Exoribonuclease Nibbler Shapes the 3″ Ends of Micrornas

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Current Biology 21, 1878–1887, November 22, 2011 ª2011 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2011.09.034 Article The 30-to-50 Exoribonuclease Nibbler Shapes the 30 Ends of MicroRNAs Bound to Drosophila Argonaute1

Bo W. Han,1 Jui-Hung Hung,2 Zhiping Weng,2 precursor miRNAs (pre-miRNAs) [8]. Pre-miRNAs comprise Phillip D. Zamore,1,* and Stefan L. Ameres1,* a single-stranded loop and a partially base-paired stem whose 1Howard Hughes Medical Institute and Department of termini bear the hallmarks of RNase III processing: a two-nucle- Biochemistry and Molecular Pharmacology otide 30 overhang, a 50 phosphate, and a 30 hydroxyl group. 2Program in Bioinformatics and Integrative Biology Nuclear pre-miRNAs are exported by Exportin 5 to the cyto- University of Massachusetts Medical School, plasm, where the RNase III enzyme Dicer liberates w22 nt 364 Plantation Street, Worcester, MA 01605, USA mature miRNA/miRNA* duplexes from the pre-miRNA stem [9–12]. Like all Dicer products, miRNA duplexes contain two- nucleotide 30 overhangs, 50 phosphate, and 30 hydroxyl groups. Summary In flies, Dicer-1 cleaves pre-miRNAs to miRNAs, whereas Dicer-2 converts long double-stranded RNA (dsRNA) into Background: MicroRNAs (miRNAs) are w22 nucleotide (nt) small interfering RNAs (siRNAs), which direct RNA interference small RNAs that control development, physiology, and pathol- (RNAi), a distinct small RNA silencing pathway required for ogy in animals and plants. Production of miRNAs involves the host defense against viral infection and somatic transposon sequential processing of primary hairpin-containing RNA poly- mobilization, as well as gene silencing triggered by exogenous merase II transcripts by the RNase III enzymes Drosha in the dsRNA [13, 14]. nucleus and Dicer in the cytoplasm. miRNA duplexes then miRNA duplexes assemble into Argonaute proteins to assemble into Argonaute proteins to form the RNA-induced form the precursor RNA-induced silencing complex (pre- silencing complex (RISC). In mature RISC, a single-stranded RISC), a process uncoupled from small RNA production [15]. miRNA directs the Argonaute protein to bind partially comple- In flies, miRNAs typically bind to Argonaute1 and siRNAs to mentary sequences, typically in the 30 untranslated regions of Argonaute2 [16]. During RISC assembly, one of the two messenger RNAs, repressing their expression. strands of a miRNA duplex is selectively retained to form an Results: Here, we show that after loading into Argonaute1 active silencing complex. Strand selection is determined by (Ago1), more than a quarter of all Drosophila miRNAs undergo the relative thermodynamic stability of the duplex ends, the 30 end trimming by the 30-to-50 exoribonuclease Nibbler identity of the 50 nucleotides, as well as the structure and (CG9247). Depletion of Nibbler by RNA interference (RNAi) length of the miRNA duplex [15]. In mature RISC, a single- reveals that miRNAs are frequently produced by Dicer-1 as in- stranded miRNA directs Ago1 to bind partially complementary termediates that are longer than w22 nt. Trimming of miRNA sequences, typically within the 30 untranslated region (30 UTR) 30 ends occurs after removal of the miRNA* strand from pre- of mRNAs [1]. RISC-binding represses mRNA expression by RISC and may be the final step in RISC assembly, ultimately accelerating its decay or inhibiting its translation [17]. enhancing target messenger RNA repression. In vivo, deple- Here, we report that more than one quarter of all miRNAs tion of Nibbler by RNAi causes developmental defects. in Drosophila S2 cells are trimmed after their loading into Conclusions: We provide a molecular explanation for the Ago1, a process that can be recapitulated in cell extracts previously reported heterogeneity of miRNA 30 ends and pro- and that we can detect in vivo in