Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles

Reyes Babiano,Gwenael Badis,Jesús De La Cruz,Abdelkader Namane,Antonio Díaz-Quintana,Micheline Fromont-Racine,Cosmin Saveanu,Antonia Doyen,Alain Jacquier

doi:10.1093/nar/gkt726

Abstract

Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7Lb within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.

Highlights

In eukaryotes, ribosome biogenesis is a complex multistep and multi-component process, which occurs primarily in the nucleolus, late steps occur in the nucleoplasm and in the cytoplasm [for reviews, see [1,2]] Most of our knowledge concerning ribosome biogenesis in eukaryotes derives from studies with Saccharomyces cerevisiae
We found that L7 mainly cross-linked to helix ES7Lb in 25S rRNA, which corresponds to an expansion segment exclusive of eukaryotic 25S rRNA [19], and a long region of 5S rRNA over H2, H4 and H5 (Figures 4 and 6, Supplementary Figures S8 and S9)
The most enriched and abundant pre-60S factors associated to Rlp7-TAP (Erb1, Nop7, Nsa3, Ytm1 and Nop15) are components of the A3 factors cluster whose loss-of-function leads to processing defects at A3 site with the subsequent accumulation of the 27SA3 precursor and depletion of its immediate 27SBS and 7S pre-rRNA products [(6,7) and references therein]

Summary

Introduction

Ribosome biogenesis is a complex multistep and multi-component process, which occurs primarily in the nucleolus, late steps occur in the nucleoplasm and in the cytoplasm [for reviews, see [1,2]] Most of our knowledge concerning ribosome biogenesis in eukaryotes derives from studies with Saccharomyces cerevisiae. Roughly 300 protein trans-acting factors, involved in ribosome biogenesis, have been identified [3,4]. These factors likely confer speed, accuracy and directionality to the ribosome synthesis process. To better understand the role of these factors in ribosome biogenesis, other experimental approaches have been developed, among them, in vivo cross-linking and cDNA analysis (CRAC). This technique allows the identification of the interaction sites between several protein trans-acting factors and pre-rRNAs or snoRNAs [e.g. This technique allows the identification of the interaction sites between several protein trans-acting factors and pre-rRNAs or snoRNAs [e.g. [7,8,9,10,11]]

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