A comprehensive view on r-protein binding and rRNA domain structuring during early eukaryotic ribosome formation

TL;DR

This study explores how ribosomal proteins and RNA domains organize during early ribosome formation in eukaryotes, revealing a complex and non-linear assembly process.

Contribution

The study provides novel insights into the role of the foot structure and r-protein binding during early ribosome assembly in eukaryotes.

Findings

Compaction of domain I of the 25S rRNA depends on the presence of foot factors.

Rlp7 depletion affects the assembly of small subunit intermediates before A1 cleavage.

r-protein incorporation in eukaryotes does not follow a linear co-transcriptional mode.

Abstract

Formation of the eukaryotic ribosomal subunits follows a strict regime to assemble ribosomal proteins (r-protein) with ribosomal RNAs (rRNA) while removing internal (ITS) and external (ETS) transcribed rRNA spacers. During the early stages of large subunit (LSU) formation, ITS2, together with six assembly factors, forms the characteristic foot structure of early nuclear pre-LSU particles. Here, we address the function of this foot structure during the early stages of ribosome assembly. We present cryo-EM structures from wild-type cells and cells depleted for the foot structure factor Rlp7. We show that compaction of domain I of the 25S rRNA is strictly dependent on the presence of foot factors, while domain II folds independently. Furthermore, Rlp7-depletion accumulated small subunit (SSU) processome intermediates prior to A1 cleavage and compaction of the individual domains of the 18S rRNA, providing also novel insights into the SSU-assembly process. SILAC labeling and affinity purification of co-transcriptionally assembled pre-ribosomes enabled us to resolve the assembly line of most early binding r-proteins step by step. This showed that incorporation of r-proteins in eukaryotes neither follows the bacterial regime nor a strict linear co-transcriptional mode. Instead, seed r-proteins might structurally define the individual rRNA domains before their compaction and fixation in the context of early pre-ribosomes.

Graphical Abstract

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