flies. Trimming of miRNAs is pose a model in which Nibbler converts miRNAs into isoforms mediated by the Mg2+-dependent 30-to-50 exoribonuclease that are compatible with the preferred length of Ago1-bound Nibbler (CG9247), a member of the DEDD family of exonucle- small RNAs. ases. Nibbler activity is required to trim Ago1-loaded miRNAs, and miRNA trimming enhances target RNA repression. Nibbler Introduction is required for normal fly development. Our results show that the 30 ends of miRNAs are not simply defined by the RNase MicroRNAs (miRNAs) regulate messenger RNA (mRNA) III enzymes Drosha and Dicer-1 but undergo exonucleolytic reshaping after their loading into Ago1. Thus, the previously stability and translation in plants, green algae, and animals 0 [1, 2]. Originally discovered in Caenorhabditis elegans, these described heterogeneity of miRNA 3 ends reflects mainly w22 nucleotide (nt) small RNAs regulate development and active trimming, rather than sloppy precursor processing. physiology and have been implicated in diseases such as cancer, diabetes, and viral infection [3–5]. Loss of proteins Results required for the production or function of miRNAs typically 0 results in severe developmental defects or lethality. The 3 End of miR-34 Is Trimmed after Its Production miRNA genes are generally transcribed by RNA polymerase II by Dicer-1 to generate 50 capped and 30 polyadenylated primary miRNAs miRBase annotates miR-34 (miR-34-5p) as 24 nt long, pairing (pri-miRNAs) that are then sequentially processed into mature to a 23 nt miR-34* strand (miR-34-3p) (Figure 1A), but high miRNA duplexes [6]. Pri-miRNAs contain one or more charac- resolution northern hybridization revealed additional, abun- teristic stem-loops that are recognized and cleaved by the dant 23, 22, and 21 nt miR-34 isoforms (Figure 1B). To test nuclear RNase III enzyme Drosha [7] to generate w70 nt long whether inaccurate processing of pre-miR-34 by Dicer-1 explains miR-34 heterogeneity, we incubated 50 32P-radiolabeled pre-miR-34 with purified, recombinant Dicer-1/Loqua- *Correspondence: [email protected] (P.D.Z.), stefan. cious PB, S2 cell lysate or 0–2 hr Drosophila embryo lysate [email protected] (S.L.A.) for 15 min (Figure 1C). In all three conditions, pre-miR-34 Nibbler Trims MicroRNAs 1879

Figure 1. miR-34 Is Trimmed after Its Production by Dicer-1 (A) Structure of pre-miR-34. miR-34 (24 nt) is shown in red, and miR-34* (23 nt) is shown in blue. (B) miR-34 isoforms detected in total RNA from S2 cells by northern hybridization. (C) 50 32P-radiolabeled pre-miR-34 was incubated with purified, recombinant Dicer-1/Loquacious-PB heterodimer (Dcr-1/Loqs-PB), S2 cell lysate, or 0–2 hr embryo lysate. Products were resolved by denaturing polyacrylamide gel electrophoresis. (D) Abundance of miR-34 isoforms detected in fly heads and S2 cells by high throughput sequencing. Only reads with the annotated miR-34 50 ends were analyzed. Genome-matching is shown in black, and prefix-matching reads are shown in gray. (E) 50 32P-radiolabeled pre-miR-34, 24 nt miR-34, or 21 nt let-7 RNA was incubated in 0–2 hr embryo lysate. Products were resolved by denaturing polyacrylamide gel electrophoresis. See also Figure S1. was rapidly converted to 24 nt (Dcr-1/Loqs-PB: 61%; S2 cell processing would be expected to generate heterogeneous lysate: 63%; embryo lysate: 60%), 25 nt (Dcr-1/Loqs-PB: 50 ends for miRNAs, like miR-34, that derive from the 50 arm 25%; S2 cell lysate: 26%; embryo lysate: 29%), and 23 nt of their pre-miRNA. We analyzed small RNA high through- (Dcr-1/Loqs-PB: 13%; S2 cell lysate: 11%; embryo lysate: put sequencing data from fly heads for reads mapping to 11%) products; we observed no isoforms shorter than 23 nt. the miR-34 genomic locus. Of those reads mapping to the Thus, the shorter isoforms of miR-34 are unlikely to reflect miR-34 locus, 98.5% began at the annotated 50 end of inaccurate processing of pre-miR-34 by Dicer-1. miR-34; for miR-34-mapping reads bound to Ago1, 99.0% Sloppy Drosha cleavage of pri-miR-34 might also gene- shared this same, unique 50 end (see Figures S1A and S1B rate the shorter miR-34 isoforms. Such inaccurate Drosha available online). Similarly, 98.8% of all miR-34 reads in total Current Biology Vol 21 No 22 1880

