RPS28B mRNA acts as a scaffold promoting cis-translational interaction of proteins driving P-body assembly

Abstract

AbstractP-bodies (PBs) are cytoplasmic mRNA-protein (mRNP) granules conserved throughout eukaryotes which are implicated in the repression, storage and degradation of mRNAs. PB assembly is driven in part by proteins with self-interacting and low-complexity protein domains. Non-translating mRNA is also required for PB assembly, however no studies to date have explored whether particular mRNA transcripts are more critical than others in facilitating PB assembly. A previous genome-wide microscopy screen in yeast revealed that rps28bΔ (Ribosomal protein subunit-28B) mutants do not form PBs under normal growth conditions. Here, we demonstrate that the RPS28B 3’UTR is important for PB assembly, consistent with the fact that this is a known binding site for the PB assembly protein Edc3. However, expression of the RPS28B 3’UTR in isolation is insufficient to drive normal PB assembly. Intriguingly, chimeric mRNA studies revealed that Rps28 protein, translated in cis from an mRNA bearing the RPS28B 3’UTR, physically interacts more strongly with Edc3 than Rps28 protein synthesized in trans. This Edc3-Rps28 interaction in turn also facilitates PB assembly. In summary, our work indicates that PB assembly may be preferentially nucleated by specific RNA “scaffolds”, which may be a common theme in RNP granule assembly. Furthermore, this is the first description in yeast to our knowledge of a cis-translated protein interacting with another protein in the 3’UTR of the mRNA which encoded it, which in turn has functional consequences for assembly of cellular structures.