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. 2009 Nov 13;36(3):445-56.
doi: 10.1016/j.molcel.2009.09.028.

Hierarchical rules for Argonaute loading in Drosophila

Benjamin Czech  1 , Rui Zhou, Yaniv Erlich, Julius Brennecke, Richard Binari, Christians Villalta, Assaf Gordon, Norbert Perrimon, Gregory J Hannon
Affiliations

Hierarchical rules for Argonaute loading in Drosophila

Benjamin Czech et al. Mol Cell. .

Abstract

Drosophila Argonaute-1 and Argonaute-2 differ in function and small RNA content. AGO2 binds to siRNAs, whereas AGO1 is almost exclusively occupied by microRNAs. MicroRNA duplexes are intrinsically asymmetric, with one strand, the miR strand, preferentially entering AGO1 to recognize and regulate the expression of target mRNAs. The other strand, miR*, has been viewed as a byproduct of microRNA biogenesis. Here, we show that miR*s are often loaded as functional species into AGO2. This indicates that each microRNA precursor can potentially produce two mature small RNA strands that are differentially sorted within the RNAi pathway. miR* biogenesis depends upon the canonical microRNA pathway, but loading into AGO2 is mediated by factors traditionally dedicated to siRNAs. By inferring and validating hierarchical rules that predict differential AGO loading, we find that intrinsic determinants, including structural and thermodynamic properties of the processed duplex, regulate the fate of each RNA strand within the RNAi pathway.

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Figures

Figure 1
Figure 1. miR*s have modified 3′ termini
(A) Pie charts represent the relative abundance of different endo-siRNA classes and microRNAs in 19- to 24-nt small RNA libraries from wild-type S2 cells. Results from a standard cloning protocol (upper diagram) and from a cloning strategy that enriches for small RNAs with modified 3′ termini (lower diagram) are shown. The fraction of miRs and miR*s is indicated for both libraries. (B) Heatmaps show the relative abundance of endo-siRNAs derived from structured loci, miRs, and miR*s in the indicated libraries (in grayscale). The ratio of normalized representation in the libraries indicates preferential association of small RNAs with either AGO1 (green) or AGO2 (red).
Figure 2
Figure 2. miR*s are preferentially loaded into AGO2
(A) Pie charts show the relative abundance of endo-siRNA classes and microRNAs libraries from AGO1 (left diagram) and AGO2 (right diagram) immunoprecipitates from S2 cells. (B) Northern blots of RNA from AGO1 and AGO2 immunoprecipitates from S2 cells. AGO-bound small RNAs were untreated (−) or subjected to β-elimination (+) prior to gel electrophoresis. The same membrane was probed for three miRs, three miR*s, and two endo-siRNAs derived from structured loci. (C) Heatmaps showing the relative abundance of endo-siRNAs derived from structured loci, miRs and miR*s in AGO1 and AGO2 libraries (grayscale). The relative association of small RNAs with AGO1 or AGO2 is indicated on a red/green scale. (D) Median base pairing (upper chart) and nucleotide composition (lower chart) of all sequences that show a relative association with AGO1 of 70% or more. Bulges on each strand were counted as mismatches (E) Analysis as in (D) but with all sequences having a relative association of 70% or more with AGO2.
Figure 3
Figure 3. Small RNA duplexes can be directed to AGO1 or AGO2
(A) Schematic drawing of the experimental procedure (Argonaute loading assay). (B) Immunoprecipitation followed by Northern blotting shows the loading of both top and bottom strands of various modified miR-276a duplexes into AGO1 or AGO2. miR-bantam and esi-2.1 served as controls. (C) Quantification of the Argonaute loading assay for modified miR-276a duplexes. The relative Argonaute loading index for each strand was normalized to that of the corresponding strand of duplex #1 (wild-type control), results were log(2) transformed and plotted. Positive numbers indicate preferential loading into AGO1, whereas negative numbers indicate favored loading into AGO2. The asterisk indicates that the bottom strand of duplex 5 had low signal and could not be reliably quantified. The inset shows the loading pattern of both individual strands of duplex #1. Duplex structures are shown to the right. (D) The relative Argonaute loading index for modified let-7 duplexes as described in (C).
Figure 4
Figure 4. Requirements for biogenesis and loading of miR*s
(A) Northern blots were probed with two miRs, two miR*s, and an endo-siRNA derived from a structured locus. Total RNAs from the indicated RNAi knockdowns were untreated (−) or subjected to β-elimination (+) prior to gel electrophoresis. 2S rRNA served as loading control. (B) Heatmaps showing the relative abundance of miRs, miR*s, and endo-siRNAs derived from structured loci in total RNA libraries of samples treated with dsRNAs against AGO1 or AGO2 (in grayscale). Preferential dependence of small RNAs on AGO1 (green) or AGO2 (red) is shown to the right.
Figure 5
Figure 5. Silencing by miR and miR* strands in S2 cells
(A) Schematic diagram showing the configuration of the sensor constructs. Three perfect match or bulged target sites for the miR or miR* strands of miR-bantam and miR-276a were placed in the 3′ UTR of the Renilla luciferase gene. A firefly luciferase construct without target sites served as a normalization control. (B) The indicated Renilla luciferase sensor constructs for miR-bantam or a control Renilla luciferase construct without target sites were co-transfected into S2 cells with a firefly luciferase construct. Cells were treated with dsRNAs targeting indicated RNAi pathway components. Fold changes in reporter activity were calculated as Renilla/firefly ratio normalized first against the control sample (cells treated with dsRNA targeting lacZ), then against cells transfected with the control construct without target sites. Shown is the average reporter activity with standard deviation (n=2). (C) Sensor activities for miR-276a as described in (B). (D) Sensor activities for over-expressed miR-bantam. Experiments were performed as described in (B) but in addition, an expression construct for miR-bantam was co-transfected with the reporter constructs. (E) Sensor activities for over-expressed miR-276a as described in (D).
Figure 6
Figure 6. Silencing by miR and miR* strands in flies
Shown are sensors for miR-bantam or miR-bantam* containing perfectly matched or bulged target sites (as indicated to the left). (A-D). β-Gal staining (red in the merged images) indicates dcr-1 mutant clones (also marked with arrows). Cells with strong β-Gal staining contain two wild-type dcr-1 genes, while cells with intermediate staining are heterozygous for dcr-1. EGFP sensor activity is shown in green. The black and white panels indicate the separate channels for β-Gal and EGFP. (E-H) Clonal analysis for ago1: Details as in (A-D). Selected regions (enclosed in white boxes) were enlarged and shown as insets within each panel to display the smaller ago1 clones.
Figure 7
Figure 7. A hierarchy of rules for small RNA loading in flies
(A) Thermodynamic properties of AGO2-associated endo-siRNAs matching the klarsicht locus (upper chart) and viral siRNAs (lower chart). All siRNA duplexes with both strands cloned were extracted bioinformatically and ratios of cloning abundances between guide and passenger strands were calculated. Average energies for up to six terminal nucleotides were plotted for strongly asymmetric (strand bias of 20:1 or higher) and weakly asymmetric duplexes (strand bias of 5:1 or lower). (B) Model for differential sorting of miRNA duplexes in flies.
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