Sequence-specific inhibition of small RNA function.

György Hutvágner,Martin J Simard,Phillip D Zamore,Craig C Mello,Gerald Joyce Gerald Joyce

doi:10.1371/journal.pbio.0020098

György Hutvágner, Martin J Simard + Show 3 more

Open Access

https://doi.org/10.1371/journal.pbio.0020098

Copy DOI

Abstract

Hundreds of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified from both plants and animals, yet little is known about their biochemical modes of action or biological functions. Here we report that 2′-O-methyl oligonucleotides can act as irreversible, stoichiometric inhibitors of small RNA function. We show that a 2′-O-methyl oligonucleotide complementary to an siRNA can block mRNA cleavage in Drosophila embryo lysates and HeLa cell S100 extracts and in cultured human HeLa cells. In Caenorhabditis elegans, injection of the 2′-O-methyl oligonucleotide complementary to the miRNA let-7 can induce a let-7 loss-of-function phenocopy. Using an immobilized 2′-O-methyl oligonucleotide, we show that the C. elegans Argonaute proteins ALG-1 and ALG-2, which were previously implicated in let-7 function through genetic studies, are constituents of a let-7-containing protein–RNA complex. Thus, we demonstrate that 2′-O-methyl RNA oligonucleotides can provide an efficient and straightforward way to block small RNA function in vivo and furthermore can be used to identify small RNA-associated proteins that mediate RNA silencing pathways.

Highlights

The endoribonuclease Dicer produces two types of small regulatory RNAs that regulate gene expression: small interfering RNAs and microRNAs (Bernstein et al 2001; Grishok et al 2001; Hutvagner et al 2001; Ketting et al 2001; Knight and Bass 2001)
To test whether 29-O-methyl oligonucleotides can act as RNA-induced silencing complex (RISC) inhibitors, we asked whether a 29-O-methyl oligonucleotide, tethered to streptavidin paramagnetic beads via a 59 biotin linkage, could be used to deplete small interfering RNA (siRNA)-programmed RISC from the reaction
The 29-Omethyl oligonucleotide completely depleted the reaction of the RISC programmed with the antisense strand of the siRNA, but not of RISC programmed with the sense strand (Figure 1B)

Summary

Introduction

The endoribonuclease Dicer produces two types of small regulatory RNAs that regulate gene expression: small interfering RNAs (siRNAs) and microRNAs (miRNAs) (Bernstein et al 2001; Grishok et al 2001; Hutvagner et al 2001; Ketting et al 2001; Knight and Bass 2001). Recent data suggest that both siRNAs and miRNAs incorporate into similar, perhaps even identical, protein complexes and that a critical determinant of mRNA destruction versus translation regulation is the degree of sequence complementary between the small RNA and its mRNA target (Hutvagner and Zamore 2002; Mourelatos et al 2002; Zeng et al 2002; Doench et al 2003; Saxena et al 2003; Zeng et al 2003). Biochemical studies in Drosophila S2 cells (Bernstein et al 2001; Hammond et al 2001a; Caudy et al 2002; Liu et al 2003) and affinity purification (Martinez et al 2002) or immunoprecipitation (Hutvagner and Zamore 2002) from cultured human HeLa cells have identified protein components of the RNAi effector complex, the RNA-induced silencing complex (RISC). Key steps in the RNAi pathway have emerged from studies of RNAi reconstituted in cell-free extracts (Tuschl et al 1999; Zamore et al 2000; Hammond et al 2001b; Nykanen et al 2001; Martinez et al 2002; Schwarz et al 2002; Tang et al 2003)

Methods

Results

Conclusion