In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei

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Chakrabarti, K., Dey, A., Klotz, K., Davis, J., Monroy-Eklund, A., Saha, A., … Laederach, A. (2021). In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei. Nucleic Acids Research. https://doi.org/doi:10.1093/nar/gkab1042
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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.

Details
Title

In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei

Author(s)

Chakrabarti, Kausik
Dey, Abishek
Klotz, Kaitlin
Davis, Justin
Monroy-Eklund, Anais
Saha, Arpita
Li, Bibo
Laederach, Alain

UNC Charlotte Affiliate Type

Faculty

Publisher

Oxford University Press

Extent

1 online resource

Date Published

2021

Material Type

Journal article

Genre

articles

Resource Type

text

Abstract

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.

Subjects--Topics

RNA
Nucleoproteins

Is Part Of

Nucleic acids research

Volume

Vol. 49, issue 21

Beginning Page Number

12445

Ending Page Number

12466

Language

English

Published Version (Please cite this version)

doi:10.1093/nar/gkab1042

Publisher's Version

https://academic.oup.com/nar/article/49/21/12445/6438038

Permalink

http://hdl.handle.net/20.500.13093/work:235

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