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Figure 2. Trimming of miR-34 Requires Ago1 and Is Limited by the Removal of the microRNA* Strand (A) 50 32P-radiolabeled pre-miR-34 was incubated in 0–2 hr embryo lysate or lysate immunodepleted of Ago1. Products were resolved by denaturing polyacrylamide gel electrophoresis. (B) Double-stranded RNA (dsRNA)-triggered RNA interference (RNAi) targeting Ago1, but not Ago2, decreased trimming of miR-34, compared to treatment with a control dsRNA targeting GFP. ‘‘Trimmed’’ indicates the fraction of all miR-34 corresponding to 21 and 22 nt isoforms. The bantam microRNA (miRNA) and 2S ribosomal RNA (rRNA) served as controls. (C) The fraction of long miR-34 isoforms, measured by high throughput sequencing, increased when S2 cells were depleted of Ago1 by RNAi. Only isoforms with the annotated miR-34 50 end were analyzed. The abundance of miR-34 in the two libraries was 3,499 ppm (control) and 4,506 ppm (ago1 RNAi). (D and E) Synthetic duplexes of 50 32P-radiolabeled miR-34 (red) paired to variants of miR-34* (D) were incubated in 0–2 hr embryo lysate (E), and the products were analyzed by denaturing polyacrylamide gel electrophoresis. (F) Mean 6 standard deviation for three independent replicates of the experiment in (E). See also Figure S2. small RNA data sets from S2 cells shared this 50 end (Fig- 23–25 nt Dcr-1 products were not trimmed. In contrast, the ure S1C). We conclude that neither Dicer-1 nor Drosha con- miR-34 cleaved from pre-miR-34 was trimmed in lysate con- tribute to the observed heterogeneity in miR-34 length. taining Ago1 (Figure 2A). Similarly, the fraction of trimmed Despite the uniformity of the 50 end of miR-34 in the high miR-34 decreased in S2 cells depleted of Ago1 by RNAi, throughput sequencing data sets, miR-34 reads showed a compared to the control, when measured by both northern length heterogeneity similar to that detected by northern hybridization (Figure 2B) and high throughput sequencing (Fig- hybridization (Figure 1B), with 21, 22, and 24 nt isoforms ure 2C). RNAi depletion of Ago2—the Argonaute protein that accounting for most of the miR-34 reads in both S2 cells and binds small interfering RNAs in the RNA interference pathway fly heads (Figure 1D; Figure S1). We conclude that the ob- [18]—had no effect on the amount of trimmed miR-34. We served miR-34 length heterogeneity reflects 30 heterogeneity conclude that trimming of miR-34 requires Ago1, presumably generated after dicing by 30 trimming. Supporting this idea, because miR-34 trimming occurs after loading into Ago1. incubation of 50 32P-radiolabeled pre-miR-34 or a mature miR-34/miR-34* duplex in 0–2 hr embryo lysate produced 21 miRNA* Strand Dissociation Limits the Rate of miRNA to 22 nt isoforms (Figure 1E). In contrast, let-7, a 21 nt miRNA Trimming that has been extensively used to study canonical miRNA A key step in the assembly of mature Ago1-RISC is the removal biogenesis, function, and turnover, was not shortened when of the miRNA* strand from the Ago1-bound, miRNA/miRNA* incubated in embryo lysate. duplex, a process that converts pre-RISC to RISC. Mis- matches between the miRNA seed sequence and the corre- miRNA Trimming Requires Ago1 sponding nucleotides in the miRNA* promote maturation of Trimming of miR-34 might occur immediately after its produc- pre-Ago1-RISC [19–21]. We performed in vitro trimming tion by Dicer-1 when miR-34 is still bound to miR-34*, after assays using three miR-34/miR-34* duplexes that differ in loading of the miR-34/miR-34* duplex into Ago1 to generate the strength of pairing of the miR-34 