Abstract
Production of eukaryotic ribosomal subunits is a highly dynamic process; pre-ribosomes undergo numerous structural rearrangements that establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome assembly. Using in vivo crosslinking, we here identify the pre-ribosomal binding sites of three RNA helicases. Our data support roles for Has1 in triggering release of the U14 snoRNP, a critical event during early 40S maturation, and in driving assembly of domain I of pre-60S complexes. Binding of Mak5 to domain II of pre-60S complexes promotes recruitment of the ribosomal protein Rpl10, which is necessary for subunit joining and ribosome function. Spb4 binds to a molecular hinge at the base of ES27 facilitating binding of the export factor Arx1, thereby promoting pre-60S export competence. Our data provide important insights into the driving forces behind key structural remodelling events during ribosomal subunit assembly.
Highlights
Production of eukaryotic ribosomal subunits is a highly dynamic process; pre-ribosomes undergo numerous structural rearrangements that establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome assembly
To define the preribosomal complexes that Has[1], Mak[5] and Spb[4] associate with and to demonstrate at which stage of ribosome biogenesis these proteins are required, we first established yeast strains in which each of these essential helicases could be transiently depleted by addition of doxycycline or by growth in the presence of glucose and pre-rRNAs were analysed by northern blotting to identify processing defects (Fig. 1b–d)
We isolated Has1, Mak5and Spb4-containing particles and investigated their pre-rRNA and protein composition using northern blotting and mass spectrometry respectively (Fig. 1e and Supplementary Table 1). These analyses revealed that Has[1] associates with the initial 35S pre-rRNA transcript and 27SB pre-rRNA as well as numerous ribosome biogenesis factors implicated in early separates the precursors of the small (SSU) and intermediate LSU biogenesis (Fig. 1e and Supplementary Table 1)
Summary
Production of eukaryotic ribosomal subunits is a highly dynamic process; pre-ribosomes undergo numerous structural rearrangements that establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome assembly. Key events during pre-60S maturation are the docking of the 5S RNP and its 180° rotation to form the central protuberance[11], assembly of the peptidyl transferase centre and polypeptide exit tunnel, and the removal of ITS2 sequences and biogenesis factors from the “foot” region[15,16] Such irreversible remodelling steps, which are typically driven by NTP-dependent enzymes including GTPases, AAA-ATPases and RNA helicases, enforce the directionality of the pathway and act as key surveillance checkpoints, ensuring the fidelity of subunit assembly[17,18,19,20]. Spb[4] binds to the hinge region at the base of a highly flexible arm formed by H63/ES27, which anchors the pre-60S export factor Arx[18] and we demonstrate that Spb[4] facilitates the association of Arx[1] to pre-60S complexes
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