Abstract
Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.
Highlights
Prokaryotic ribosomes consist of three ribosomal RNAs and,55 ribosomal proteins (r-proteins)
To analyse possible hierarchical relationships between recruitment of small ribosomal subunit (SSU) processome sub-modules to yeast pre-ribosomal RNAs (rRNAs) and rprotein assembly events we constructed a set of yeast conditional rprotein gene mutants expressing epitope tagged variants of SSU processome components
We wanted to test how preribosome association of the UTP-A member Utp4p and the UTPB member Pwp2p is affected in strains depleted of rpS11, rpS9, rpS22, rpS13, and rpS5 or in strains depleted of rpS15 and rpS14 which bind to different regions of the SSU rRNA ([31,32], see Fig. 1)
Summary
Prokaryotic ribosomes consist of three ribosomal RNAs (rRNAs) and ,55 ribosomal proteins (r-proteins). Each of the three major secondary structure domains of the 16S rRNA forms distinct morphological features of the SSU: the 59 domain forms the shoulder and the foot, the central domain forms the platform and the 39 major domain forms the head. These three major SSU rRNA domains can largely assemble in vitro with corresponding r-proteins independently of each other [5,6,7]. Time resolved hydroxyl radical footprinting analyses showed that some of the contacts of rproteins with the 16S rRNA can already be observed very soon after initiating prokaryotic SSU in vitro assembly reactions [8]. The establishment of other contacts, was substantially slower, probably driven by induced fit mechanisms
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