Anti-sense oligonucleotide probing as a structural platform for studying ribonucleoprotein complex assembly
Abstract Investigating the intricate and rapid folding kinetics of large RNA-protein complexes (RNPs), like the bacterial ribosome, remains a formidable challenge in structural biology. Previous genetic approaches to probe assembly have focused on modulating the expression of either r-proteins or as...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-61640-1 |
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| Summary: | Abstract Investigating the intricate and rapid folding kinetics of large RNA-protein complexes (RNPs), like the bacterial ribosome, remains a formidable challenge in structural biology. Previous genetic approaches to probe assembly have focused on modulating the expression of either r-proteins or assembly factors. Here, anti-sense oligonucleotides (ASOs) were used to disrupt native RNA/RNA and RNA/protein interactions, in order to generate previously uncharacterized folding intermediates. In an in vitro co-transcriptional ribosome assembly assay, 10 assembly inhibitor ASOs were identified. Using cryo-electron microscopy, 38 intermediate structures were determined resulting from the specific inhibitions generated by 6 inhibitory ASOs. A notable intermediate class provided compelling evidence for independent rRNA domain folding before proper interdomain docking. Three PNAs targeting domain-I of 23S rRNA further subdivide the previously identified assembly core into smaller blocks representing the earliest steps in assembly. The resulting assembly graph reveals template-directed RNA docking of defined regions as foldons, and domain consolidation, which provides a hierarchical view of the RNP assembly process. This approach not only identifies potential targets for antibiotic development but also establishes a platform for probing the structure and dynamics of RNP assemblies. |
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| ISSN: | 2041-1723 |