DICER-ARGONAUTE2 complex in continuous fluorogenic assays of RNA interference enzymes.

Mark A Bernard,Leyu Wang,Souvenir D Tachado,Christophe Antoniewski

doi:10.1371/journal.pone.0120614

Abstract

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (K m=74 nM) with substrate inhibition kinetics (K i=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy.

Highlights

RNA interference brings about silencing of specific genes by sequence-specific degradation of mRNA [1]
By contrast for control conditions, melting was observed that was indistinguishable from dsRNA substrate alone (Fig. 5B, D, F, H, J; Control). These results demonstrate that magnesium-dependent catalysis by DICER+AGO2 enzymes cleaves the dsRNA DICER substrates and fluorogenic short interfering RNA (siRNA) to yield products that cannot persist as duplexes at assay temperature (37°C)
DICER and AGO2 enzymes form a functional equimolar complex with high affinity. This minimal RNA-Induced Silencing Complex (RISC) complex supports siRNA loading onto AGO2, whereas AGO2 does not efficiently self-load/cleave a double-stranded siRNA

Summary

Introduction

RNA interference brings about silencing of specific genes by sequence-specific degradation of mRNA [1]. Micro RNA precursors are derived from primary RNA transcripts that can form intramolecular hairpins (pri-miRNA), which can be cleaved in the nucleus by the endonuclease DROSHA to produce hairpin loops with a 3’ dinucleotide overhang called pre-microRNAs [2]. Pre-miRNAs are exported via nuclear export receptor, Exportin-5 into the cytoplasm [2,3] for endonucleolytic processing by the RNA-Induced Silencing Complex (RISC) consisting of DICER, ARGONAUTE and the human immunodeficiency virus-1 transactivating response RNA-binding protein TRBP [4]. The cytoplasmic endonuclease, DICER binds the 3’-overhang and cleaves in a staggered fashion to yield an additional 3’ dinucleotide overhang on the opposite end in which each strand of the resulting double-stranded short interfering RNA (siRNA) is 21- to 23-nt long [5,6]. The endonuclease ARGONAUTE2 (AGO2) binds one of the strands as the Guide Strand and is programmed to make single-strand cleavages in mRNA at a site complementary to the Guide Strand residing in the active site of AGO2 [10]

Methods

Results

Discussion

Conclusion