seed sequence to the pre-RISC or following the eviction of miR-34* from pre-RISC seed match in miR-34* (Figure 2D). One duplex contained a to create miR-34-guided Ago1-RISC. To distinguish among mismatch within the miR-34 seed sequence. A second duplex these possibilities, we monitored pre-miR-34 processing and included two locked nucleic acid (LNA) ribose modifications miRNA trimming in 0–2 hr embryo lysate immunodepleted of within the seed match of miR-34*; LNA modifications increase Ago1 (Figure 2A). Although pre-miR-34 was efficiently con- the strength of base pairing by favoring the C30 endo ribose verted into miR-34 in the absence of Ago1, the resulting conformation found in RNA helices. None of the modifications Nibbler Trims MicroRNAs 1881

within the miR-34* strand are predicted to alter the rela- Nibbler homologs include Mut-7 in C. elegans and EXD3 in tive thermodynamic stability of the miR-34 versus miR-34* humans (Figure S3A). mut-7, which was one of the very first 50 ends and therefore preserve the preference to load miR-34 genes discovered to act in the RNAi pathway, is required for rather than miR-34* into Ago1. The mismatch miR-34* transposon silencing, RNAi, and cosuppression in worms more than doubled the rate of miR-34 trimming (kobs = 5.8 3 [26, 31–33], but no role for mut-7 in miRNA biogenesis has 25 10 nM/sec), compared to the canonical miR-34* (kobs = been reported. Like Mut-7 and EXD3, Nibbler belongs to the 2.7 3 1025 nM/sec). In contrast, the miR-34* containing LNA DEDD family of exoribonucleases, which are part of a larger modifications more than halved the rate of trimming (kobs = superfamily that includes DNA exonucleases as well as the 1.1 3 1025 nM/sec; Figures 2E and 2F). proofreading domains of many DNA polymerases [34]. DEDD Assembly of miRNA/miRNA* duplexes into pre-RISC pro- exonucleases contain three characteristic sequence motifs ceeds normally at 15C, but low temperature slows the transi- (Figure S3A), which include four invariant acidic amino acids tion from pre-RISC to mature RISC [19]. For all of the three (DEDD) (Figure 3B) [34, 35]. The structure of DNA polymerase miR-34/miR-34* duplexes, incubation at 15C further reduced suggests that these four amino acids organize two divalent the fraction of miR-34 that was trimmed (Figure S2A). The rate metal ions at the catalytic center [36]. Consistent with the of destruction of a miRNA* strand reflects the rate at which the view that Nibbler is a metal-dependent DEDD exoribonucle- miRNA and miRNA* strands dissociate. Mismatches between ase, miR-34 trimming in fly lysate was inhibited by EDTA; add- miR-34 and miR-34* accelerated the rate of destruction of ing additional Mg2+ rescued the inhibition (Figure S3B) [37, 38]. miR-34*, whereas the addition of LNA modifications to miR- We changed two of the four invariant amino acids of the 34* slowed the decay of miRNA*, compared to an unmodified Nibbler DEDD motif to alanine (D435A and E437A; Figure 3B), miR-34* RNA (Figure S2B). Thus, miR-34* modifications that mutations predicted to block exonuclease activity, then rein- accelerate RISC assembly also accelerated trimming, whereas troduced wild-type or mutant nibbler open reading frame into modifications that slow RISC assembly also slowed trimming. S2 cells depleted of endogenous nibbler using dsRNA Our results suggest that miR-34 is first loaded into Ago1 as targeting its 30 UTR (dsRNA 30 UTR; Figure 3B). nibbler comple- a 24 nt RNA and is only converted into shorter isoforms after mentary DNA (cDNA) expression was driven by the constitutive miR-34* is removed from pre-RISC. The majority of 24 nt Actin5C promoter (Figure S3C). In these experiments, deple- miR-34 likely corresponds to miR-34 bound to miR-34* in tion of endogenous nibbler in control S2 cells decreased the pre-RISC, because the 24 nt isoform, unlike the 21–23 nt iso- fraction of trimmed miR-34 from 54% to 32% (Figure 3D); forms, is