The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.

Sohail Khoshnevis,Katrin Karbstein,Isabel Askenasy,Crystal L Young-Erdos,Maria D Dattolo,Matthew C Johnson,M Elizabeth Stroupe,Jamie Cate

doi:10.1371/journal.pbio.1002480

Sohail Khoshnevis, Katrin Karbstein + Show 6 more

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https://doi.org/10.1371/journal.pbio.1002480

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Abstract

DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed “helicases,” their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.

Highlights

IntroductionRibosome assembly involves the transcription, modification, and processing of a large precursor rRNA, encoding 18S, 5.8S, and 25S rRNAs separated by spacer regions (internal transcribed spacers [ITSs]) (S1A and S1B Fig)
Ribosome assembly involves the transcription, modification, and processing of a large precursor rRNA, encoding 18S, 5.8S, and 25S rRNAs separated by spacer regions (S1A and S1B Fig)
We show that a complex of Rrp5, Rok1, and adenosine triphosphate (ATP) binds small subunits or mimics of ribosomal RNA more tightly than does a complex of Rrp5, Rok1, and adenosine diphosphate (ADP)

Summary

Introduction

Ribosome assembly involves the transcription, modification, and processing of a large precursor rRNA, encoding 18S, 5.8S, and 25S rRNAs separated by spacer regions (internal transcribed spacers [ITSs]) (S1A and S1B Fig). These processes are integrated with the binding of ribosomal proteins and rRNA folding via the activities of a large machinery comprising ~200 assembly factors (AFs), most of which are essential [1]. A 193 kDa protein, is one of three AFs that function in the biogenesis of both subunits It consists of 12 tandem repeats of the S1 domain, followed by seven tetratricopeptide (TPR) repeats (S1C Fig). TPR motifs are known as protein-protein interaction sites [8]

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