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
Related in: MedlinePlus
Mentions: The ChemModSeq analyses revealed distinct differences in the pattern of nucleotide SHAPE reactivity between 40S assembly intermediates. Pre-40S particles that contained Rio2, Tsr1, Ltv1 and Enp1 were generally more flexible compared to late pre-40S complexes that lacked these proteins. Many of these flexible nucleotides in early-middle pre-40S complexes clustered near assembly factor binding sites, including Ltv1 and Enp1 binding sites in the 3′ major domain. Interestingly, our ChemModSeq analyses on in vitro refolded 18S rRNA (Figure 8) demonstrated that many of these highly flexible regions could spontaneously fold into, what appeared to be, relatively stable structures. We propose that assembly factors function by providing the necessary energy to maintain a more flexible conformation in the pre-rRNA (Figure 9A). More specifically, we hypothesize that Ltv1 and Enp1, which bind to the head domain, act as RNA chaperones by delaying folding steps in the 3′ major domain (Figure 9A). This would be required to allow earlier pre-rRNA folding steps to be completed and/or to safeguard correct folding by preventing the accumulation of kinetically trapped intermediates. A similar function has been proposed for bacterial RimM, which regulates 3′ major domain formation by holding the rRNA in a conformation that allows proper folding of the beak structure and H43 (46). Similarly, the U3 and snR30 snoRNAs regulate the formation of long-range rRNA interactions in the 18S rRNA. The U3-rRNA-5′18S rRNA base-pairing interaction blocks the formation of the universally conserved central pseudoknot (47), whereas snR30-rRNA base-pairing interferes with long-range interactions between two rRNA expansion segments (48). At some point during the maturation pathway, certain assembly factors are no longer required and need to be released. This can be achieved through phosphorylation of proteins (such as Hrr25-dependent phosphorylation of Enp1 and Ltv1, Figure 9B; (7)), which presumably reduces their affinity for RNA, or through the activity of other energy-dependent remodelers, such as RNA helicases and GTPases (e.g. see (49,50)). Release of these factors would allow completion of RNA folding steps, trigger binding of late assembling r-proteins (our work) or trigger remodeling steps (7,51).
Affiliation: Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3JD, UK.