Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

Abstract

In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character-as is found in small interfering RNAs (siRNAs)-directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.

FIGURE 1.

miRNA*s are loaded in Ago2. (A) Relative abundance of miRNA, miRNA*, and endo-siRNAs among total fly head small RNA, Ago1-bound small RNAs—inferred from co-immunoprecipitation with Ago1, and Ago2-bound small RNAs—inferred from their presence in an oxidized small RNA library. (B) Box plots illustrating the enrichment scores for all miRNA and miRNA associated with Ago1 (i.e., in the Ago1 immunoprecipitate) or Ago2 (i.e., in the oxidized library) and for miRNA and miRNA* that were significantly (P ≤ 0.01) associated with Ago1 or Ago2. For miRNA* enriched in Ago2, six outliers with enrichment scores greater than 150 are not shown: miR-92a* (score = 1206), miR-308* (score = 649), miR-998* (score = 598), miR-315* (score = 514), miR-2a-2* (score = 309), and miR-33* (score = 304). (C) Box plots illustrating the abundance of Ago2-enriched miRNA* and white exo-siRNAs in the total RNA library. For miRNA* enriched in Ago2, 18 outliers with abundance greater than 250 ppm are not shown, including miR-8* (2748 ppm) and miR-34* (1747 ppm).

FIGURE 2.

Exemplary miRNA and miRNA* duplexes. Typical miRNA/miRNA* duplexes load their miRNA strands into Ago1 and their miRNA* strands into Ago2. The examples here correspond to duplexes whose miRNA strand was significantly (P ≤ 0.01) enriched in Ago1 and whose miRNA* strand was enriched in Ago2. These duplexes present different structures to the Ago1 and Ago2 sorting machinery, as the prospective guide strand occupies a unique position during Argonaute loading. When viewed with the miRNA strand as the guide and the miRNA* strand as the passenger, the duplex presents a structure with central bulges, mismatches, and G:U wobbles, but when the miRNA* strand will become the guide and the miRNA strand serves as the passenger, the duplexes present more stably paired central regions. The duplexes are drawn using the guide isoform that was most abundant for the specific Argonaute protein paired to the most abundant passenger sequence detected in the total small RNA library. Red text, seed sequence; shaded bars highlight position that are significantly different between Ago1- and Ago2-loaded guides (see Fig. 4).

FIGURE 3.

Association of miRNA* with Ago2 relies on the Ago2-loading machinery. (A) Efficient loading into Ago2 of miRNA and miRNA* strands—measured by their abundance in an oxidized small RNA library—was diminished in heads from dcr-2^L811fsX and r2d2¹ mutants for miRNA and miRNA* normally enriched in Ago2, but the abundance of Ago1-enriched miRNAs was unaltered, as measured in the total small RNA library. Box plots illustrate the fold-change between mutant and wild-type. (B,C) The requirement for Dcr-2 and R2D2 for Ago2 loading was well correlated for miRNA and miRNA* strands preferentially loaded into Ago2. (D) The overall abundance of Ago2-enriched miRNA and miRNA*—measured in the total small RNA library—decline in ago2 mutant heads. Box plots illustrate the fold-change between mutant and wild-type in total small RNA libraries.

FIGURE 4.

Pairing profiles of Ago1- and Ago2-loaded small RNA guides. (A) Box plots illustrate the predicted double-stranded character of each nucleotide position, 1–19, for all Ago1- or Ago2-enriched miRNA or miRNA* strands. (B) The Wilcoxon test P-value for each comparison was used to identify nucleotide positions that were significantly different between Ago1-enriched miRNA plus miRNA* compared with Ago2-enriched miRNA plus miRNA*. The red line indicates P = 0.01. Gray circles, nonsignificant; black circles, significant. (C) Box plots illustrate the differences in double-stranded character for each position that was significantly different in double-stranded character between Ago1- and Ago2-loaded miRNA plus miRNA* in B. (D) The data in A–C suggest that miRNA duplexes with less stable 5′ ends and central mismatches act as guides for Ago1 and miRNA duplexes with less stable 3′ ends act as guides for Ago2.

FIGURE 5.

miRNAs and miRNA*s show an Argonaute-specific first nucleotide bias. miRNAs and miRNA*s associated with Ago1 or Ago2 differ in the bias of their first nucleotide. miRNAs generally begin with uridine; this bias increased for the subset of miRNA that were Ago1-bound (measured in the Ago1 immunoprecipitate library), and increased further for the subset of Ago1-enriched miRNAs (measured in the total small RNA library). In contrast, Ago2-enrcihed miRNAs were depleted of 5′ uridine in the oxidized small RNA library. miRNA* strands generally began with adenosine or cytidine. All miRNA* strands detected in the oxidized library (i.e., loaded in Ago2) or those enriched in Ago2, were significantly more likely to begin with cytidine, whereas those miRNA*s enriched in Ago1 were depleted of a 5′ cytidine. A 5′ cytidine bias was also observed for white exo-siRNAs and was diminished in r2d2¹, a mutant defective in Ago2 loading.

FIGURE 6.

Ago1 prefers to load miRNAs that begin with a 5′ uridine, while Ago2 prefers siRNAs that begin with a 5′ cytidine. (A) Four small RNA duplexes were incubated with embryo lysate and then cross-linked with shortwave UV to identify small RNA-bound proteins. Representative data are shown. (B) Kinetic analysis of miRNA association with Ago1, monitored by UV cross-linking. (C) Kinetic analysis of siRNA association with Ago2, monitored by UV cross-linking. In B and C, each data point represents the average ± standard deviation for three trials.

FIGURE 7.

miRNA and miRNA* can switch seeds between Ago1 and Ago2. Depicted are miRNA/miRNA* duplexes that load distinct isoforms of their miRNA or miRNA* between Ago1 and Ago2, resulting in seed switching between Argonautes. The duplexes are drawn pairing the most abundant guide isoform associated with the particular Argonaute to the most abundant passenger strand isoform in total head small RNA library. Reads in parts per million represent the sum of all isoforms that share the same seed as detected in the total small RNA library. Ratio reports the relative number of reads for the isoform enriched in Ago1: the number of reads for the isoform enriched in Ago2 as detected within either the library prepared from Ago1 immunoprecipitated small RNAs (Ago1 ratio) or oxidized small RNAs (Ago2 ratio). Red text, seed sequence; shaded bars, determinative positions for small RNA sorting between Ago1 and Ago2; N.D., detected in wild-type, but not detected in the ago2⁴¹⁴ mutant.

FIGURE 8.

A model for small RNA sorting. Sorting of small RNA into an Argonaute is governed by structure and first nucleotide identity. Consequently, a single miRNA/miRNA* duplex derived from a single pre-miRNA can present two distinct structures to the Argonaute-loading machinery. From one end, the duplex can act as a favorable substrate for loading Ago1, while from the other end, its structure and sequence can favor entry into the RNAi—i.e., the Ago2—pathway.