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Abstract

The ‘birth’ of the eukaryotic ribosome is preceded by RNA folding and processing reactions that depend on assembly factors and snoRNAs. The 90S (SSU-processome) is the earliest pre-ribosome structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5’>3’, but this directionality may not be accurate. Here, by analyzing the structure of series of novel 90S assembly intermediates isolated from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. This revealed that large parts of the 18S rRNA 3’ and central domains assemble first into the 90S, before the 5’ domain is stably integrated. This final incorporation depends on a physical contact between a heterotrimer Enp2-Bfr2-Lcp1 recruited to the flexible 5’ domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S pre-ribosome. Keeping the 5’ domain temporarily segregated from the 90S scaffold could provide an extra time to complete the multifaceted 5’ domain folding, which depends on a distinct set of snoRNAs and processing factors.