Abstract
Ribosome assembly in eukaryotes involves the activity of hundreds of assembly factors that direct the hierarchical assembly of ribosomal proteins and numerous ribosomal RNA folding steps. However, detailed insights into the function of assembly factors and ribosomal RNA folding events are lacking. To address this, we have developed ChemModSeq, a method that combines structure probing, high-throughput sequencing and statistical modeling, to quantitatively measure RNA structural rearrangements during the assembly of macromolecular complexes. By applying ChemModSeq to purified 40S assembly intermediates we obtained nucleotide-resolution maps of ribosomal RNA flexibility revealing structurally distinct assembly intermediates and mechanistic insights into assembly dynamics not readily observed in cryo-electron microscopy reconstructions. 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.
Highlights
Ribosome synthesis in eukaryotes is an incredibly complex process that requires around 200 ribosome assembly factors to facilitate the modification, folding and processing of rRNA precursors and their ordered assembly with r-proteins [1]
In the yeast Saccharomyces cerevisiae, ribosome assembly starts in the nucleolus, where a 35S rRNA precursor is cleaved at sites A0, A1 and A2 within a large, 90S-sized complex
We developed a protocol, dubbed ChemModSeq that uses reverse transcription (RT) with oligonucleotides that randomly hybridize to the RNA template (Figure 1D), allowing us to quickly generate an overview of chemically modified sites in rRNA molecules in a single RT reaction
Summary
Ribosome synthesis in eukaryotes is an incredibly complex process that requires around 200 ribosome assembly factors to facilitate the modification, folding and processing of rRNA precursors (pre-rRNA) and their ordered assembly with r-proteins [1]. In the yeast Saccharomyces cerevisiae, ribosome assembly starts in the nucleolus, where a 35S rRNA precursor is cleaved at sites A0, A1 and A2 within a large, 90S-sized complex. Just before D-site cleavage, an aminocarboxypropyl (acp) group is added to a hypermodified uridine at position 1191 in the P-site of the 18S rRNA decoding center [3]. This hypermodification is important for efficient translation and D-site cleavage [4]
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