In vivo and in vitro search for rRNA expansion segment interaction partners

Abstract

Expansion segments (ESs) are highly flexible ribosomal RNA elements and cover the majority of the ribosomal surface. The two most prominent ones are ES7 and ES27 of the large ribosomal subunit. So far only few interaction partners of these ESs are known, confirmed and functional connections established. In this study the in vivo RaPID approach for RNA-protein interaction detection and an in vitro pulldown using ESs as bait were employed in search for ES7 and ES27 interaction partners in two model organisms, yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. RaPID, developed for human cells, proved to be not readily adaptable for use in the model organism Saccharomyces cerevisiae. Meanwhile, with the in vitro pulldown approach potential ES interaction partners were identified. Besides confirming the binding of Map1 and Map2 to S. cerevisiae ES27, conservation of MAP-2 binding in C. elegans was demonstrated, and ES27 binding of W08E12.7, the C. elegans ortholog of human Map2-like ES27 interactor Ebp1, was shown. Moreover, the data suggest a role of ESs in several metabolic pathways, from acetyl-CoA to carbon metabolism, by contacting involved proteins. The proposition of ES7 as central docking site for aminoacyl-tRNA synthases could be strengthened as all components of the yeast multi-aminoacyl-tRNA synthetase complex were found interacting with the ES. Furthermore, data from this study indicate, that ESs assist in grouping functionally related factors, such as Tef1, its nucleotide exchange factor Cam1 and its modifier Efm1, possibly regulating activity by facilitating contact. This study provides results towards understanding the ES functions. The identified ES interaction partners build a basis for follow-up studies and pave the way for deeper understanding of these still mysterious ribosomal RNA elements.