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Mutagenisis of Argonaute 2 in studying microRNA loading
Author(s) -
Doxtader Katelyn Ashley,
Androsavich John,
Walter Nils
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.944.1
Subject(s) - argonaute , microrna , gene silencing , biology , microbiology and biotechnology , rna induced silencing complex , drosha , repressor , rna interference , rna , non coding rna , genetics , gene expression , gene
MicroRNAs (miRNA) are small, non‐coding RNAs that play a critical role in post‐transcriptional gene regulation by acting as a guide for directing the repressor protein Argonaute 2 (Ago2) and its RNA‐induced silencing complex (RISC) to mRNA targets. Recently, we reported on the dynamic movements and localizations of individual miRNAs in living cells, thus demonstrating the feasibility and potential of using single molecule analysis to study non‐coding RNAs in the cellular environment. Our goal is to now extend these studies to the Ago2 protein, the central effecter of the miRNA pathway. Rudell et al (2011) recently found that mutation of the conserved tyrosine Y529 located in the 5′‐end‐binding pocket of Ago2 to a negatively charged glutamate mimics phosphorylation by an as‐yet‐unknown kinase and inhibits small RNA binding. We plan to incorporate this mutation using site‐directed mutagenesis into a plasmid encoding a human Ago2‐EGFP fusion. Using fluorescence microscopy, we will then monitor and compare the localizations and diffusive properties of EGFP‐Ago2 Y529E to its wild‐type counterpart in HeLa cell lines under transient expression. Since EGFP‐Ago2 Y529E is incapable of binding miRNA, it will enable us to distinguish the diffusive motions of miRNA‐loaded RISC from unloaded‐RISC, and to characterize and potentially quantify miRNA loading, a critical step in the miRNA pathway.