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SUMOylation of Argonaute-2 regulates RNA interference activity.

ABSTRACT: Post-translational modification of substrate proteins by small ubiquitin-like modifier (SUMO) regulates a vast array of cellular processes. SUMOylation occurs through three sequential enzymatic steps termed E1, E2 and E3. Substrate selection can be determined through interactions between the target protein and the SUMO E2 conjugating enzyme Ubc9 and specificity can be enhanced by substrate interactions with E3 ligase enzymes. We used the putative substrate recognition (PINIT) domain from the SUMO E3 PIAS3 as bait to identify potential SUMO substrates. One protein identified was Argonaute-2 (Ago2), which mediates RNA-induced gene silencing through binding small RNAs and promoting degradation of complimentary target mRNAs. We show that Ago2 can be SUMOylated in mammalian cells by both SUMO1 and SUMO2. SUMOylation occurs primarily at K402, and mutation of the SUMO consensus site surrounding this lysine reduces Ago2-mediated siRNA-induced silencing in a luciferase-based reporter assay. These results identify SUMOylation as a potential regulator of Ago2 activity and open new avenues for research into the mechanisms underlying the regulation of RNA-induced gene silencing.

SUBMITTER: Josa-Prado F

PROVIDER: S-EPMC4624959 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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SUMOylation of Argonaute-2 regulates RNA interference activity.

Josa-Prado Fernando F Henley Jeremy M JM Wilkinson Kevin A KA

Biochemical and biophysical research communications 20150717 4

Post-translational modification of substrate proteins by small ubiquitin-like modifier (SUMO) regulates a vast array of cellular processes. SUMOylation occurs through three sequential enzymatic steps termed E1, E2 and E3. Substrate selection can be determined through interactions between the target protein and the SUMO E2 conjugating enzyme Ubc9 and specificity can be enhanced by substrate interactions with E3 ligase enzymes. We used the putative substrate recognition (PINIT) domain from the SUM ...[more]

PMID: 26188511

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Project description:RNA interference (RNAi) is a gene-silencing mechanism that plays an important role in gene regulation in a number of eukaryotic organisms. Two core components, Dicer and Argonaute, are central in the RNAi machinery. However, the physiological roles of Dicer and Argonaute in the entomopathogenic fungus Metarhizium robertsii have remained unclear. Here, the roles of genes encoding Dicer (M. robertsiidcl1 [Mrdcl1] and Mrdcl2) and Argonaute (Mrago1 and Mrago2) proteins in M. robertsii were investigated. The results showed that the Dicer-like protein MrDCL2 and Argonaute protein MrAGO1 are the major components of the RNAi process occurring in M. robertsii The Dicer and Argonaute genes were not involved in the regulation of growth and diverse abiotic stress response in M. robertsii under the tested conditions. Moreover, our results showed that the Dicer and Argonaute gene mutants demonstrated reduced abilities to produce conidia, compared to the wild type (WT) and the gene-rescued mutant. In particular, the conidial yields in the Δdcl2 and Δago1 mutants were reduced by 55.8% and 59.3%, respectively, compared with those from the control strains. Subsequently, for the WT and Δdcl2 mutant strains, digital gene expression (DGE) profiling analysis of the stage of mycelium growth and conidiogenesis revealed that modest changes occur in development or metabolism processes, which may explain the reduction in conidiation in the Δdcl2 mutant. In addition, we further applied high-throughput sequencing technology to identify small RNAs (sRNAs) that are differentially expressed in the WT and the Δdcl2 mutant and found that 4 known microRNA-like small RNAs (milRNAs) and 8 novel milRNAs were Mrdcl2 dependent in M. robertsiiIMPORTANCE The identification and characterization of components in RNAi have contributed significantly to our understanding of the mechanism and functions of RNAi in eukaryotes. Here, we found that Dicer and Argonaute genes play an important role in regulating conidiation in M. robertsii Our study also demonstrates that diverse small RNA pathways exist in M. robertsii The study provides a theoretical platform for exploration of the functions of Dicer and Argonaute genes involved in RNAi in fungi.

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SUMOylation of Argonaute-2 regulates RNA interference activity.

Publications

SUMOylation of Argonaute-2 regulates RNA interference activity.

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