Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast

Mariam Jaafar,Carine Dominique,Nicholas J Watkins,Carmen Velasco,Yves Henry,Raghida Abou Merhi,Anthony K Henras,Markus T Bohnsack,Odile Humbert,Olga Rodríguez-Galán,Régine Capeyrou,Jesús De La Cruz,Julia Contreras,Katherine E Bohnsack,Sara Martín-Villanueva,Eduardo Villalobo,Patrice Vitali

doi:10.1038/s41467-021-26207-w

Abstract

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.

Highlights

Introduction of mutations in boxA, box B or the internal stem of SNR190 in the pCH32 plasmid containing the SNR190-U14 expression cassette was achieved using the PCR-based In-Fusion mutagenesis system and appropriate primers for each mutation (Supplementary Table 4)
We made use of the yeast strain NOY89131, in which all the chromosomal rDNA genes are deleted and the 35S pre-ribosomal RNA (rRNA) is expressed from a plasmid driven by the galactoseinducible GAL7 promoter[31]
Transformants were screened on selective SD medium for faster-growing clones, condition where neither pNOY353 nor the GAL1::HA-DBP7 construct are expressed (Supplementary Fig. 3)

Summary

Introduction

Introduction of mutations in boxA (snr190-[mut.A]), box B (snr190-[mut.B]) or the internal stem (snr190-[mut.S]) of SNR190 in the pCH32 plasmid containing the SNR190-U14 expression cassette was achieved using the PCR-based In-Fusion mutagenesis system and appropriate primers for each mutation (Supplementary Table 4). To obtain rRNA suppressors able to bypass the growth defect of Dbp[7] depletion, we first integrated at the DBP7 locus, a GAL::HA-DBP7 construct in the strain NOY891 (MATa ade[] ura[] leu[2,3] leu[2-112] his[3,4,5,6,7,8,9,10,11] can[1-100] rdnΔΔ::HIS3). Western blotting confirmed the presence of a single protein band of the expected molecular mass when candidates are grown in SGal medium (Supplementary Fig. 3)

Methods

Results

Conclusion