not susceptible to target RNA-directed destruction, the presence of a stable, wild-type Nibbler transgene en- a process that requires extensive base pairing between the hanced miR-34 trimming (73% trimmed), even after depletion small RNA and its RNA target [22, 23]. of endogenous nibbler (78% trimmed miR-34; Figure 3D). Enhanced miR-34 trimming likely reflects the greater abun- The 30-to-50 Exoribonuclease Nibbler Trims miR-34 dance of Nibbler protein in the stable transgenic cell line, To identify the exoribonuclease that trims miR-34, we because nibbler mRNA levels were w100 times higher than in performed a candidate RNAi screen in S2 cells using long control S2 cells (data not shown). In contrast, expression of double-stranded RNA targeting genes with sequence similarity the D435A,E437A mutant Nibbler protein reduced miR-34 trim- to known or suspected exoribonucleases. Our screen included ming. The fraction of trimmed miR-34 decreased to 16% when Drosophila homologs of exonucleases previously implicated in transgenic, D435A,E437A mutant Nibbler was expressed along small RNA silencing pathways, such as the small RNA degrad- with endogenous Nibbler. The fraction of trimmed miR-34 ing nucleases (SDN) of plants [24], Enhancer of RNAi-1 (Eri-1) decreased to 7% when D435A,E437A mutant Nibbler was ex- [25], and Mut-7 in C. elegans [26], as well as components of pressed and endogenous Nibbler was depleted by RNAi. the general cellular RNA decay machinery such as RRP4, a In cultured Drosophila S2 cells, trimming of miR-34 by core component of the exosome, the SKI-2 ortholog Twister, Nibbler enhanced its target mRNA silencing activity. We and the general 50-to-30 exonuclease Pacman (XRN1) [27]. compared the repression of a miR-34-regulated Renilla renifor- RRP4, Twister, and Pacman were previously proposed to mis luciferase reporter in S2 cells stably expressing wild-type degrade the mRNA products generated by RNAi [28], and Nibbler to cells expressing D435A,E437A mutant Nibbler. S2 Xrn-1 was implicated in miRNA turnover [29, 30]. The miRNA cells expressing transgenic wild-type Nibbler produced mostly bantam, which does not undergo detectable trimming, served the 21 nt miR-34 isoform, whereas S2 cells stably expressing as a control for general destabilization of miRNAs. mutant Nibbler produce predominantly the 24 nt miR-34 iso- Among the exonucleases we tested, only depletion of form (Figure 3D). For each cell line, we compared the level of CG9247 decreased the fraction of trimmed miR-34 (frac- reporter expression when the cells were transfected with a tion trimmed = 20%), compared to control RNAi (fraction of control anti-miRNA 20-O-methyl oligonucleotide to the reporter miR-34 trimmed = 56%; Figure 3A). We observed a similar expression when the cells were transfected with an anti-miR-34 loss of miR-34 trimming for two additional, nonoverlapping 20-O-methyl oligonucleotide. The ratio of anti-miR-34 to control dsRNAs targeting different regions within the second exon indicated the extent of repression. We observed significantly and the 30 untranslated region of CG9247 (Figures 3B and 3C). (p = 0.003, n = 6) greater repression of the miR-34 reporter in In all cases, trimming of miR-34 was reduced by more than the cells expressing wild-type Nibbler, compared to those half. To reflect its role in 30 shortening of miRNAs, we named expressing the mutant protein, indicating that trimming of CG9247 nibbler (nbr). miR-34 to shorter isoforms enhances its activity. We conclude (We note that RNAi depletion of snipper [snp; CG42257] that trimming of long miRNAs by the Mg2+-dependent, 30-to-50 decreased full length 2S ribosomal RNA (rRNA) and caused exoribonuclease Nibbler enhances miRNA function. the accumulation of higher molecular weight isoforms of 2S rRNA, suggesting that Snipper plays a previously unknown Nibbler Trims Many miRNAs role in the maturation of 2S rRNA, which is generated by the To assess the role of Nibbler in the production of other processing of 5.8S rRNA in flies.) miRNAs, we sequenced 18–30 nt small RNAs from S2 cells Current Biology Vol 21 No 22 1882

