principles of the cell life-cycle, the knowledge that we will win in this project will help understanding the role of ribose methylation in disease and possibly contribute to the discovery of new pathologies where defects in RNA methylation are involved.
DFG Programme
Priority Programmes
