Modulation of conformational changes in helix 69 mutants by pseudouridine modifications

Jiang, Jun; Kharel, Daya Nidhi; Chow, Christine S.

doi:10.1016/j.bpc.2015.03.001

Cited by 8 publications

(9 citation statements)

References 44 publications

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“…The consecutive Ψ38 and Ψ39 residues in tRNA His provide stacking stabilization for bases on the 3′ side of the tRNA anticodon loop 17 . Also, the conserved Ψ modifications in helix 69 (H69) from large subunit ribosomal RNA have been shown to modulate the structure and conformational behavior of the stem-loop region by stabilizing the loop-closing base pair and promoting base stacking in the 3′ half of the loop 18,19 and an additional A-Ψ base pair within the loop 20 . The UV melting data revealed that the Ψ residues in the stem region provided thermodynamic stabilization to the RNA, whereas individual Ψs located in the loop region contributed to a slight destabilization 21,22 .…”

Section: Introductionmentioning

confidence: 99%

Computational and NMR studies of RNA duplexes with an internal pseudouridine-adenosine base pair

Deb

Popenda

Sarzyñska

et al. 2019

Sci Rep

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Pseudouridine (Ψ) is the most common chemical modification present in RNA. In general, Ψ increases the thermodynamic stability of RNA. However, the degree of stabilization depends on the sequence and structural context. To explain experimentally observed sequence dependence of the effect of Ψ on the thermodynamic stability of RNA duplexes, we investigated the structure, dynamics and hydration of RNA duplexes with an internal Ψ-A base pair in different nearest-neighbor sequence contexts. The structures of two RNA duplexes containing 5′-GΨC/3′-CAG and 5′-CΨG/3′-GAC motifs were determined using NMR spectroscopy. To gain insight into the effect of Ψ on duplex dynamics and hydration, we performed molecular dynamics (MD) simulations of RNA duplexes with 5′-GΨC/3′-CAG, 5′-CΨG/3′-GAC, 5′-AΨU/3′-UAA and 5′-UΨA/3′-AAU motifs and their unmodified counterparts. Our results showed a subtle impact from Ψ modification on the structure and dynamics of the RNA duplexes studied. The MD simulations confirmed the change in hydration pattern when U is replaced with Ψ. Quantum chemical calculations showed that the replacement of U with Ψ affected the intrinsic stacking energies at the base pair steps depending on the sequence context. The calculated intrinsic stacking energies help to explain the experimentally observed sequence dependent changes in the duplex stability from Ψ modification.

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Section: Introductionmentioning

confidence: 99%

Computational and NMR studies of RNA duplexes with an internal pseudouridine-adenosine base pair

Deb

Popenda

Sarzyñska

et al. 2019

Sci Rep

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“…Since previous chemical probing studies were consistent with conformational changes in the H69 loop upon subunit association, 35 this result suggests crosstalk between the H69 stem and loop regions on the ribosome level, which was previously observed in H69 model systems. 41,43 Such conformational rearrangements appear to affect the binding scaffold for aminoglycosides within the stem region of H69, as shown by the differences in DMS reactivity at G1922 ( i.e ., protection by neomycin or paromomycin) between isolated subunits and complete ribosomes.…”

Section: Resultsmentioning

confidence: 99%

Pseudouridine modifications influence binding of aminoglycosides to helix 69 of bacterial ribosomes

Sakakibara

Chow

2017

Org. Biomol. Chem.

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Development of antibiotics that target new regions of functionality is a possible way to overcome antibiotic resistance. In this study, the interactions of aminoglycoside antibiotics with helix 69 of the E. coli 23S rRNA in the context of complete 70S ribosomes or the isolated 50S subunit were investigated by using chemical probing and footprinting analysis. Helix 69 is a dynamic RNA motif that plays major roles in bacterial ribosome activity. Neomycin, paromomycin, and gentamicin interact with the stem region of helix 69 in complete 70S ribosomes, but have diminished binding to the isolated 50S subunit. Pseudouridine modifications in helix 69 were shown to impact the aminoglycoside interactions. These results suggest a requirement for a specific conformational state of helix 69 for efficient aminoglycoside binding, and imply that this motif may be a suitable target for mechanism-based therapeutics.

