Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution.
Bottom Line: However, detailed insights into the function of assembly factors and ribosomal RNA folding events are lacking.We show that RNA restructuring events coincide with the release of assembly factors and predict that completion of the head domain is required before the Rio1 kinase enters the assembly pathway.Collectively, our results suggest that 40S assembly factors regulate the timely incorporation of ribosomal proteins by delaying specific folding steps in the 3' major domain of the 20S pre-ribosomal RNA.
Affiliation: Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3JD, UK.Show MeSH
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Mentions: To address whether the observed changes in nucleotide flexibility between early-middle and late pre-40S particles could be the result of ribosomal protein binding events, we performed 1M7 probing experiments on 18S rRNA that was in vitro refolded under the same conditions used to purify pre-40S complexes (Figure 8A–C, compare lanes 7 and 8). Comparison of the SHAPE reactivity profiles revealed that large regions of the 3′ major domain in the refolded rRNA adopted secondary structures similar to 18S and 20S rRNA molecules in purified particles (Figure 8A–C). Surprisingly, the majority of the highly SHAPE reactive nucleotides in early and middle pre-40S particles that clustered near assembly factor binding sites (H33, H35, H37, H40 and H41; see Figure 7) did not show the same degree of flexibility in in vitro refolded RNA (Figure 8D, red colored nucleotides in regions indicated with roman numbers). This suggests that under the conditions used, these regions can fold into, what appear to be, relatively stable structures independently of proteins in vitro. In the yeast 80S crystal structure, the nucleotides in the H37 terminal loop and the internal loop in H41 (U1514–U1517) that do not form Watson–Crick base-pairs, form a large network of base-stacking interactions, including many long-range interactions (Figure 8D, indicated with dotted lines). These presumably help stabilize the structure of the rRNA. Many of these stacking interactions are s53 interactions (5′ side of one base with 3′ side of other base) that are generally less reactive to SHAPE reagents (44), The relatively low SHAPE reactivity of these nucleotides in the in vitro refolded 18S rRNA suggests that at least some of these base-stacking interactions form in the absence of proteins. Based on these results, we hypothesize that assembly factors that associate with early and middle pre-40S particles function by providing the energy necessary to maintain a flexible 3′ major domain conformation.
Affiliation: Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3JD, UK.