microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein

Abstract

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.

Figure 1

Smaug co-immunoprecipitates with Ago1 and Ago2. Embryo extracts collected 0–2 h post egg laying were immunoprecipitated with anti-Smaug antibody or non-immune guinea-pig serum. Western blots to assay for the indicated proteins in crude extracts and the indicated immunoprecipitates are shown. Where indicated, immunoprecipitations were performed in the presence of RNase A. Embryo extract lanes represent 5% of the material used in the immunoprecipitations. Ago, Argonaute.

Figure 2

Ago1 represses the translation of unlocalized nos mRNA. Wild-type, smaug, Ago1 or Ago2 embryos were fixed and stained with anti-Nos (A,B) or anti-Osk (C) antibodies. Embryos containing eight nuclei, as assayed via staining with picogreen, are shown. (D) Extracts derived from embryos collected 0–2 h post egg laying were assayed for indicated proteins by western blots. (E) Northern blot analysis was performed on total RNA extracted from wild-type or Ago1 mutant embryos collected at the indicated times post egg laying. (F,G) Northern blots were exposed to PhosphorImager screens and quantified with ImageQuant Software. nos mRNA levels from three independent experiments were quantified and normalized using RpLP2 mRNA as a loading control. Error bars indicate s.e.m. Ago, Argonaute; nos, nanos; Osk, Oskar.

Figure 3

Ago1 interacts with nos mRNA in a Smaug-dependent manner. Crosslinked embryo extracts collected 0–2 h post egg laying were immunoprecipitated with anti-Ago1 antibody or normal mouse serum, and levels of specific mRNAs were quantified by quantitative real-time PCR. (A) The fold enrichment of nos and bicoid mRNAs in Ago1 immunoprecipitates versus normal mouse serum immunoprecipitates. (B) The fold enrichment of nos mRNA in Ago1 immunoprecipitates versus normal mouse serum immunoprecipitates normalized to bicoid mRNA levels (n=5). Error bars represent s.e.m. calcuated from five biological replicates, the dotted line indicates onefold (i.e., no) enrichment and ** indicates a significant difference between the indicated samples as judged by the t-test (P<0.007). (C) Crosslinked extracts from wild-type and smaug mutant embryos were collected 0–2 h post egg laying and western blots were used to assay the levels of Ago1 protein in both the starting extracts and in material immunoprecipitated from these extracts using anti-Ago1 antibody. Dp1 serves as a loading control for the embryo extracts. Note that similar levels of Ago1 protein are immunprecipitated from smaug mutant extract compared with wild-type (97.2%±7.0%, n=2). Ago, Argonaute; IP, immunoprecipitation; nos, nanos.

Figure 4

Smaug mediates miRNA-independent recruitment of Ago1 to nos mRNA. (A) Embryo extracts collected 0–3 h post egg laying were incubated with biotinylated SRE⁺ or SRE⁻ RNAs immobilized on streptavidin particles. Western blots to assay for the indicated proteins in crude extracts as well as the indicated purification are shown. For Smaug Westerns, equivalent amounts of material were run in extract lanes and capture lanes while the embryo extract lanes for the other Westerns are equivalent to 2% of the material run in the capture lanes. (B) Embryo extracts collected 0–2 h post egg laying from wild-type or transgene-carrying flies were incubated with anti-FLAG beads. Western blots to assay for the indicated proteins in crude extracts as well as the indicated purification are shown. Embryo extract lanes represent 5% of the material used in the immunoprecipitations. Dp1 serves as a loading control for the embryo extracts. (C) Purifications similar to (B) using crosslinked embryo samples were assayed for indicated miRNAs by northern blot. Embryo extract lanes represent 5% of the material used in the immunoprecipitations. (D) Purifications similar to (B) using crosslinked embryo samples were assayed for the indicated mRNAs by quantitative real-time PCR. The fold enrichment of the indicated mRNAs in transgenic embryos versus wild-type embryos carrying no transgene normalized to bicoid mRNA levels is shown. Error bars represent s.e.m. calculated from five biological replicates, the dotted line indicates onefold (i.e., no) enrichment and ** indicates a significant difference between the indicated samples as judged by the t-test (P<0.0011). Ago, Argonaute; miRNA, microRNA; IP, immunoprecipitation; nos, nanos.

Figure 5

Smaug recruits Ago1 to nos mRNA in a miRNA-independent manner. Ago1 is typically recruited to a target mRNA through base pairing between a miRNA and the target transcript. In contrast, our data indicate that Smaug directly recruits Ago1 to nos mRNA without the need for a targeting miRNA. While a miRNA is not required for targeting, Ago1 recruited by Smaug could be assoicated with a miRNA and, as described in more detail in the Discussion, this bound miRNA could be required for translational repression. Ago, Argonaute; miRNA, microRNA; nos, nanos.