High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down

Xiaoyi Li; Yuri Pritykin; Carla P. Concepcion; Yuheng Lu; Gaspare La Rocca; Minsi Zhang; Bryan King; Peter J. Cook; Yu Wah Au; Olesja Popow; Joao A. Paulo; Hannah G. Otis; Chiara Mastroleo; Paul Ogrodowski; Ryan Schreiner; Kevin M. Haigis; Doron Betel; Christina S. Leslie; Andrea Ventura

doi:10.1016/j.molcel.2020.05.009

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down

Xiaoyi Li, Yuri Pritykin, Carla P. Concepcion, Yuheng Lu, Gaspare La Rocca, Minsi Zhang, Bryan King, Peter J. Cook, Yu Wah Au, Olesja Popow, Joao A. Paulo, Hannah G. Otis, Chiara Mastroleo, Paul Ogrodowski, Ryan Schreiner, Kevin M. Haigis, Doron Betel, Christina S. Leslie, Andrea Ventura

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

25 Scopus citations

Abstract

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

Original language	English (US)
Pages (from-to)	167-179.e11
Journal	Molecular Cell
Volume	79
Issue number	1
DOIs	https://doi.org/10.1016/j.molcel.2020.05.009
State	Published - Jul 2 2020
Externally published	Yes

All Science Journal Classification (ASJC) codes

Molecular Biology
Cell Biology

Keywords

Ago2
CLIP
HaloTag
conditional
microRNAs
mouse
non-coding RNAs
targets

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10.1016/j.molcel.2020.05.009

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Li, X., Pritykin, Y., Concepcion, C. P., Lu, Y., La Rocca, G., Zhang, M., King, B., Cook, P. J., Au, Y. W., Popow, O., Paulo, J. A., Otis, H. G., Mastroleo, C., Ogrodowski, P., Schreiner, R., Haigis, K. M., Betel, D., Leslie, C. S., & Ventura, A. (2020). High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down. Molecular Cell, 79(1), 167-179.e11. https://doi.org/10.1016/j.molcel.2020.05.009

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title = "High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down",

abstract = "The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.",

keywords = "Ago2, CLIP, HaloTag, conditional, microRNAs, mouse, non-coding RNAs, targets",

author = "Xiaoyi Li and Yuri Pritykin and Concepcion, {Carla P.} and Yuheng Lu and {La Rocca}, Gaspare and Minsi Zhang and Bryan King and Cook, {Peter J.} and Au, {Yu Wah} and Olesja Popow and Paulo, {Joao A.} and Otis, {Hannah G.} and Chiara Mastroleo and Paul Ogrodowski and Ryan Schreiner and Haigis, {Kevin M.} and Doron Betel and Leslie, {Christina S.} and Andrea Ventura",

note = "Publisher Copyright: {\textcopyright} 2020 Elsevier Inc.",

year = "2020",

month = jul,

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doi = "10.1016/j.molcel.2020.05.009",

language = "English (US)",

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Li, X, Pritykin, Y, Concepcion, CP, Lu, Y, La Rocca, G, Zhang, M, King, B, Cook, PJ, Au, YW, Popow, O, Paulo, JA, Otis, HG, Mastroleo, C, Ogrodowski, P, Schreiner, R, Haigis, KM, Betel, D, Leslie, CS & Ventura, A 2020, 'High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down', Molecular Cell, vol. 79, no. 1, pp. 167-179.e11. https://doi.org/10.1016/j.molcel.2020.05.009

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T1 - High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down

AU - Li, Xiaoyi

AU - Pritykin, Yuri

AU - Concepcion, Carla P.

AU - Lu, Yuheng

AU - La Rocca, Gaspare

AU - Zhang, Minsi

AU - King, Bryan

AU - Cook, Peter J.

AU - Au, Yu Wah

AU - Popow, Olesja

AU - Paulo, Joao A.

AU - Otis, Hannah G.

AU - Mastroleo, Chiara

AU - Ogrodowski, Paul

AU - Schreiner, Ryan

AU - Haigis, Kevin M.

AU - Betel, Doron

AU - Leslie, Christina S.

AU - Ventura, Andrea

PY - 2020/7/2

Y1 - 2020/7/2

N2 - The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

AB - The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

KW - Ago2

KW - CLIP

KW - HaloTag

KW - conditional

KW - microRNAs

KW - mouse

KW - non-coding RNAs

KW - targets

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AN - SCOPUS:85087530351

SN - 1097-2765

VL - 79

SP - 167-179.e11

JO - Molecular Cell

JF - Molecular Cell

IS - 1

ER -

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down

Abstract

All Science Journal Classification (ASJC) codes

Keywords

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