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. 2011 Jan;17(1):54-63.
doi: 10.1261/rna.2498411. Epub 2010 Nov 24.

Target RNA-directed tailing and trimming purifies the sorting of endo-siRNAs between the two Drosophila Argonaute proteins

Stefan L Ameres  1 , Jui-Hung Hung, Jia Xu, Zhiping Weng, Phillip D Zamore
Affiliations

Target RNA-directed tailing and trimming purifies the sorting of endo-siRNAs between the two Drosophila Argonaute proteins

Stefan L Ameres et al. RNA. 2011 Jan.

Abstract

In flies, 22-23-nucleotide (nt) microRNA duplexes typically contain mismatches and begin with uridine, so they bind Argonaute1 (Ago1), whereas 21-nt siRNA duplexes are perfectly paired and begin with cytidine, promoting their loading into Ago2. A subset of Drosophila endogenous siRNAs-the hairpin-derived hp-esiRNAs-are born as mismatched duplexes that often begin with uridine. These would be predicted to load into Ago1, yet accumulate at steady-state bound to Ago2. In vitro, such hp-esiRNA duplexes assemble into Ago1. In vivo, they encounter complementary target mRNAs that trigger their tailing and trimming, causing Ago1-loaded hp-esiRNAs to be degraded. In contrast, Ago2-associated hp-esiRNAs are 2'-O-methyl modified at their 3' ends, protecting them from tailing and trimming. Consequently, the steady-state distribution of esiRNAs reflects not only their initial sorting between Ago1 and Ago2 according to their duplex structure, length, and first nucleotide, but also the targeted destruction of the single-stranded small RNAs after their loading into an Argonaute protein.

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Figures

FIGURE 1.
FIGURE 1.
Hairpin-derived endo-siRNAs (hp-esiRNAs) assemble in vitro into Ago1 and Ago2 RISC. (A) Overview of the model for small silencing RNA production and sorting into Argonaute effector complexes in Drosophila. (B) The top row depicts the structures and sequences of the let-7/let-7* miRNA duplex, a canonical siRNA (let-7 siRNA), as well as the most abundant hp-esiRNAs from the esi-2 (esi-2.1) and esi-1 loci (esi-1.1 and esi-1.2). Below each small RNA duplex are representative data for a time course of assembly of the small RNA into Ago1 and Ago2 as determined by 254 nm UV crosslinking.
FIGURE 2.
FIGURE 2.
The 5′ nucleotide of the guide strand and the length of the small RNA duplex determine sorting between Ago1 and Ago2. miRNA or siRNA duplexes that differ from each other only in their 5′ nucleotide (A) and length (B), but not their duplex structure, were assessed by UV crosslinking for their assembly into Ago1 or Ago2 RISC in vitro. Mean ± SD of three replicates is reported. (C) The 5′ nucleotide distribution for the hp-esiRNAs bound to Ago1 and Ago2 was deduced by analyzing the small RNA immunoprecipitated with Ago1 and Ago2 from S2 cell lysates or the small RNAs from fly heads immunoprecipitated with Ago1 and inferred to be bound to Ago2 by their resistance to oxidation.
FIGURE 3.
FIGURE 3.
Mutations in the RNAi machinery change the repertoire of hp-esiRNAs. (A) Size distribution and abundance of hp-esiRNAs in dicer-2 and r2d2 mutant fly heads compared to their heterozygous siblings or ago2414 mutants compared to Oregon R fly heads. (B) Decrease in hp-esiRNAs in dicer-2L811fsX, r2d21, and ago2414 mutant fly heads. (C) Change in full-length esi-2.1, esi-1.1, or esi-1.2 in dicer-2L811fsX, r2d21, and ago2414 mutant fly heads. (D) Relative abundance of hp-esiRNAs starting with each possible 5′ nucleotide as deduced from sequencing of small RNAs from the heads of ago2414 and r2d21 heterozygote or homozygote or Oregon R flies.
FIGURE 4.
FIGURE 4.
The RNAi loading complex loads hp-esiRNAs starting with C into Ago2. (A) Correlation analysis of the change in abundance of hp-esiRNAs in r2d21 mutants and ago2414 mutants (r = 0.47; P = 0.0002). The most abundant hp-esiRNA guide (red) and passenger strands (blue), as well as a trimmed species of esi-2.1 (green) are indicated. (B) Same data as in A, but with 5′ nucleotide identity indicated by color: green, 5′ A; blue, 5′ C; yellow, 5′ G; red, 5′ U. (C) The 5′ nucleotide composition of the hp-esiRNAs species in (B) ranked by the change in abundance (<y-fold, top panel; >y-fold, bottom panel) in ago2414 and r2d21. (<l.d.) Below the limit of detection.
FIGURE 5.
FIGURE 5.
Ago1- but not Ago2-bound hp-esiRNAs are destabilized by highly complementary target RNAs. (A) Size distribution of genome-matching (top) and prefix-matching (bottom) esi-2.1 sequences associated with Ago1 (left) or Ago2 (right) in S2 cell extracts. The most abundant 3′ nontemplated, added nucleotides are noted. (B) The abundance of esi-2.1 (top) or all other small RNAs from the esi-2 locus (bottom) associated with Ago1 (red) or Ago2 (blue) in S2 cell extracts. (C) Size distribution of small RNAs, other than esi-2.1, derived from the esi-2 locus and associated with Ago1 (top) or Ago2 (bottom) in S2 cell extracts. (D) Size distribution of genome-matching (top) and prefix-matching (bottom) small RNAs from the esi-1 locus associated with Ago1 (left) or Ago2 (right) in S2 cell extracts. The most abundant 3′ nontemplated, added nucleotides are noted.
FIGURE 6.
FIGURE 6.
Hen1 stabilizes hp-esiRNAs. (A) The size distribution of genome-matching (top) and prefix-matching (bottom) sequences corresponding to esi-2.1 (left) or derived from the esi-1 locus (right) in the heads of hen1f00810 homozygous (red) or hen1f00810/CyO heterozygous (black) flies. The most abundant 3′ nontemplated, added nucleotides are noted. (B) The distribution of 5′ nucleotides among genome-matching hp-esiRNAs in hen1f00810 heterozygotes or homozygous mutant fly heads.
FIGURE 7.
FIGURE 7.
A model for the sorting of Drosophila hp-esiRNAs between Ago1 and Ago2 and their subsequent purification by target-directed tailing and trimming.
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