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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| Format: | Article |
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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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| author | Kai Sheng Xiyu Dong Sriram Aiyer Joan Lee Selena Đorđević-Marquardt Dmitry Lyumkis James R. Williamson |
| author_facet | Kai Sheng Xiyu Dong Sriram Aiyer Joan Lee Selena Đorđević-Marquardt Dmitry Lyumkis James R. Williamson |
| author_sort | Kai Sheng |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-b3748c22c6574462beb13a41efd62c8d |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-b3748c22c6574462beb13a41efd62c8d2025-08-20T04:02:54ZengNature PortfolioNature Communications2041-17232025-07-0116111410.1038/s41467-025-61640-1Anti-sense oligonucleotide probing as a structural platform for studying ribonucleoprotein complex assemblyKai Sheng0Xiyu Dong1Sriram Aiyer2Joan Lee3Selena Đorđević-Marquardt4Dmitry Lyumkis5James R. Williamson6Department of Integrative Structural and Computational Biology, The Scripps Research InstituteDepartment of Integrative Structural and Computational Biology, The Scripps Research InstituteLaboratory of Genetics, The Salk Institute for Biological StudiesDepartment of Integrative Structural and Computational Biology, The Scripps Research InstituteLaboratory of Genetics, The Salk Institute for Biological StudiesDepartment of Integrative Structural and Computational Biology, The Scripps Research InstituteDepartment of Integrative Structural and Computational Biology, The Scripps Research InstituteAbstract 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.https://doi.org/10.1038/s41467-025-61640-1 |
| spellingShingle | Kai Sheng Xiyu Dong Sriram Aiyer Joan Lee Selena Đorđević-Marquardt Dmitry Lyumkis James R. Williamson Anti-sense oligonucleotide probing as a structural platform for studying ribonucleoprotein complex assembly Nature Communications |
| title | Anti-sense oligonucleotide probing as a structural platform for studying ribonucleoprotein complex assembly |
| title_full | Anti-sense oligonucleotide probing as a structural platform for studying ribonucleoprotein complex assembly |
| title_fullStr | Anti-sense oligonucleotide probing as a structural platform for studying ribonucleoprotein complex assembly |
| title_full_unstemmed | Anti-sense oligonucleotide probing as a structural platform for studying ribonucleoprotein complex assembly |
| title_short | Anti-sense oligonucleotide probing as a structural platform for studying ribonucleoprotein complex assembly |
| title_sort | anti sense oligonucleotide probing as a structural platform for studying ribonucleoprotein complex assembly |
| url | https://doi.org/10.1038/s41467-025-61640-1 |
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