Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Dey, Abhishek, Monroy-Eklund, Anais, Klotz, Kaitlin, Saha, Arpita, Davis, Justin, Li, Bibo, Laederach, Alain, Chakrabarti, Kausik
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
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
_version_ 1784609908833910784
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
work_keys_str_mv AT deyabhishek invivoarchitectureofthetelomerasernacatalyticcoreintrypanosomabrucei
AT monroyeklundanais invivoarchitectureofthetelomerasernacatalyticcoreintrypanosomabrucei
AT klotzkaitlin invivoarchitectureofthetelomerasernacatalyticcoreintrypanosomabrucei
AT sahaarpita invivoarchitectureofthetelomerasernacatalyticcoreintrypanosomabrucei
AT davisjustin invivoarchitectureofthetelomerasernacatalyticcoreintrypanosomabrucei
AT libibo invivoarchitectureofthetelomerasernacatalyticcoreintrypanosomabrucei
AT laederachalain invivoarchitectureofthetelomerasernacatalyticcoreintrypanosomabrucei
AT chakrabartikausik invivoarchitectureofthetelomerasernacatalyticcoreintrypanosomabrucei