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Figure 3. The 30-to-50 Exoribonuclease Nibbler (CG9247) Trims miR-34, Enhancing miR-34 Function in S2 Cells (A) S2 cells were transfected with dsRNA against a panel of predicted exonucleases and the effect on miR-34 length analyzed by high resolution northern hybridization. bantam and 2S rRNA served as controls. The fraction of miR-34 trimmed to 21 to 22 nt is indicated below each lane. (B) The predicted structure of the nibbler (CG9247) gene, messenger RNA (mRNA), and protein. (C) S2 cells were transfected with three dsRNAs targeting the second exon or the 30 UTR of nibbler as indicated in (B). All four dsRNAs decreased miR-34 trimming, relative to a control dsRNA targeting firefly luciferase. bantam and 2S rRNA served as controls. (D) S2 cells stably expressing wild-type or D435A,E437A mutant Nibbler were transfected with dsRNA targeting the 30 UTR of endogenous nibbler and the effect on miR-34 trimming measured. bantam and 2S rRNA served as controls. (E) Reporter construct used in (F). The three miR-34 binding sites pair with miR-34 nucleotides 2–8 and 13–15, mimicking typical animal miRNA binding sites [55]. The following abbreviation is used: Rr luc, Renilla reniformis luciferase. (F) Nibbler trimming of miR-34 enhances miRNA function. Repression by miR-34 in S2 cells expressing wild-type or D435A,E437A mutant Nibbler was measured by blocking miR-34 using a 20-O-methyl-modified anti-miRNA oligonucleotide and measuring the increase in Rr luciferase expression compared to a control oligonucleotide targeting let-7, a miRNA not normally expressed in S2 cells. See also Figure S3.

treated with nibbler dsRNA and from S2 cells treated with a in mean length suggests that miR-34 is not the only miRNA control dsRNA. S2 cells produce 36 distinct miRNAs that trimmed by Nibbler in S2 cells. were detected at >200 parts per million (ppm) in our high In fact, of the 36 abundantly expressed S2 cell miRNAs, 28 throughput sequencing. Among the isoforms of these 36 increased in mean length. Of these, 13 increased by more miRNAs, we detected a small but statistically signiﬁcant than 0.1 nt and nine by more than 0.33 nt. We used a chi- increase in the overall mean length of miRNAs when Nibbler square test to assess the signiﬁcance of the change in the was depleted: 21.96 nt in the control versus 22.11 nt in nibbler distributions of isoform lengths in the nibbler (RNAi) S2 cells (RNAi) (p = 3.9 3 1025, Wilcoxon signed rank test). If all of for each miRNA (Table S1). An increase of w0.2 nt in mean miR-34 were 22 nt long in the control and became 24 nt in length was the smallest change we could corroborate by the nibbler dsRNA-treated cells, the mean miRNA length northern hybridization, an admittedly less sensitive method would be expected to increase by 0.056. Thus, a 0.15 increase than high throughput sequencing. Using the 0.2 nt mean length Nibbler Trims MicroRNAs 1883

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Figure 4. Nibbler Trims a Quarter of All miRNAs in S2 Cells (A) Analysis of mean miRNA and miRNA* length in S2 cells transfected with dsRNA targeting nibbler or a control dsRNA targeting firefly luciferase. miRNA is shown in red, miRNA* is shown in blue, and filled circles indicate miRNAs with a significant increase in mean length. In S2 cells, miR-7 (green) does not match our conservative criteria for Nibbler substrates, but in flies, miR-7 is trimmed by Nibbler (Figure S5C). (B) Nibbler trimming explains miRNA 30 heterogeneity. 