Abstract
Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA3 pre-rRNA to generate the 5′ end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps.
Highlights
Ribosome biogenesis requires folding, modification and processing of four ribosomal RNAs and association of $80 ribosomal proteins (r-proteins) to generate mature 40S and 60S subunits of eukaryotic ribosomes [1]
Trans-acting assembly factors have been characterized by the pre-ribosomal RNAs (rRNAs) processing steps that are blocked in their absence
Has1 is necessary for processing of 27SA3 and 27SB pre-rRNAs for 60S subunit biogenesis
Summary
Modification and processing of four ribosomal RNAs (rRNAs) and association of $80 ribosomal proteins (r-proteins) to generate mature 40S and 60S subunits of eukaryotic ribosomes [1]. These events are coupled and occur in a hierarchical manner in the context of pre-ribosomes (Figure 1A, 90S, 66S and 43S pre-ribosomes). Pre-rRNAs undergo precise removal of internal and external transcribed spacer (ITS, ETS) sequences to generate mature rRNAs (Figure 1A) These cleavage and processing events are irreversible, suggesting that they represent points of regulatory control to ensure that only properly assembled intermediates proceed to the step. How assembly factors participate in RNP maturation, including RNA conformational changes, stable association of r-proteins with rRNA and binding and release of assembly factors with pre-ribosomes to facilitate these processing steps, has been less well characterized [2,3,4]
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