Unambiguous Identification of miRNA: Target site interactions by different types of ligation reactions

Stefanie Grosswendt; Andrei Filipchyk; Mark Manzano; Filippos Klironomos; Marcel Schilling; Margareta Herzog; Eva Gottwein; Nikolaus Rajewsky

doi:10.1016/j.molcel.2014.03.049

Unambiguous Identification of miRNA: Target site interactions by different types of ligation reactions

Stefanie Grosswendt, Andrei Filipchyk, Mark Manzano, Filippos Klironomos, Marcel Schilling, Margareta Herzog, Eva Gottwein, Nikolaus Rajewsky^*

^*Corresponding author for this work

Microbiology-Immunology

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

224 Scopus citations

Abstract

To exert regulatory function, miRNAs guide Argonaute (AGO) proteins to partially complementary sites on target RNAs. Crosslinking and immunoprecipitation (CLIP) assays are state-of-the-art to map AGO binding sites, but assigning the targeting miRNA to these sites relies on bioinformatics predictions and is therefore indirect. To directly and unambiguously identify miRNA:target site interactions, we modified our CLIP methodology in C.elegans to experimentally ligate miRNAs to their target sites. Unexpectedly, ligation reactions also occurred in the absence of the exogenous ligase. Our invivo data set and reanalysis of published mammalian AGO-CLIP data for miRNA-chimeras yielded ~17,000 miRNA:target site interactions. Analysis of interactions and extensive experimental validation of chimera-discovered targets of viral miRNAs suggest that our strategy identifies canonical, noncanonical, and nonconserved miRNA:targets. About 80% of miRNA interactions have perfect or partial seed complementarity. In summary, analysis of miRNA:target chimeras enables the systematic, context-specific, invivo discovery of miRNA binding.

Original language	English (US)
Pages (from-to)	1042-1054
Number of pages	13
Journal	Molecular cell
Volume	54
Issue number	6
DOIs	https://doi.org/10.1016/j.molcel.2014.03.049
State	Published - Jun 19 2014

Funding

We thank F. Slack for the GFP::ALG-1 transgenic strain and M. Simard for the ALG-1 antibody. We thank all members of the Rajewsky lab for discussions and support. We acknowledge P. Shamulailatpam for technical assistance in validation experiments for viral miRNA interactions. C. Langnick and M. Feldkamp (W. Chen lab, MDC) performed sequencing runs. A.F. thanks the DFG Graduate School “Computational Systems Biology” CBS-GRK 1772 for a fellowship. F.K. acknowledges funding from DEEP (Deutsches Epigenom Programm), and M.S. acknowledges funding from the e:Bio program. Validation experiments for viral miRNA interactions in this publication were supported by the National Cancer Institute of the National Institutes of Health under Award Number U54CA143869. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

ASJC Scopus subject areas

Molecular Biology
Cell Biology

Access to Document

10.1016/j.molcel.2014.03.049

Cite this

@article{3d4bef4828f8449c8b0a0a89a3950c4f,

title = "Unambiguous Identification of miRNA: Target site interactions by different types of ligation reactions",

abstract = "To exert regulatory function, miRNAs guide Argonaute (AGO) proteins to partially complementary sites on target RNAs. Crosslinking and immunoprecipitation (CLIP) assays are state-of-the-art to map AGO binding sites, but assigning the targeting miRNA to these sites relies on bioinformatics predictions and is therefore indirect. To directly and unambiguously identify miRNA:target site interactions, we modified our CLIP methodology in C.elegans to experimentally ligate miRNAs to their target sites. Unexpectedly, ligation reactions also occurred in the absence of the exogenous ligase. Our invivo data set and reanalysis of published mammalian AGO-CLIP data for miRNA-chimeras yielded ~17,000 miRNA:target site interactions. Analysis of interactions and extensive experimental validation of chimera-discovered targets of viral miRNAs suggest that our strategy identifies canonical, noncanonical, and nonconserved miRNA:targets. About 80% of miRNA interactions have perfect or partial seed complementarity. In summary, analysis of miRNA:target chimeras enables the systematic, context-specific, invivo discovery of miRNA binding.",

author = "Stefanie Grosswendt and Andrei Filipchyk and Mark Manzano and Filippos Klironomos and Marcel Schilling and Margareta Herzog and Eva Gottwein and Nikolaus Rajewsky",

