The Rio1p ATPase hinders premature entry into translation of late pre-40S pre-ribosomal particles

Abstract

Cytoplasmic maturation of precursors to the small ribosomal subunit in yeast requires the intervention of a dozen assembly factors (AFs), the precise roles of which remain elusive. One of these is Rio1p that seems to intervene at a late step of pre-40S particle maturation. We have investigated the role played by Rio1p in the dynamic association and dissociation of AFs with and from pre-40S particles. Our results indicate that Rio1p depletion leads to the stalling of at least 4 AFs (Nob1p, Tsr1p, Pno1p/Dim2p and Fap7p) in 80S-like particles. We conclude that Rio1p is important for the timely release of these factors from 80S-like particles. In addition, we present immunoprecipitation and electron microscopy evidence suggesting that when Rio1p is depleted, a subset of Nob1p-containing pre-40S particles associate with translating polysomes. Using Nob1p as bait, we purified pre-40S particles from cells lacking Rio1p and performed ribosome profiling experiments which suggest that immature 40S subunits can carry out translation elongation. We conclude that lack of Rio1p allows premature entry of pre-40S particles in the translation process and that the presence of Nob1p and of the 18S rRNA 3′ extension in the 20S pre-rRNA is not incompatible with translation elongation.

Good Vibrations: Structural Remodeling of Maturing Yeast Pre-40S Ribosomal Particles Followed by Cryo-Electron Microscopy

“…The sedimentation profile showed that the majority of Tsr1-FPZ pre-40S particles presented a peak position very similar to that of mature 40S ribosomal subunits, thus migrating as "free" pre-40S subunits. A smaller peak of heavier fractions indicates that Tsr1-FPZ could be found in bigger complexes, some of them probably being 80S-like particles [29,30,34,35] (Figure 1a, upper panel). Transmission electron microscopy (TEM) observations after negative staining confirmed that the particles sedimenting on lighter fractions had the overall size and shape of 40S subunits, while heavier fractions contained larger, rounder objects, with dimensions compatible with 80S-like particles (Figure 1a, lower panel).…”

“…Pre-ribosomal particles were purified from a BY4742 Saccharomyces cerevisiae strain harboring a C-terminal tandem affinity purification tag on Tsr1 (Tsr1-FPZ). This FPZ tag is composed of a Flag peptide, a PreScission cleavage site and a Z domain derived from S. aureus protein A [35]. 12 L of yeast cells were grown at 30 • C on YPD medium (1% yeast extract, 1% peptone, supplemented with 2% glucose) to an optical density between 1.2 and 1.8 (600 nm), and harvested by centrifugation at 4000 rpm for 10 min at 4 • C. Pellets were washed twice with ddH2O, and flash frozen in liquid nitrogen.…”

A kinase-dependent checkpoint prevents escape of immature ribosomes into the translating pool

“…Rio1 is an essential aspartate kinase bound to very late cytoplasmic pre-40S subunits that have shed all bound assembly factors except Nob1 and Pno1 (Ferreira-Cerca et al, 2014;Hector et al, 2014;Turowski et al, 2014;Vanrobays et al, 2003;Widmann et al, 2012). Depletion of Rio1 or overexpression of a catalytically-inactive Rio1 mutant leads to the accumulation of 20S pre-rRNA and assembly factors in 80S-like ribosomes (Belhabich-Baumas et al, 2017;Ferreira-Cerca et al, 2014;Vanrobays et al, 2001;Widmann et al, 2012). However, the role Rio1 plays in 18S rRNA maturation and ribosome assembly remains unknown, despite its interest as a target for the development of anti-cancer drugs (Kiburu and LaRonde-LeBlanc, 2012;Kubinski et al, 2017;Mielecki et al, 2013;Read et al, 2013;Weinberg et al, 2017), and the observation that RIOK1 mutations accumulate in human cancers (TCGA Research Network:…”

Maturation of pre‐40S particles in yeast and humans

“…When Rio1p enters the maturation pathway is unclear. Lack of Rio1p (Belhabich‐Baumas et al, ) or overexpression of a Rio1p dominant negative catalytic mutant (Ferreira‐Cerca, Kiburu, Thomson, LaRonde, & Hurt, ) impairs the release of Pno1p/Dim2p, Nob1p, Tsr1p, Dim1p, and Fap7p from 80S‐like particles, suggesting that Rio1p joins the maturation pathway prior to Fap7p intervention in the cytoplasm.…”