Poster abstracts
Poster number 30 submitted by Stefanie Hedayati
The IDR of the rRNA methyltransferase Nop2 is critical for ribosome biogenesis
Stefanie Hedayati (Biological Sciences, Carnegie Mellon University ), Geqi Li (Biological Sciences, Carnegie Mellon University ), Nadia Ghetie (Biological Sciences, Carnegie Mellon University ), John L Woolford Jr. (Biological Sciences, Carnegie Mellon University )
Abstract:
Eukaryotic ribosome biogenesis occurs in a step-wise fashion in the nucleolus, nucleoplasm and cytoplasm. About 200 assembly factors (AFs) are required for maturation of yeast ribosomes. Cryo-EM structures of co-transcriptional to cytoplasmic pre-ribosome intermediates have provided information about the composition and conformation of assembly factors, ribosomal proteins and rRNA in these complexes. However, about half of the yeast AFs (110/206) contain intrinsically disordered regions (IDRs), which are very rarely visualized by cryo-EM. IDRs have emerged as important participants in numerous cellular processes, such as facilitating macromolecular interactions or sensing cellular physiology. Additionally, many RNA-binding proteins possess IDRs, often containing long stretches of negatively-charged amino acids. Despite the wealth of information on IDRs, their roles in ribosome biogenesis have been investigated for only a handful of AFs. Therefore, we have an incomplete understanding of the role of IDRs, and thus assembly factors, in ribosome biogenesis. To address this question, we deleted the non-visible IDRs in select AFs and assessed growth, polysome profiles and pre-ribosome composition. We found that even partial deletion of the 196-amino acid long N-terminal IDR of the rRNA methyltransferase Nop2, an essential 60S AF, leads to a dominant-negative slow-growth phenotype, reduced levels of 60S subunits and mature ribosomes, and a block in ribosome assembly immediately prior to the release of Nop2 from pre-ribosomes. We hypothesize that the Nop2 IDR facilitates release of Nop2 from its rRNA ligand in pre-ribosomes to enable its recycling as well as subsequent remodeling steps. We are testing whether the negatively-charged IDR of Nop2 expedites its release by competing with rRNA for binding to Nop2. To further investigate this hypothesis, we are using cryo-EM analysis of nop2 mutants and detailed RNA-binding assays in vitro.
Keywords: IDR, ribosome biogenesis, rRNA methyltransferase