Abstract
Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3' end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-electron microscopy analysis of late human pre-40S particles purified using a catalytically inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3' end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.
Highlights
Synthesis of eukaryotic ribosomes relies on a large array of ribosome biogenesis factors (RBFs) that coordinate the multiple steps of pre-ribosomal RNA modification, cleavage and folding, together with ribosomal protein (RP) assembly (Bohnsak and Bohnsack, 2019)
Pre-40S particles purified with a catalytically-deficient form of RIO1 display pre- and post18S rRNA processing structural states In order to characterize the final cytoplasmic maturation steps undergone by human pre-40S particles, we purified pre-40S particles from a human cell line overexpressing a tagged version of a catalytically-inactive form of RIO1 mutated on aspartic acid 324 (D324A) (Widmann et al, 2012)
These purified pre-40S particles contain all ribosomal proteins of the small subunit (RPSs), including the late binding RPS10 and RPS26, but only a handful of ribosome biogenesis factors, namely RIO1, DIM2, NOB1 and TSR1 (Figure 1 and figure supplement 2)
Summary
Synthesis of eukaryotic ribosomes relies on a large array of ribosome biogenesis factors (RBFs) that coordinate the multiple steps of pre-ribosomal RNA (pre-rRNA) modification, cleavage and folding, together with ribosomal protein (RP) assembly (Bohnsak and Bohnsack, 2019) Progression through this process, defined by the timely association or dissociation of RBFs and RPs to pre-ribosomal particles and the gradual maturation of pre-rRNAs, is tightly monitored from one stage to the next. Pre-40S particles would be especially prone to premature interactions with the translation apparatus without the presence of several RBFs (Bystin/ENP1, LTV1, RIO2, TSR1, DIM2, NOB1), that occupy the binding sites of translation partners near their “head” and “platform” domains (Larburu et al, 2016; Strunk et al, 2012) These two structural domains undergo several remodeling steps in the cytoplasm leading to the gradual release of these RBFs (Ameismeier et al, 2018; Zemp et al, 2009; Zemp et al, 2014). Interfering with this late stage can result in the incorporation of immature 40S subunits in the translation pool (Belhabich-Baumas et al, 2017; Parker et al, 2019)
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