Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Nov 1;40(20):10452-62.
doi: 10.1093/nar/gks748. Epub 2012 Aug 16.

The slicing activity of miRNA-specific Argonautes is essential for the miRNA pathway in C. elegans

Samir Bouasker  1 , Martin J Simard
Affiliations

The slicing activity of miRNA-specific Argonautes is essential for the miRNA pathway in C. elegans

Samir Bouasker et al. Nucleic Acids Res. .

Abstract

Among the set of Argonautes proteins encoded by metazoan genomes, some have conserved amino acids important for catalytic or slicing activity. The functional significance of these residues in microRNA (miRNA)-specific Argonautes in animals is still unclear since miRNAs do not induce site-specific cleavage of targeted messenger RNAs (mRNAs), unlike small interfering RNAs (siRNAs). Here, we report that miRNA-specific ALG-1 and ALG-2 Argonautes from Caenorhabditis elegans possess the slicing activity normally implicated in the siRNA-silencing pathway. We also find that ALG-1/2 can bind and use a Dicer-processed miRNA duplex to target mRNAs, suggesting an ability to displace RNA strands. Importantly, the slicing activity of ALG-1 or ALG-2 is essential for the miRNA pathway in vivo, as shown by the accumulation of truncated miRNA precursors and altered miRNA-induced silencing complex (miRISC) formation. Taken together, our data demonstrate that the slicing activity of Argonautes contributes to a new and unexpected step in the canonical miRNA pathway that occurs prior to miRISC loading in animals.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Purified ALG-1 and ALG-2 Argonautes have the slicing activity. (A) Schematic of the assay. (B) A representation of the RNA molecules used for the enzymatic assay. A 32 nucleotide (nt) 5′-end radiolabeled (32P) RNA target (in black) and fully complementary (P) or bulged next to the cleavage site (M) 21 nt ssRNA are shown in gray. (C) The radiolabeled RNA target is incubated with GST, GST-ALG-1 or GST-ALG-2 proteins with (+) the fully complementary 21-nt ssRNAs. Reaction performed with (+) or without (–) 10 mM EDTA to sequester magnesium ions essential for the enzymatic reaction are indicated. (D) Alteration of base pairing at the cleavage site (ssRNA M) abrogates target specific cleavage. The radiolabeled RNA target is incubated with either GST or GST-ALG-1 proteins. Hydroxylation of the RNA target (OH) is used as size marker (the 3′-end nucleotides of the radiolabeled RNA fragment are indicated). (E) Point mutations of the DDH motif in the PIWI domain of GST-ALG-1 abrogate cleavage activity. Amounts (nM) of recombinant proteins used in the assay are indicated.
Figure 2.
Figure 2.
Recombinant ALG-1 can bind and cleave small RNA duplexes. (A) Representation of the small RNA duplexes used for the assay. Strands complementary to the target RNA are shown in gray. (B) Determination of the affinity of ALG-1 for different types of small RNAs. Filter binding assays were performed with increasing amounts of GST-ALG-1 AAA (nM) incubated with different 5′-radiolabeled (32P) 21-nt RNAs illustrated in A (ssRNA is shown in Figure 1). The error bars represent a 95% confidence interval from three independent experiments. (C) Schematic of the slicing assay. (D) ALG-1 is able to cleave a perfectly paired small RNA duplex. Increasing amounts of GST-ALG-1 (nM) are incubated with different 5′ radiolabeled (32P) small RNA duplexes (as shown in A). Hydroxylation of the 21-nt RNA (OH) is used as size marker (the 3′ end nucleotides of the radiolabeled RNA fragment are indicated). 8G and 7C represent other cleavage products.
Figure 3.
Figure 3.
ALG-1 can use a miRNA-like duplex to induce target cleavage. (A) Schematic of the assay. (B)The radiolabeled (32P) 32 nt RNA target was incubated with different concentration of GST-ALG-1 protein (nM) along with RNA duplexes containing zero (duplex), one (d1M) or two mismatches (d2M). (C) Comparative analysis of the cleavage induced with either single stranded RNA (ssRNA) or miRNA small RNA duplex (d2M). Cleavage product sizes are indicated as Figure 1.
Figure 4.
Figure 4.
The presence of the slicing-competent ALG-1 or ALG-2 is essential for the production of let-7 and lin-4 miRNAs. (A and B) Northern blot detection of miRNAs in animals expressing ALG-1 and ALG-2 transgenes. Upon total RNA extraction from different transgenic animals carrying either wild-type (wt) or catalytically defective (AAA) alg-1 and alg-2 genes, lin-4 and let-7 were detected using probes complementary to mature miRNA (antisense) and to the complementary strand (sense). SL1 RNA was probed and used as loading control (the detection of SL1 is only shown for one blot because the same membrane was stripped and re-used to detect RNA species from both miRNA). Precursor (pre), truncated precursor (pre short) and mature miRNA forms are indicated. The genotype and RNAi (below) as well as transgene (above) found in each animal strain are indicated. (C) Representation of the RNA molecules detected by Northern blotting.
Figure 5.
Figure 5.
Slicing ALG-1 is required to generate functional miRISC. (A) Detection of lin-4 in bound (IP) or unbound (FT) fractions after ALG-1 immunoprecipitations. Transgenically expressed ALG-1 wild-type (wt) or catalytically defective (AAA) protein were IPed from alg-1(lf); alg-2(RNAi) animals. The presence of lin-4 strands was detected by Northern blotting (left panel) and 10% of immunopurified complexes were used for detection of ALG-1 (right panel). (B) The quantification of three independent experiments is shown in the lower panel. Errors bars represent a 95% confidence interval and a Student’s two-sided t-test was applied to obtain P values.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell. Biol. 2009;10:126–139. - PubMed
    1. Hutvagner G, Simard MJ. Argonaute proteins: key players in RNA silencing. Nat. Rev. Mol. Cell. Biol. 2008;9:22–32. - PubMed
    1. Lingel A, Simon B, Izaurralde E, Sattler M. Nucleic acid 3'-end recognition by the Argonaute2 PAZ domain. Nat. Struct. Mol. Biol. 2004;11:576–577. - PubMed
    1. Frank F, Sonenberg N, Nagar B. Structural basis for 5'-nucleotide base-specific recognition of guide RNA by human AGO2. Nature. 2010;465:818–822. - PubMed
    1. Wang Y, Juranek S, Li H, Sheng G, Tuschl T, Patel DJ. Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature. 2008;456:921–926. - PMC - PubMed

Publication types

MeSH terms