30 heterogeneity was determined for all S2 cell miRNAs that were more abundant than 200 ppm in high throughput sequencing data. The 11 Nibbler substrates identified in this study are shown in red. Boxplots illustrate 30 heterogeneity of Nibbler substrate miRNAs (red) versus all other miRNAs (black). P was determined using the Mann-Whitney U test. (C) The mean length of Nibbler substrate miRNAs is longer than nonNibbler substrate miRNAs in S2 cells treated with nibbler dsRNA. P was determined using the Mann-Whitney U test. (D and E) Synthetic miRNA/miRNA* duplexes comprising a 24 or 22 nt 50 32P-radiolabeled miR-305 RNA and the corresponding miRNA* strand (D) were incubated in embryo lysate, and the products were analyzed by denaturing polyacrylamide gel electrophoresis (E). See also Figure S4 and Tables S1 and S2. increase as a conservative threshold, 11 S2 cell miRNAs corre- Ago2-bound small RNAs are not Nibbler substrates (Fig- spond to Nibbler substrates (red filled circles, Figure 4A and ure S4C). We also analyzed the effect of Nibbler depletion on Figure S4A). Thus, R30% of S2 cell miRNAs are trimmed by the length of the 32 miRNA* strands for which we de- Nibbler after their production by Dicer-1. tected >10 ppm by high throughput sequencing. The overall Nibbler substrates included both miRNAs derived from the miRNA* mean length changed from 22.00 to 22.02 nt (p = 50 arm of their pre-miRNA (four miRNAs) and miRNAs derived 0.04, Wilcoxon signed-rank test), but only two miRNA* strands from the 30 arm of their pre-miRNA (seven miRNAs). miRNAs showed a significant increase in the nibbler dsRNA-treated S2 trimmed by Nibbler account for most of the previously identi- cells when analyzed using the chi-square test; neither of the fied 30 heterogeneity of S2 cell miRNAs, because Nibbler- two miRNA* strands increased more than 0.1 nt (Table S1). substrates exhibit significantly higher 30 heterogeneity than Consistent with the proposal that miRNA trimming occurs after nonsubstrate miRNAs (p < 0.0001, Mann-Whitney U-test, Fig- miRNA* strands depart from pre-RISC, those miRNA* strands ure 4B). In contrast, 50 heterogeneity, which is generally low whose miRNAs were Nibbler substrates did not change sig- because of the purification process associated with Argonaute nificantly in length when compared to all other miRNA*s loading [39], was unaffected by the depletion of Nibbler by (Figure S4D). RNAi (Figure S4B). What destines miRNAs for trimming by Nibbler? Perhaps The 11 Nibbler substrate miRNAs were significantly longer many Nibbler substrate miRNAs are initially produced by in S2 cells treated with nibbler dsRNA than nonNibbler sub- Dicer-1 as long isoforms that are trimmed to a more typical strate miRNAs: the median of the mean lengths was 23.0 nt miRNA length. To test this idea, we incubated synthetic miR- for Nibbler substrates versus 21.8 nt for all others (p = 0.02, 305/miR-305* duplexes (Figure 4C) in Drosophila embryo Mann-Whitney U test, Figure 4C). In contrast to Nibbler lysate and monitored their trimming. In vivo in flies, miR-305 substrate miRNAs, the length of endogenous siRNAs did not is efficiently trimmed (Figure S5A, adult males). Moreover, change after depletion of Nibbler by RNAi, suggesting that miR-305 is abundantly expressed and efficiently trimmed in Current Biology Vol 21 No 22 1884

0–2 hr embryos: among the 3,668 ppm miR-305 reads de- exclude the possibility that this pupal lethality reflects a tected in the total small RNAs of 0–2 hr embryos, 23 nt (5%) requirement for Nibbler in processing substrates other than and 24 nt (14%) miR-305 isoforms represent just 19% of all miRNAs. miR-305 reads, whereas the shorter, trimmed 21 nt (45%) and 22 nt (32%) isoforms represent 77% of all miR-305 reads. Discussion When a 24 nt 50 32P-radiolabeled miR-305, paired to a 23 nt miR-305* strand, was incubated overnight in embryo lysate, miRNA 30 heterogeneity [39, 41, 42] has been attributed to 17% was trimmed to shorter isoforms: 10% accumulated as inaccurate processing by Dicer or Drosha. Our data suggest 23 nt, 5% as 22 nt, and 2% as 21 nt (Figure 4D). In contrast, that much of the 30 diversity of