Release of the ribosome biogenesis factor Bud23 from small subunit precursors in yeast

Abstract

Ribosomes are the universally conserved ribonucleoprotein complexes that synthesize proteins. The two subunits of the eukaryotic ribosome are produced through a quasi-independent assembly-line-like pathway involving the hierarchical actions of numerous trans-acting biogenesis factors and the incorporation of ribosomal proteins. The factors work together to shape the nascent subunits through a series of intermediate states into their functional architectures. The earliest intermediate of the small subunit (SSU or 40S) is the SSU Processome which is subsequently transformed into the pre-40S intermediate. This transformation is, in part, facilitated by the binding of the methyltransferase Bud23. How Bud23 is released from the resultant pre-40S is not known. The ribosomal proteins Rps0, Rps2, and Rps21, termed the Rps0-cluster proteins, and several biogenesis factors are known to bind the pre-40S around the time that Bud23 is released, suggesting that one or more of these factors induce Bud23 release. Here, we systematically examined the requirement of these factors for the release of Bud23 from pre-40S particles. We found that the Rps0-cluster proteins are needed but not sufficient for Bud23 release. The atypical kinase/ATPase Rio2 shares a binding site with Bud23 and is thought to be recruited to pre-40S after the Rps0-cluster proteins. Depletion of Rio2 prevented the release of Bud23 from the pre-40S. More importantly, the addition of recombinant Rio2 to pre-40S particles affinity-purified from Rio2-depleted cells was sufficient for Bud23 release in vitro. The ability of Rio2 to displace Bud23 was independent of nucleotide hydrolysis. We propose a novel role for Rio2 in which its binding to the pre-40S actively displaces Bud23 from the pre-40S, and we suggest a model in which the binding of the Rps0-cluster proteins and Rio2 promote the release of Bud23.

Quality control ensures fidelity in ribosome assembly and cellular health

“…Release of U3 snoRNA and binding of Bud23 are both required for the binding of Rps2 (uS5), Rps0 (uS2), and Rps21 (eS21). Consistently, the absence of Bud23 impairs pre-40S nuclear export, leading to reduced production of mature 40S subunits ( White et al, 2008 ) and a severe growth defect, which can be suppressed by weakly binding mutants of these AFs and overexpression of Rps2 ( Black et al, 2020 ; Black and Johnson, 2022 ). Binding of Rps2, Rps0, and Rps21 in turn is required for Bud23 release ( Black and Johnson, 2022 ) promoted by the kinase Rio2, which then takes the position of Bud23 ( Black and Johnson, 2022 ; Linnemann et al, 2019 ).…”

“…Consistently, the absence of Bud23 impairs pre-40S nuclear export, leading to reduced production of mature 40S subunits ( White et al, 2008 ) and a severe growth defect, which can be suppressed by weakly binding mutants of these AFs and overexpression of Rps2 ( Black et al, 2020 ; Black and Johnson, 2022 ). Binding of Rps2, Rps0, and Rps21 in turn is required for Bud23 release ( Black and Johnson, 2022 ) promoted by the kinase Rio2, which then takes the position of Bud23 ( Black and Johnson, 2022 ; Linnemann et al, 2019 ). Surprisingly, even though Rio2, Rps2, Rps0, and Rps21 promote Bud23 release in a partially reconstituted system ( Black and Johnson, 2022 ), Rio2 binding is independent of these RPs ( Linnemann et al, 2019 ; Black and Johnson, 2022 ).…”

“…Another example of an assembly step that features aspects of QC is the exchange of Bud23 (WBSCR22 in humans) for Rio2 (RIOK2 in humans) after the ribosome neck has formed ( Black and Johnson, 2022 ), enabling pre-40S nuclear export. Acting as a hinge during head rotation, the neck plays a crucial function for tRNA translocation during translation ( Korostelev et al, 2008 ; Mohan et al, 2014 ).…”

Impact of the yeast S0/uS2-cluster ribosomal protein rpS21/eS21 on rRNA folding and the architecture of small ribosomal subunit precursors

“…To gain more insights into functional interrelationships between r-protein assembly and pre-rRNA folding events, we analysed here the impact of the assembly of the S0-cluster of r-proteins on the pre-rRNA fold and the general architecture of late nucleoplasmic to early cytoplasmic yeast SSU precursors. Previous biochemical studies indicated that S0-cluster formation is required for the production and accumulation of yeast SSUs [33,[40][41][42][43][44][45]. Effects were observed on the dynamics of their nuclear export, the cytoplasmic release of the ITS1, the recruitment of factors such as Nob1, and the release of other factors such as Slx9, Rrp12, and Bud23.…”

Allele-specific alternative splicing of Drosophila Ribosomal protein S21 suppresses a lethal mutation in the Phosphorylated adaptor for RNA export (Phax) gene

“…RpS21 is component of the small subunit of the ribosome with functions in translation initiation and ribosome biogenesis ( Török et al 1999 ; Sato et al 2003 ; Tabb-Massey et al 2003 ; Black and Johnson 2022 ). In Drosophila , haploinsufficient RpS21 mutants exhibit the classic Minute phenotype of short and thin bristles, prolonged development, and impaired viability ( Török et al 1999 ; Marygold et al 2007 ).…”