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In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei
Telomerase is a unique ribonucleoprotein (RNP) reverse transcriptase that utilizes its cognate RNA molecule as a template for telomere DNA repeat synthesis. Telomerase contains the reverse transcriptase protein, TERT and the template RNA, TR, as its core components. The 5’-half of TR forms a highly...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8643685/ https://www.ncbi.nlm.nih.gov/pubmed/34850114 http://dx.doi.org/10.1093/nar/gkab1042 |
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author | Dey, Abhishek Monroy-Eklund, Anais Klotz, Kaitlin Saha, Arpita Davis, Justin Li, Bibo Laederach, Alain Chakrabarti, Kausik |
author_facet | Dey, Abhishek Monroy-Eklund, Anais Klotz, Kaitlin Saha, Arpita Davis, Justin Li, Bibo Laederach, Alain Chakrabarti, Kausik |
author_sort | Dey, Abhishek |
collection | PubMed |
description | Telomerase is a unique ribonucleoprotein (RNP) reverse transcriptase that utilizes its cognate RNA molecule as a template for telomere DNA repeat synthesis. Telomerase contains the reverse transcriptase protein, TERT and the template RNA, TR, as its core components. The 5’-half of TR forms a highly conserved catalytic core comprising of the template region and adjacent domains necessary for telomere synthesis. However, how telomerase RNA folding takes place in vivo has not been fully understood due to low abundance of the native RNP. Here, using unicellular pathogen Trypanosoma brucei as a model, we reveal important regional folding information of the native telomerase RNA core domains, i.e. TR template, template boundary element, template proximal helix and Helix IV (eCR4-CR5) domain. For this purpose, we uniquely combined in-cell probing with targeted high-throughput RNA sequencing and mutational mapping under three conditions: in vivo (in WT and TERT(−/−) cells), in an immunopurified catalytically active telomerase RNP complex and ex vivo (deproteinized). We discover that TR forms at least two different conformers with distinct folding topologies in the insect and mammalian developmental stages of T. brucei. Also, TERT does not significantly affect the RNA folding in vivo, suggesting that the telomerase RNA in T. brucei exists in a conformationally preorganized stable structure. Our observed differences in RNA (TR) folding at two distinct developmental stages of T. brucei suggest that important conformational changes are a key component of T. brucei development. |
format | Online Article Text |
id | pubmed-8643685 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-86436852021-12-06 In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei Dey, Abhishek Monroy-Eklund, Anais Klotz, Kaitlin Saha, Arpita Davis, Justin Li, Bibo Laederach, Alain Chakrabarti, Kausik Nucleic Acids Res RNA Prot Comp Telomerase is a unique ribonucleoprotein (RNP) reverse transcriptase that utilizes its cognate RNA molecule as a template for telomere DNA repeat synthesis. Telomerase contains the reverse transcriptase protein, TERT and the template RNA, TR, as its core components. The 5’-half of TR forms a highly conserved catalytic core comprising of the template region and adjacent domains necessary for telomere synthesis. However, how telomerase RNA folding takes place in vivo has not been fully understood due to low abundance of the native RNP. Here, using unicellular pathogen Trypanosoma brucei as a model, we reveal important regional folding information of the native telomerase RNA core domains, i.e. TR template, template boundary element, template proximal helix and Helix IV (eCR4-CR5) domain. For this purpose, we uniquely combined in-cell probing with targeted high-throughput RNA sequencing and mutational mapping under three conditions: in vivo (in WT and TERT(−/−) cells), in an immunopurified catalytically active telomerase RNP complex and ex vivo (deproteinized). We discover that TR forms at least two different conformers with distinct folding topologies in the insect and mammalian developmental stages of T. brucei. Also, TERT does not significantly affect the RNA folding in vivo, suggesting that the telomerase RNA in T. brucei exists in a conformationally preorganized stable structure. Our observed differences in RNA (TR) folding at two distinct developmental stages of T. brucei suggest that important conformational changes are a key component of T. brucei development. Oxford University Press 2021-11-24 /pmc/articles/PMC8643685/ /pubmed/34850114 http://dx.doi.org/10.1093/nar/gkab1042 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | RNA Prot Comp Dey, Abhishek Monroy-Eklund, Anais Klotz, Kaitlin Saha, Arpita Davis, Justin Li, Bibo Laederach, Alain Chakrabarti, Kausik In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei |
title |
In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei |
title_full |
In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei |
title_fullStr |
In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei |
title_full_unstemmed |
In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei |
title_short |
In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei |
title_sort | in vivo architecture of the telomerase rna catalytic core in trypanosoma brucei |
topic | RNA Prot Comp |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8643685/ https://www.ncbi.nlm.nih.gov/pubmed/34850114 http://dx.doi.org/10.1093/nar/gkab1042 |
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