HITS-CLIP analysis of human ALKBH8 reveals interactions with fully processed substrate tRNAs and with specific noncoding RNAs

Ivana Cavallin; Marek Bartosovic; Tomas Skalicky; Praveenkumar Rengaraj; Martin Demko; Martina Christina Schmidt-Dengler; Aleksej Drino; Mark Helm; Stepanka Vanacova

doi:10.1261/rna.079421.122

HITS-CLIP analysis of human ALKBH8 reveals interactions with fully processed substrate tRNAs and with specific noncoding RNAs

Ivana Cavallin
, Marek Bartosovic
, Tomas Skalicky
, Praveenkumar Rengaraj
, Martin Demko
, Martina Christina Schmidt-Dengler
, Aleksej Drino
, Mark Helm
, Stepanka Vanacova

Research output: Contribution to journal › Article › peer-review

5 Scopus citations

Abstract

Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U–containing tRNAs obtain 5-methoxycar-bonylmethyluridine (mcm⁵U₃₄) or 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U₃₄), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U–containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.

Original language	English
Pages (from-to)	1568-1581
Number of pages	14
Journal	RNA
Volume	28
Issue number	12
DOIs	https://doi.org/10.1261/rna.079421.122
State	Published - Dec 2022
Externally published	Yes

Bibliographical note

Publisher Copyright:
© 2022 Cold Spring Harbor Laboratory Press. All rights reserved.

Funding

We want to thank Karolina Vavrouskova for excellent technical support, Dasa Zigackova for help with cell fractionation and Hana Macickova Cahova for helpful advice on tRNA modifications. This work was supported by the Czech Science Foundation (19-21829S to S.V.; T.S. was supported by 20-19617S), the Ministry of Education, Youth and Sports within program INTER-COST (LTC18052) and the institutional support of CEITEC 2020 (LQ1601). M.B. was supported by Brno City Municipality Scholarship for Talented Ph.D. Students. We further acknowledge the CELLIM core facility supported by MEYS CR (LM2018129 Czech-BioImaging). Computational resources were provided by the project “e-Infrastruktura CZ” (e-INFRA LM2018140) provided within the program Projects of Large Research, Development, and Innovations Infrastructures.

Funders	Funder number
CEITEC 2020	LQ1601
INTER-COST	LTC18052
Innovations Infrastructures
Ministerstvo Školství, Mládeže a Tělovýchovy	LM2018129, LM2018140
Grantová Agentura České Republiky	20-19617S, 19-21829S

Keywords

ALKBH8
HITS-CLIP
Trm9
mcmU
mcmsU
wobble uridine modification

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10.1261/rna.079421.122

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title = "HITS-CLIP analysis of human ALKBH8 reveals interactions with fully processed substrate tRNAs and with specific noncoding RNAs",

abstract = "Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U–containing tRNAs obtain 5-methoxycar-bonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U–containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.",

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author = "Ivana Cavallin and Marek Bartosovic and Tomas Skalicky and Praveenkumar Rengaraj and Martin Demko and Schmidt-Dengler, \{Martina Christina\} and Aleksej Drino and Mark Helm and Stepanka Vanacova",

year = "2022",

month = dec,

doi = "10.1261/rna.079421.122",

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volume = "28",

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AU - Cavallin, Ivana

AU - Bartosovic, Marek

AU - Skalicky, Tomas

AU - Rengaraj, Praveenkumar

AU - Demko, Martin

AU - Schmidt-Dengler, Martina Christina

AU - Drino, Aleksej

AU - Helm, Mark

AU - Vanacova, Stepanka

PY - 2022/12

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N2 - Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U–containing tRNAs obtain 5-methoxycar-bonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U–containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.

AB - Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U–containing tRNAs obtain 5-methoxycar-bonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U–containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.

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HITS-CLIP analysis of human ALKBH8 reveals interactions with fully processed substrate tRNAs and with specific noncoding RNAs

Abstract

Bibliographical note

Funding

Keywords

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