Abstract
Argonaute (AGO) proteins play a central role in the RNA interference (RNAi) pathway, which is a cytoplasmic mechanism important for post-transcriptional regulation of gene expression. In Drosophila, AGO2 also functions in the nucleus to regulate chromatin insulator activity and transcription. Although there are a number of studies focused on AGO2 function, the regulation of AGO2 turnover is not well understood. We found that mutation of T1149 or R1158 in the conserved PIWI domain causes AGO2 protein instability, but only T1149 affects RNAi activity. Mass spec analysis shows that several proteasome components co-purify with both wildtype and mutant AGO2, and knockdown of two proteasome pathway components results in AGO2 protein accumulation. Finally, AGO2 protein levels increase after treatment with the proteasome inhibitor MG132. Our results indicate that the ubiquitin-proteasome pathway is involved in AGO2 protein turnover.
Keywords:
Argonaute; Proteasome; Protein stability; RNAi; Ubiquitin.
Published by Elsevier Inc.
Publication types
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Research Support, N.I.H., Intramural
MeSH terms
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Amino Acid Sequence
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Amino Acid Substitution
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Animals
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Animals, Genetically Modified
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Argonaute Proteins / chemistry
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Argonaute Proteins / genetics
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Argonaute Proteins / metabolism*
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Drosophila Proteins / antagonists & inhibitors
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Drosophila Proteins / chemistry
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Drosophila Proteins / genetics
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Drosophila Proteins / metabolism*
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Drosophila melanogaster / genetics
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Drosophila melanogaster / metabolism*
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Gene Knockdown Techniques
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Genes, Insect
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Leupeptins / pharmacology
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Mutagenesis, Site-Directed
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Proteasome Endopeptidase Complex / metabolism*
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Proteasome Inhibitors / pharmacology
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Protein Stability
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RNA Interference
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Sequence Homology, Amino Acid
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Signal Transduction
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Transcription Factors / antagonists & inhibitors
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Ubiquitin-Conjugating Enzymes / antagonists & inhibitors
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Ubiquitin-Conjugating Enzymes / genetics
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Ubiquitin-Conjugating Enzymes / metabolism
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Ubiquitins / metabolism*
Substances
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AGO2 protein, Drosophila
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Argonaute Proteins
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Drosophila Proteins
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Leupeptins
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Proteasome Inhibitors
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Transcription Factors
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Ubiquitins
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Ubiquitin-Conjugating Enzymes
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Uev1a protein, Drosophila
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Proteasome Endopeptidase Complex
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benzyloxycarbonylleucyl-leucyl-leucine aldehyde