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Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution.

Hector RD, Burlacu E, Aitken S, Le Bihan T, Tuijtel M, Zaplatina A, Cook AG, Granneman S - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3JD, UK.

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Ribosomal protein rRNA binding sites are highly flexible in early and middle pre-40S complexes. (A and B) Nucleotides in the head domain reactive to SHAPE chemicals (A) cluster near assembly factor UV cross-linking sites (B). (C–F) SHAPE-modified nucleotides (blue) in the head domain coincide with Rps3, Rps15, Rps17 and Rps20 binding sites. Relevant nucleotide positions are indicated. Green dots highlight the 2′-OH. (G and H) Ribosomal proteins Rps3 and Rps17 form salt-labile interactions with pre-40S complexes. Immunoprecipitations were performed using strains expressing FLAG-tagged r-proteins (indicated above the panels). Co-precipitated 20S was detected by northern hybridization with oligo 004, 18S rRNA was detected by ethidium bromide staining (EtBr). Input indicates 1% of total RNA extracted from cell lysates. (H) Quantification of results shown in (G). Error bars indicate standard deviations obtained from two biological replicates.
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Figure 7: Ribosomal protein rRNA binding sites are highly flexible in early and middle pre-40S complexes. (A and B) Nucleotides in the head domain reactive to SHAPE chemicals (A) cluster near assembly factor UV cross-linking sites (B). (C–F) SHAPE-modified nucleotides (blue) in the head domain coincide with Rps3, Rps15, Rps17 and Rps20 binding sites. Relevant nucleotide positions are indicated. Green dots highlight the 2′-OH. (G and H) Ribosomal proteins Rps3 and Rps17 form salt-labile interactions with pre-40S complexes. Immunoprecipitations were performed using strains expressing FLAG-tagged r-proteins (indicated above the panels). Co-precipitated 20S was detected by northern hybridization with oligo 004, 18S rRNA was detected by ethidium bromide staining (EtBr). Input indicates 1% of total RNA extracted from cell lysates. (H) Quantification of results shown in (G). Error bars indicate standard deviations obtained from two biological replicates.

Mentions: Many of the highly flexible nucleotides in early-middle pre-40S complexes clustered near previously identified assembly factor UV cross-linking sites (see (5)) (Figure 7A and B). This indicates that these factors play a role in maintaining a more flexible rRNA conformation. In late pre-40S particles, including the Rio1 particle, the 3′ major domain was significantly less reactive to SHAPE chemicals (Figure 6B–D, Supplementary Figure S7A–E, regions indicated with roman numbers). The observed structural rearrangements in the 3′ major domain therefore presumably occur before Rio1 enters the assembly pathway and may coincide with the release of assembly factors Rio2, Tsr1, Ltv1 and Enp1. Although Nob1 assembles into pre-ribosomes at a very early stage (41), and cross-links to the 3′ major domain (5), it does not appear to contribute to these RNA restructuring events, as pre-40S complexes that accumulated in cells lacking Nob1 had all the hallmarks of late pre-40S particles (Supplementary Figure S8: see regions indicated with roman numbers).


Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution.

Hector RD, Burlacu E, Aitken S, Le Bihan T, Tuijtel M, Zaplatina A, Cook AG, Granneman S - Nucleic Acids Res. (2014)

Ribosomal protein rRNA binding sites are highly flexible in early and middle pre-40S complexes. (A and B) Nucleotides in the head domain reactive to SHAPE chemicals (A) cluster near assembly factor UV cross-linking sites (B). (C–F) SHAPE-modified nucleotides (blue) in the head domain coincide with Rps3, Rps15, Rps17 and Rps20 binding sites. Relevant nucleotide positions are indicated. Green dots highlight the 2′-OH. (G and H) Ribosomal proteins Rps3 and Rps17 form salt-labile interactions with pre-40S complexes. Immunoprecipitations were performed using strains expressing FLAG-tagged r-proteins (indicated above the panels). Co-precipitated 20S was detected by northern hybridization with oligo 004, 18S rRNA was detected by ethidium bromide staining (EtBr). Input indicates 1% of total RNA extracted from cell lysates. (H) Quantification of results shown in (G). Error bars indicate standard deviations obtained from two biological replicates.
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Related In: Results  -  Collection

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Figure 7: Ribosomal protein rRNA binding sites are highly flexible in early and middle pre-40S complexes. (A and B) Nucleotides in the head domain reactive to SHAPE chemicals (A) cluster near assembly factor UV cross-linking sites (B). (C–F) SHAPE-modified nucleotides (blue) in the head domain coincide with Rps3, Rps15, Rps17 and Rps20 binding sites. Relevant nucleotide positions are indicated. Green dots highlight the 2′-OH. (G and H) Ribosomal proteins Rps3 and Rps17 form salt-labile interactions with pre-40S complexes. Immunoprecipitations were performed using strains expressing FLAG-tagged r-proteins (indicated above the panels). Co-precipitated 20S was detected by northern hybridization with oligo 004, 18S rRNA was detected by ethidium bromide staining (EtBr). Input indicates 1% of total RNA extracted from cell lysates. (H) Quantification of results shown in (G). Error bars indicate standard deviations obtained from two biological replicates.
Mentions: Many of the highly flexible nucleotides in early-middle pre-40S complexes clustered near previously identified assembly factor UV cross-linking sites (see (5)) (Figure 7A and B). This indicates that these factors play a role in maintaining a more flexible rRNA conformation. In late pre-40S particles, including the Rio1 particle, the 3′ major domain was significantly less reactive to SHAPE chemicals (Figure 6B–D, Supplementary Figure S7A–E, regions indicated with roman numbers). The observed structural rearrangements in the 3′ major domain therefore presumably occur before Rio1 enters the assembly pathway and may coincide with the release of assembly factors Rio2, Tsr1, Ltv1 and Enp1. Although Nob1 assembles into pre-ribosomes at a very early stage (41), and cross-links to the 3′ major domain (5), it does not appear to contribute to these RNA restructuring events, as pre-40S complexes that accumulated in cells lacking Nob1 had all the hallmarks of late pre-40S particles (Supplementary Figure S8: see regions indicated with roman numbers).

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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh, EH9 3JD, UK.

Show MeSH
Related in: MedlinePlus