Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit

Abstract

Abstract Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a ‘minimally invasive’ system for placement of a non-natural, clickable nucleobase within the total cellular RNA.

Autoren

Larissa Bessler
Navpreet Kaur
Lea-Marie Vogt
Laurin Flemmich
Carmen Siebenaller
Marie-Luise Winz
Francesca Tuorto
Ronald Micura
Ann E Ehrenhofer-Murray
Mark Helm

DOI

10.1093/nar/gkac822

eISSN

1362-4962

ISSN

0305-1048

Ausgabe der Veröffentlichung

18

Zeitschrift

Nucleic Acids Research

Sprache

en

Online publication date

2022

Paginierung

10785 - 10800

Datum der Veröffentlichung

2022

Status

Published

Herausgeber

Oxford University Press (OUP)

Herausgeber URL

http://dx.doi.org/10.1093/nar/gkac822

Datum der Datenerfassung

2022

Titel

Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit

Ausgabe der Zeitschrift

50

Data source: Crossref

Abstract

Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a 'minimally invasive' system for placement of a non-natural, clickable nucleobase within the total cellular RNA.

Addresses

Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, 55128 Mainz, Germany.

Autoren

Larissa Bessler
Navpreet Kaur
Lea-Marie Vogt
Laurin Flemmich
Carmen Siebenaller
Marie-Luise Winz
Francesca Tuorto
Ronald Micura
Ann E Ehrenhofer-Murray
Mark Helm

DOI

10.1093/nar/gkac822

eISSN

1362-4962

Externe Identifier

PubMed Identifier: 36169220
PubMed Central ID: PMC9561289

Funding acknowledgements

Deutsche Forschungsgemeinschaft: TRR-319 TP B05
Austrian Science Fund FWF: F8011-B
Austrian Science Fund FWF: P31691
Johannes Gutenberg University:
Deutsche Forschungsgemeinschaft: SPP1784
Deutsche Forschungsgemeinschaft: TRR-319 TP A06
Deutsche Forschungsgemeinschaft: HE 3397/13-2
Deutsche Forschungsgemeinschaft: HE 3397/14-2
Deutsche Forschungsgemeinschaft: DFG SPP1784
Deutsche Forschungsgemeinschaft: TRR-319 TP C03

Open access

true

ISSN

0305-1048

Ausgabe der Veröffentlichung

18

Zeitschrift

Nucleic acids research

Schlüsselwörter

Humans
Escherichia coli
Schizosaccharomyces
Azides
Biotin
5-Methylcytosine
tRNA Methyltransferases
RNA, Transfer
RNA, Transfer, Asp
Nucleoside Q
Fluorescent Dyes

Sprache

eng

Medium

Print

Open access status

Open Access

Paginierung

10785 - 10800

Datum der Veröffentlichung

2022

Status

Published

Publisher licence

CC BY

Datum der Datenerfassung

2022

Titel

Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit.

Sub types

Research Support, Non-U.S. Gov't
research-article
Journal Article

Ausgabe der Zeitschrift

50

Files

https://academic.oup.com/nar/article-pdf/50/18/10785/46501429/gkac822.pdf https://europepmc.org/articles/PMC9561289?pdf=render

Data source: Europe PubMed Central

Abstract

Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a 'minimally invasive' system for placement of a non-natural, clickable nucleobase within the total cellular RNA.

Date of acceptance

2022

Autoren

Larissa Bessler
Navpreet Kaur
Lea-Marie Vogt
Laurin Flemmich
Carmen Siebenaller
Marie-Luise Winz
Francesca Tuorto
Ronald Micura
Ann E Ehrenhofer-Murray
Mark Helm

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/36169220

DOI

10.1093/nar/gkac822

eISSN

1362-4962

Externe Identifier

PubMed Central ID: PMC9561289

Ausgabe der Veröffentlichung

18

Zeitschrift

Nucleic Acids Res

Schlüsselwörter

5-Methylcytosine
Azides
Biotin
Escherichia coli
Fluorescent Dyes
Humans
Nucleoside Q
RNA, Transfer
RNA, Transfer, Asp
Schizosaccharomyces
tRNA Methyltransferases

Sprache

eng

Country

England

Paginierung

10785 - 10800

PII

6725769

Datum der Veröffentlichung

2022

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

2022

Titel

Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit.

Sub types

Journal Article
Research Support, Non-U.S. Gov't

Ausgabe der Zeitschrift

50

Data source: PubMed

Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit

Files

Tools