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“…Helix 69 participates in almost every step of translation and establishes direct contacts with multiple translation factors . This helix was also identified as an antibiotic binding site. , With synthetic 19-mer RNA constructs of H69, the contribution of each residue to global thermodynamic stability was determined, allowing for a deeper understanding of the effects of mutations or loss of modifications in H69. , …”

Section: Modifications In H31 H44 and H69mentioning

confidence: 99%

“…In human H69, Ψ residues 3731 and 3733 (1915 and 1917 in E. coli ) are less destabilizing than their bacterial counterparts, with the difference in thermodynamic contributions of the modifications being attributed to the loop sequence (A1918 in bacteria compared with G1918 in eukaryotes) . Later studies in our laboratory revealed stem–loop crosstalk in H69, indicating that differences in the stem sequences of bacterial and human H69s can be propagated to the loop regions …”

Section: Modifications In H31 H44 and H69mentioning

confidence: 99%

“…40,41 With synthetic 19-mer RNA constructs of H69, the contribution of each residue to global thermodynamic stability was determined, 29 allowing for a deeper understanding of the effects of mutations or loss of modifications in H69. 31,42 The differential contributions of each Ψ to the global thermodynamic stability of E. coli H69 were quantified using three separate synthetic RNA constructs (Figure 7A). 29 Residue Ψ1911 is stabilizing, whereas Ψ1915 and Ψ1917 are destabilizing.…”

Section: ■ Ribosomal Rna Modificationsmentioning

confidence: 99%

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Post-transcriptional Modifications Modulate rRNA Structure and Ligand Interactions

2016

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Post-transcriptional modifications play important roles in modulating the functions of RNA species. The presence of modifications in RNA may directly alter its interactions with binding partners or cause structural changes that indirectly affect ligand recognition. Given the rapidly growing list of modifications identified in noncoding and mRNAs associated with human disease, as well as the dynamic control over modifications involved in various physiological processes, it is imperative to understand RNA structural modulation by these modifications. Among the RNA species, rRNAs provide numerous examples of modification types located in differing sequence and structural contexts. In addition, the modified rRNA motifs participate in a wide variety of ligand interactions, including those with RNA, protein, and small molecules. In fact, several classes of antibiotics exert their effects on protein synthesis by binding to functionally important and highly modified regions of the rRNAs. These RNA regions often display conservation in sequence, secondary structure, tertiary interactions, and modifications, trademarks of ideal drug-targeting sites. Furthermore, ligand interactions with such regions often favor certain modification-induced conformational states of the RNA. Our laboratory has employed a combination of biophysical methods such as nuclear magnetic resonance spectroscopy (NMR), circular dichroism, and UV melting to study rRNA modifications in functionally important motifs, including helix 31 (h31) and helix h44 (h44) of the small subunit rRNA and helix 69 (H69) of the large subunit rRNA. The modified RNA oligonucleotides used in these studies were generated by solid-phase synthesis with a variety of phosphoramidite chemistries. The natural modifications were shown to impact thermal stability, dynamic behavior, and tertiary structures of the RNAs, with additive or cooperative effects occurring with multiple, clustered modifications. Taking advantage of the structural diversity offered by specific modifications in the chosen rRNA motifs, phage display was used to select peptides that bind with moderate (low micromolar) affinity and selectivity to modified h31, h44, and H69. Interactions between peptide ligands and RNAs were monitored by biophysical methods, including electrospray ionization mass spectrometry (ESI-MS), NMR, and surface plasmon resonance (SPR). The peptides compare well with natural compounds such as aminoglycosides in their binding affinities to the modified rRNA constructs. Some candidates were shown to exhibit specificity toward different modification states of the rRNA motifs. The selected peptides may be further optimized for improved RNA targeting or used in screening assays for new drug candidates. In this Account, we hope to stimulate interest in bioorganic and biophysical approaches, which may be used to deepen our understanding of other functionally important, naturally modified RNAs beyond the rRNAs.

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Modulation of conformational changes in helix 69 mutants by pseudouridine modifications

Cited by 8 publications

References 44 publications

Computational and NMR studies of RNA duplexes with an internal pseudouridine-adenosine base pair

Computational and NMR studies of RNA duplexes with an internal pseudouridine-adenosine base pair

Pseudouridine modifications influence binding of aminoglycosides to helix 69 of bacterial ribosomes

Post-transcriptional Modifications Modulate rRNA Structure and Ligand Interactions

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