Conformational proofreading of distant 40S ribosomal subunit maturation events by a long-range communication mechanism

Abstract

Eukaryotic ribosomes are synthesized in a hierarchical process driven by a plethora of assembly factors, but how maturation events at physically distant sites on pre-ribosomes are coordinated is poorly understood. Using functional analyses and cryo-EM, we show that ribosomal protein Rps20 orchestrates communication between two multi-step maturation events across the pre-40S subunit. Our study reveals that during pre-40S maturation, formation of essential contacts between Rps20 and Rps3 permits assembly factor Ltv1 to recruit the Hrr25 kinase, thereby promoting Ltv1 phosphorylation. In parallel, a deeply buried Rps20 loop reaches to the opposite pre-40S side, where it stimulates Rio2 ATPase activity. Both cascades converge to the final maturation steps releasing Rio2 and phosphorylated Ltv1. We propose that conformational proofreading exerted via Rps20 constitutes a checkpoint permitting assembly factor release and progression of pre-40S maturation only after completion of all earlier maturation steps.

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

“…Our bottom-up analyses revealed phosphorylation marks on S172, however, like for Ltv1, the amount of phosphorylated peptide seems to be low (2.1% abundance compared to the non-phosphorylated corresponding peptide); other amino acids of Enp1 also bore phosphorylation marks, but in negligible amounts ( Table 1). As these modifications are thought to trigger Ltv1 and Enp1 release from cytoplasmic pre-40S particles [12,23,37], the small amount of phosphorylated Ltv1 and Enp1 associated to Tsr1-FPZ pre-40S particles suggests that their phosphorylation might be rapidly followed by the release of these RBFs from pre-ribosomal particles, as recently suggested [19].…”

“…The corresponding non-phosphorylated peptide could not be found, suggesting that 100% of Tsr1 would harbor such a modification; the kinase responsible for this phosphorylation remains to be identified. Hrr25 is the kinase that phosphorylates Ltv1 in both human and yeast cells [11,19,37], and systematic point mutations identified Ltv1 Serines S336, S339 and S342 as its putative phosphorylation targets [23]. Two of these sites were retrieved in our bottom-up analyses, which revealed phosphorylation marks on S336 or S339, though for a small fraction (6.5%) of the considered peptides (Table 1).…”

“…Furthermore, most of the identified RBFs physically block the association of the translation machinery by occupying functional sites on the 40S subunit. Dim1, which is responsible for base methylation of adenosines 1781 and 1782 on the 18S rRNA [18], was located on the intersubunit side under the platform region by low resolution cryo-EM studies, but seems to be absent or structurally instable in most of the high resolution 3D structures of yeast pre-40S particles published to date [16,17,19], suggesting an early dissociation from cytoplasmic pre-40S particles. Nob1, the endonuclease responsible for the final cleavage of the 20S pre-rRNA into mature 18S rRNA [20], could not be precisely located on any EM structures of yeast pre-40S particles.…”

“…Moreover, this would also permit Rps3, located between the head and the beak of the pre-40S, to be fitted into its mature position [24,25]. This structural modeling of the beak region would allow the pursuit of the small subunit maturation [19].…”

“…Interestingly, a study based on high-throughput rRNA structure probing (ChemModSeq) revealed that pre-40S particles containing Rio2, Tsr1, Ltv1 and Enp1 harbored a 3 major domain (corresponding to the beak and platform domains of the small ribosomal subunit) with a higher degree of flexibility than late pre-40S particles, which lacked these proteins [28]. Furthermore, a very recent cryo-EM study performed on pre-40S particles with a truncated form of Rps20 revealed that these pre-40S particles could be trapped in at least two structural classes, one with immature beak and platform domains, and the second with a fully matured head and a highly dynamic platform [19]. Taken together, this suggests that pre-40S particles might undergo various states ranging from structurally stable to highly dynamic, and that the platform might be the last domain to be matured.…”