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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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A model for structural rearrangements during late stages of 40S subunit maturation. (A) Proposed model for the function of ribosome assembly factors in 40S assembly. Binding of assembly factors (AF) to RNA is required to maintain a more flexible conformation. Their presence allows the assembly of ribosomal proteins (RP) but prevent that these RPs adopt their final conformation. At some point during the maturation pathway, the system receives a signal that certain ribosome assembly/RNA folding steps have been completed and AFs are no longer needed. Their release could be triggered through phosphorylation (such as Hrr25-dependent phosphorylation of Enp1 and Ltv1). This allows completion of RNA folding steps and binding of late RPs. (B and C) Model for rearrangements that take place in the head domain during late stages of 40S assembly. In early and middle pre-40S complexes, binding of Ltv1 and Enp1 to the head domain is required to maintain a more flexible conformation in the 3′ major domain. Rps3, Rps17, Rps20 and Rps17 assemble into pre-ribosomes but do not adopt their final conformation. Hrr25 phosphorylation of Ltv1 and Enp1 triggers their release, allowing late RNA folding steps and assembly of head domain r-proteins to be completed. We propose that Rio2 and Tsr1 are released shortly afterward (B) and Rio1 enters the assembly pathway. The exact function of Rio1, however, remains unclear.
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Figure 9: A model for structural rearrangements during late stages of 40S subunit maturation. (A) Proposed model for the function of ribosome assembly factors in 40S assembly. Binding of assembly factors (AF) to RNA is required to maintain a more flexible conformation. Their presence allows the assembly of ribosomal proteins (RP) but prevent that these RPs adopt their final conformation. At some point during the maturation pathway, the system receives a signal that certain ribosome assembly/RNA folding steps have been completed and AFs are no longer needed. Their release could be triggered through phosphorylation (such as Hrr25-dependent phosphorylation of Enp1 and Ltv1). This allows completion of RNA folding steps and binding of late RPs. (B and C) Model for rearrangements that take place in the head domain during late stages of 40S assembly. In early and middle pre-40S complexes, binding of Ltv1 and Enp1 to the head domain is required to maintain a more flexible conformation in the 3′ major domain. Rps3, Rps17, Rps20 and Rps17 assemble into pre-ribosomes but do not adopt their final conformation. Hrr25 phosphorylation of Ltv1 and Enp1 triggers their release, allowing late RNA folding steps and assembly of head domain r-proteins to be completed. We propose that Rio2 and Tsr1 are released shortly afterward (B) and Rio1 enters the assembly pathway. The exact function of Rio1, however, remains unclear.

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).


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)

A model for structural rearrangements during late stages of 40S subunit maturation. (A) Proposed model for the function of ribosome assembly factors in 40S assembly. Binding of assembly factors (AF) to RNA is required to maintain a more flexible conformation. Their presence allows the assembly of ribosomal proteins (RP) but prevent that these RPs adopt their final conformation. At some point during the maturation pathway, the system receives a signal that certain ribosome assembly/RNA folding steps have been completed and AFs are no longer needed. Their release could be triggered through phosphorylation (such as Hrr25-dependent phosphorylation of Enp1 and Ltv1). This allows completion of RNA folding steps and binding of late RPs. (B and C) Model for rearrangements that take place in the head domain during late stages of 40S assembly. In early and middle pre-40S complexes, binding of Ltv1 and Enp1 to the head domain is required to maintain a more flexible conformation in the 3′ major domain. Rps3, Rps17, Rps20 and Rps17 assemble into pre-ribosomes but do not adopt their final conformation. Hrr25 phosphorylation of Ltv1 and Enp1 triggers their release, allowing late RNA folding steps and assembly of head domain r-proteins to be completed. We propose that Rio2 and Tsr1 are released shortly afterward (B) and Rio1 enters the assembly pathway. The exact function of Rio1, however, remains unclear.
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Related In: Results  -  Collection

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Figure 9: A model for structural rearrangements during late stages of 40S subunit maturation. (A) Proposed model for the function of ribosome assembly factors in 40S assembly. Binding of assembly factors (AF) to RNA is required to maintain a more flexible conformation. Their presence allows the assembly of ribosomal proteins (RP) but prevent that these RPs adopt their final conformation. At some point during the maturation pathway, the system receives a signal that certain ribosome assembly/RNA folding steps have been completed and AFs are no longer needed. Their release could be triggered through phosphorylation (such as Hrr25-dependent phosphorylation of Enp1 and Ltv1). This allows completion of RNA folding steps and binding of late RPs. (B and C) Model for rearrangements that take place in the head domain during late stages of 40S assembly. In early and middle pre-40S complexes, binding of Ltv1 and Enp1 to the head domain is required to maintain a more flexible conformation in the 3′ major domain. Rps3, Rps17, Rps20 and Rps17 assemble into pre-ribosomes but do not adopt their final conformation. Hrr25 phosphorylation of Ltv1 and Enp1 triggers their release, allowing late RNA folding steps and assembly of head domain r-proteins to be completed. We propose that Rio2 and Tsr1 are released shortly afterward (B) and Rio1 enters the assembly pathway. The exact function of Rio1, however, remains unclear.
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).

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