note = "Funding Information: We thank F. Slack for the GFP::ALG-1 transgenic strain and M. Simard for the ALG-1 antibody. We thank all members of the Rajewsky lab for discussions and support. We acknowledge P. Shamulailatpam for technical assistance in validation experiments for viral miRNA interactions. C. Langnick and M. Feldkamp (W. Chen lab, MDC) performed sequencing runs. A.F. thanks the DFG Graduate School “Computational Systems Biology” CBS-GRK 1772 for a fellowship. F.K. acknowledges funding from DEEP (Deutsches Epigenom Programm), and M.S. acknowledges funding from the e:Bio program. Validation experiments for viral miRNA interactions in this publication were supported by the National Cancer Institute of the National Institutes of Health under Award Number U54CA143869. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. ",

year = "2014",

month = jun,

day = "19",

doi = "10.1016/j.molcel.2014.03.049",

language = "English (US)",

volume = "54",

pages = "1042--1054",

journal = "Molecular cell",

issn = "1097-2765",

publisher = "Cell Press",

number = "6",

}

TY - JOUR

T1 - Unambiguous Identification of miRNA

T2 - Target site interactions by different types of ligation reactions

AU - Grosswendt, Stefanie

AU - Filipchyk, Andrei

AU - Manzano, Mark

AU - Klironomos, Filippos

AU - Schilling, Marcel

AU - Herzog, Margareta

AU - Gottwein, Eva

AU - Rajewsky, Nikolaus

N1 - Funding Information: We thank F. Slack for the GFP::ALG-1 transgenic strain and M. Simard for the ALG-1 antibody. We thank all members of the Rajewsky lab for discussions and support. We acknowledge P. Shamulailatpam for technical assistance in validation experiments for viral miRNA interactions. C. Langnick and M. Feldkamp (W. Chen lab, MDC) performed sequencing runs. A.F. thanks the DFG Graduate School “Computational Systems Biology” CBS-GRK 1772 for a fellowship. F.K. acknowledges funding from DEEP (Deutsches Epigenom Programm), and M.S. acknowledges funding from the e:Bio program. Validation experiments for viral miRNA interactions in this publication were supported by the National Cancer Institute of the National Institutes of Health under Award Number U54CA143869. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

PY - 2014/6/19

Y1 - 2014/6/19

N2 - To exert regulatory function, miRNAs guide Argonaute (AGO) proteins to partially complementary sites on target RNAs. Crosslinking and immunoprecipitation (CLIP) assays are state-of-the-art to map AGO binding sites, but assigning the targeting miRNA to these sites relies on bioinformatics predictions and is therefore indirect. To directly and unambiguously identify miRNA:target site interactions, we modified our CLIP methodology in C.elegans to experimentally ligate miRNAs to their target sites. Unexpectedly, ligation reactions also occurred in the absence of the exogenous ligase. Our invivo data set and reanalysis of published mammalian AGO-CLIP data for miRNA-chimeras yielded ~17,000 miRNA:target site interactions. Analysis of interactions and extensive experimental validation of chimera-discovered targets of viral miRNAs suggest that our strategy identifies canonical, noncanonical, and nonconserved miRNA:targets. About 80% of miRNA interactions have perfect or partial seed complementarity. In summary, analysis of miRNA:target chimeras enables the systematic, context-specific, invivo discovery of miRNA binding.

AB - To exert regulatory function, miRNAs guide Argonaute (AGO) proteins to partially complementary sites on target RNAs. Crosslinking and immunoprecipitation (CLIP) assays are state-of-the-art to map AGO binding sites, but assigning the targeting miRNA to these sites relies on bioinformatics predictions and is therefore indirect. To directly and unambiguously identify miRNA:target site interactions, we modified our CLIP methodology in C.elegans to experimentally ligate miRNAs to their target sites. Unexpectedly, ligation reactions also occurred in the absence of the exogenous ligase. Our invivo data set and reanalysis of published mammalian AGO-CLIP data for miRNA-chimeras yielded ~17,000 miRNA:target site interactions. Analysis of interactions and extensive experimental validation of chimera-discovered targets of viral miRNAs suggest that our strategy identifies canonical, noncanonical, and nonconserved miRNA:targets. About 80% of miRNA interactions have perfect or partial seed complementarity. In summary, analysis of miRNA:target chimeras enables the systematic, context-specific, invivo discovery of miRNA binding.

UR - http://www.scopus.com/inward/record.url?scp=84903616729&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84903616729&partnerID=8YFLogxK

U2 - 10.1016/j.molcel.2014.03.049

DO - 10.1016/j.molcel.2014.03.049

M3 - Article

C2 - 24857550

AN - SCOPUS:84903616729

SN - 1097-2765

VL - 54

SP - 1042

EP - 1054

JO - Molecular cell

JF - Molecular cell

IS - 6

ER -

Unambiguous Identification of miRNA: Target site interactions by different types of ligation reactions

Abstract

Funding

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this