Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq
Abstract Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-pseudoknot (t/PK) and the three-way junction (CR4/5). These hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are essential for telomeras...
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Nature Portfolio
2025-01-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-56149-6 |
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| author | Nicholas M. Forino Jia Zheng Woo Arthur J. Zaug Arcelia Gonzalez Jimenez Eva Edelson Thomas R. Cech Silvi Rouskin Michael D. Stone |
| author_facet | Nicholas M. Forino Jia Zheng Woo Arthur J. Zaug Arcelia Gonzalez Jimenez Eva Edelson Thomas R. Cech Silvi Rouskin Michael D. Stone |
| author_sort | Nicholas M. Forino |
| collection | DOAJ |
| description | Abstract Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-pseudoknot (t/PK) and the three-way junction (CR4/5). These hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are essential for telomerase activity. Here, we probe hTR structure in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features required for hTERT binding. The proportion of hTR CR4/5 folded into the primary functional conformation is independent of hTERT expression levels. Mutations that stabilize the alternative CR4/5 conformation are detrimental to telomerase assembly and activity. Moreover, the alternative CR4/5 conformation is not found in purified telomerase RNP complexes, supporting the hypothesis that only the primary CR4/5 conformer is active. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded, suggesting that kinetic RNA folding traps studied in vitro may also hinder ribonucleoprotein assembly in vivo. |
| format | Article |
| id | doaj-art-12ade4e8ad2c4b2fa81159e4c521dbb8 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
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| series | Nature Communications |
| spelling | doaj-art-12ade4e8ad2c4b2fa81159e4c521dbb82025-01-26T12:42:39ZengNature PortfolioNature Communications2041-17232025-01-0116111510.1038/s41467-025-56149-6Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseqNicholas M. Forino0Jia Zheng Woo1Arthur J. Zaug2Arcelia Gonzalez Jimenez3Eva Edelson4Thomas R. Cech5Silvi Rouskin6Michael D. Stone7Department of Molecular, Cell, and Developmental Biology, University of CaliforniaDepartment of Microbiology, Harvard Medical SchoolDepartment of Biochemistry, University of ColoradoDepartment of Chemistry and Biochemistry, University of CaliforniaDepartment of Microbiology and Environmental Toxicology, University of CaliforniaDepartment of Biochemistry, University of ColoradoDepartment of Microbiology, Harvard Medical SchoolDepartment of Chemistry and Biochemistry, University of CaliforniaAbstract Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-pseudoknot (t/PK) and the three-way junction (CR4/5). These hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are essential for telomerase activity. Here, we probe hTR structure in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features required for hTERT binding. The proportion of hTR CR4/5 folded into the primary functional conformation is independent of hTERT expression levels. Mutations that stabilize the alternative CR4/5 conformation are detrimental to telomerase assembly and activity. Moreover, the alternative CR4/5 conformation is not found in purified telomerase RNP complexes, supporting the hypothesis that only the primary CR4/5 conformer is active. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded, suggesting that kinetic RNA folding traps studied in vitro may also hinder ribonucleoprotein assembly in vivo.https://doi.org/10.1038/s41467-025-56149-6 |
| spellingShingle | Nicholas M. Forino Jia Zheng Woo Arthur J. Zaug Arcelia Gonzalez Jimenez Eva Edelson Thomas R. Cech Silvi Rouskin Michael D. Stone Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq Nature Communications |
| title | Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq |
| title_full | Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq |
| title_fullStr | Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq |
| title_full_unstemmed | Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq |
| title_short | Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq |
| title_sort | telomerase rna structural heterogeneity in living human cells detected by dms mapseq |
| url | https://doi.org/10.1038/s41467-025-56149-6 |
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