Funktionelle und strukturelle Analyse von eukaryotischen snoRNP Komplexen, die die Ribose-Methylierung von rRNA katalysieren.
Biochemie
Zellbiologie
Final Report Abstract
Ribosomal RNA (rRNA) carries extensive 2’-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. In humans, deregulation of 2’-O-methylation is involved in several diseases, including cancer and neuropathologies. 2’-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive functional and structural information exists for the archaeal enzyme, but the available data is partially in conflict with each other. In contrast, no structural and little functional data is available for the eukaryotic enzyme. The goal of this project was to characterize the structure and molecular mechanism of the yeast enzyme, the Box C/D RNP, which consists of a guide-RNA, guiding the methylation to specific sites of the ribosome, the RNA- primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58 and the enzymatic module Nop1. We had planned an interdisciplinary approach consisting of molecular engineering, HPLC/MS-based quantification of ribose methylation, structural, biophysical and biochemical analysis of reconstituted and natively isolated yeast enzymes. The project was planned as a collaboration between the Carlomagno laboratory (in vitro studies) and the Entian laboratory (in vivo studies). Here, I summarize the results from the Carlomagno laboratory. The reconstitution of a functional eukaryotic Box C/D 2’-O-methylation enzyme remained out of reach, which precluded a thorough structural and functional characterization of the enzyme in vitro. Nevertheless, the work done towards the achievement of this goal led to important discoveries. First, we discovered that, in contrast to the archaeal enzyme, the eukaryotic enzyme does not have a bipartite symmetry, but features two structurally distinct protein assembly sites. In particular, the in vitro assembly of proteins at the second guide-RNA site is challenging and prevents the reconstitution of a functional complex. We propose that the correct folding of this site requires accessory factors and may happen only transiently in the cells. Second, the two scaffold proteins Nop56 and Nop58 differ mostly in the N-terminal domain, which, in eukaryotes, contains an insert of ~25 amino acid with respect to the archaeal protein. This insert forms additional contacts with the methyltransferase Nop1 but is also subject to structural changes when it enters interactions with accessory factors. Thus, the additional plasticity of the N-terminal domain of the eukaryotic scaffold proteins Nop56 and Nop58 points to a more complex mechanism of methylation regulation in eukaryotes with respect to archaea, likely including the participation of other proteins. All in all, the Box C/D snoRNP is likely not to exist as an independent stable unit in eukaryotes as in archaea, but to work in concert with other cellular factors. Our findings are the result of structural analyses of two complexes by X-ray crystallography, extensive biochemical and biophysical characterization of in vitro assembled complexes, biochemical binding assays, mutational analysis and in vitro functional assays.
Publications
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“High-resolution structure of eukaryotic Fibrillarin interacting with Nop56 N-terminal domain” RNA 2021 27, 496–512
S. Höfler, P. Lukat, W.Blankenfeldt, T. Carlomagno
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“Two non-symmetric protein assembly sites in eukaryotic Box C/D methylation machinery” Scientific reports 2021 11, 17561
S. Höfler, P. Lukat, W.Blankenfeldt, T. Carlomagno
