Functional Centers of the Eukaryotic Ribosome: From Assembly to Quality Control

Abstract

Ribosomes are the central molecular machineries that translate the genetic message encoded by mRNAs into proteins. Precise and efficient ribosome synthesis represents a demanding task for all cells. In the eukaryotic model organism budding yeast, more than 2000 ribosomes are manufactured every minute. A growing yeast cell needs to ensure that only correctly assembled ribosomal particles enter the pool of translation competent ribosomes. Ribosome biogenesis in eukaryotes is facilitated by more than 200 assembly factors that drive the compaction of the transcribed rRNA, incorporate ribosomal proteins, modify the rRNA, facilitate export through the nuclear pore complex, and finally check the integrity of maturing pre-ribosomal particles. Suboptimal temperatures pose a challenge to the cell by promoting mis-folding and kinetical trapping of ribosomal RNA (rRNA). Here we show by proteomic profiling that specific RNA binding proteins involved in ribosome biogenesis are up regulated at low temperatures. The upregulated assembly factors are located in rRNA rich functional centers of the pre-ribosomal particles. Using biochemical and cell biological assays, we identified the binding site of the atypical Pumilio domain containing protein Puf6 close to the P-site and E-site within the subunit interface. Puf6 depleted cells exhibit a severe growth defect at low temperatures and accumulate the large ribosomal subunits in the nucleus. The subunit interface is a preferred binding platform for downstream factors that facilitate the export of the pre-ribosomal particles. We show that Puf6 prepares the binding site for the GTPase Nog2 (alias Nug2) and primes the pre-60S subunit to achieve export competence. In a collaborative project with the Ban laboratory, we show that insertion of the C-terminal domain of Rei1 probes the conductivity of the polypeptide exit tunnel and is necessary to initiate final maturation of the large ribosomal subunit. Using genetic and cell biological assays in yeast I showed that failure to insert the C-terminus into the polypeptide exit tunnel halts progression of the cytoplasmic maturation and prevents potentially corrupt large ribosomal subunits to acquire translation competence. This study deciphered the precise role of a cytoplasmic maturation event that probes the correct assembly of the polypeptide exit tunnel at near-atomic resolution, and stimulated the community to investigate tunnel assembly during early maturation events.