Functional analysis of three Arabidopsis argonautes using slicer-defective mutants

Alberto Carbonell, Noah Fahlgren, Hernan Garcia Ruiz, Kerrigan B. Gilbert, Taiowa A. Montgomery, Tammy Nguyen, Josh T. Cuperus, James C. Carrington

Research output: Contribution to journalArticle

118 Citations (Scopus)

Abstract

In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicerindependent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicerdefective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.

Original languageEnglish (US)
Pages (from-to)3613-3629
Number of pages17
JournalPlant Cell
Volume24
Issue number9
DOIs
StatePublished - Sep 1 2012

Fingerprint

Arabidopsis
MicroRNAs
RNA
mutants
microRNA
slicing
Small Interfering RNA
small interfering RNA
Antiviral Agents
Genome
Arabidopsis Proteins
Mosaic Viruses
Brassica napus
Turnip mosaic virus
genome
RNA Interference
assays
active sites
Catalytic Domain
Arabidopsis thaliana

ASJC Scopus subject areas

  • Plant Science
  • Cell Biology

Cite this

Carbonell, A., Fahlgren, N., Garcia Ruiz, H., Gilbert, K. B., Montgomery, T. A., Nguyen, T., ... Carrington, J. C. (2012). Functional analysis of three Arabidopsis argonautes using slicer-defective mutants. Plant Cell, 24(9), 3613-3629. https://doi.org/10.1105/tpc.112.099945

Functional analysis of three Arabidopsis argonautes using slicer-defective mutants. / Carbonell, Alberto; Fahlgren, Noah; Garcia Ruiz, Hernan; Gilbert, Kerrigan B.; Montgomery, Taiowa A.; Nguyen, Tammy; Cuperus, Josh T.; Carrington, James C.

In: Plant Cell, Vol. 24, No. 9, 01.09.2012, p. 3613-3629.

Research output: Contribution to journalArticle

Carbonell, A, Fahlgren, N, Garcia Ruiz, H, Gilbert, KB, Montgomery, TA, Nguyen, T, Cuperus, JT & Carrington, JC 2012, 'Functional analysis of three Arabidopsis argonautes using slicer-defective mutants', Plant Cell, vol. 24, no. 9, pp. 3613-3629. https://doi.org/10.1105/tpc.112.099945
Carbonell A, Fahlgren N, Garcia Ruiz H, Gilbert KB, Montgomery TA, Nguyen T et al. Functional analysis of three Arabidopsis argonautes using slicer-defective mutants. Plant Cell. 2012 Sep 1;24(9):3613-3629. https://doi.org/10.1105/tpc.112.099945
Carbonell, Alberto ; Fahlgren, Noah ; Garcia Ruiz, Hernan ; Gilbert, Kerrigan B. ; Montgomery, Taiowa A. ; Nguyen, Tammy ; Cuperus, Josh T. ; Carrington, James C. / Functional analysis of three Arabidopsis argonautes using slicer-defective mutants. In: Plant Cell. 2012 ; Vol. 24, No. 9. pp. 3613-3629.
@article{931fb65e140f4786af3c88982e1c5b69,
title = "Functional analysis of three Arabidopsis argonautes using slicer-defective mutants",
abstract = "In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicerindependent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicerdefective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.",
author = "Alberto Carbonell and Noah Fahlgren and {Garcia Ruiz}, Hernan and Gilbert, {Kerrigan B.} and Montgomery, {Taiowa A.} and Tammy Nguyen and Cuperus, {Josh T.} and Carrington, {James C.}",
year = "2012",
month = "9",
day = "1",
doi = "10.1105/tpc.112.099945",
language = "English (US)",
volume = "24",
pages = "3613--3629",
journal = "Plant Cell",
issn = "1040-4651",
publisher = "American Society of Plant Biologists",
number = "9",

}

TY - JOUR

T1 - Functional analysis of three Arabidopsis argonautes using slicer-defective mutants

AU - Carbonell, Alberto

AU - Fahlgren, Noah

AU - Garcia Ruiz, Hernan

AU - Gilbert, Kerrigan B.

AU - Montgomery, Taiowa A.

AU - Nguyen, Tammy

AU - Cuperus, Josh T.

AU - Carrington, James C.

PY - 2012/9/1

Y1 - 2012/9/1

N2 - In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicerindependent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicerdefective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.

AB - In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicerindependent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicerdefective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.

UR - http://www.scopus.com/inward/record.url?scp=84868089450&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84868089450&partnerID=8YFLogxK

U2 - 10.1105/tpc.112.099945

DO - 10.1105/tpc.112.099945

M3 - Article

VL - 24

SP - 3613

EP - 3629

JO - Plant Cell

JF - Plant Cell

SN - 1040-4651

IS - 9

ER -