miRNAs reflects their trimming only 2% of a duplex comprising the 22 nt isoform of miR-305 by a novel processing step catalyzed by the 30-to-50 exoribo- paired to a 22 nt miR-305* strand was converted to a 21 nt nuclease Nibbler. Figure 5G presents a revised model for the form; no species shorter than 21 nt were detectable. We production of mature miRNAs from pre-miRNAs in flies. First, conclude that miRNA trimming is triggered, at least in part, Dicer-1 converts pre-miRNAs to miRNA/miRNA* duplexes. by the length of the miRNA, with w24 nt miRNAs being con- These are then sorted between Ago1 and Ago2 to generate verted by Nibbler into the 21 to 22 nt length, which is more Ago1- and Ago2-pre-RISC complexes, with Ago1 selec- typical for miRNAs at steady-state. ting R22 nt miRNAs that begin with an unpaired U or A and containing an unpaired region centered on position 9 [19–21, Nibbler Trims miRNAs In Vivo 23, 43–47]. The Ago1 sorting process helps restrict the diver- To test the role of Nibbler in vivo, we obtained two publicly sity of 50 ends of miRNAs. Next, the miRNA* strand dissociates available Drosophila strains bearing a transposon insertion from pre-RISC to produce RISC. We imagine that the 30 ends of in nibbler: nibblerEY04057, corresponding to a P element inser- ‘‘long’’ miRNAs bound to Ago1 are available for trimming by tion in the 50 UTR of nibbler and nibblerf02257, corresponding to Nibbler because they spend less time bound to the Ago1 a piggyBac insertion in the first exon of nibbler (Figure 3B). PAZ domain than do 22 nt miRNAs. Once Nibbler has short- nibblerEY04057 was homozygous viable and showed no change ened a long miRNA to 22 nt, its 30 end can bind the PAZ in nibbler mRNA abundance compared to Oregon R or w1118 domain, protecting it from further trimming. For miR-34, we control flies (Figure S5B). However, our preliminary data sug- observed that trimming enhanced miRNA activity. gests that the nibbler mRNAs in nibblerEY04057 originate within Depletion of Nibbler in S2 cells (Figures 3A and 3C) and in the P element (data not shown) and may therefore not pro- flies (Figures 5A, 5C, and 5E; Figure S5) resulted in the appear- duce wild-type levels of Nibbler protein. nibblerf02257 was ance of higher molecular weight species, reminiscent of tailed homozygous lethal, and nibblerf02257 heterozygotes produced small RNAs rather than bona fide Dicer products. Such non- 38% 6 24% of the amount of nibbler mRNA present in w1118 templated addition of nucleotides to the 30 ends of mature control flies (p = 0.008, Figure S5B). The fraction of miR-34 miRNAs has been implicated in miRNA turnover in plants that was trimmed was reduced, albeit slightly, in both mu- [48] and animals [22, 23, 49] and may indicate that Nibbler- tants: 57% of miR-34 was trimmed in Sp/CyO control flies, substrate miRNAs are marked for decay when not properly whereas 51% was trimmed in nibblerEY04057/CyO and 52% trimmed. In contrast to Ago1-bound miRNAs, Ago2-bound was trimmed in nibblerf02257/CyO (Figure 5A). Trimmed miR-34 small RNAs would be protected from Nibbler, perhaps accounted for only 40% of all miR-34 isoforms in nibblerEY04057 because of their shorter mean length of w21 nt (Figure S4C) homozygotes, and in nibblerEY04057/nibblerf02257 transhetero- or because they are 20-O-methyl modified by Hen1 [50–52]. zygotes, just 21% of miR-34 was trimmed (Figure 5A). We This model does not invoke specific recruitment of Nibbler conclude that trimming of miR-34 requires Nibbler in vivo. to Ago1-RISC and is consistent with our preliminary experi- Similarly, the two nibbler mutations recapitulated the effect ments, in which we were unable to detect epitope-tagged, on 11 miRNAs identified as Nibbler substrates in S2 cells overexpressed Nibbler bound to immunoprecipitated Ago1 (Figure S5C). (data not shown). However, such a simple model cannot nibblerf02257 likely corresponds to a strong allele, but this explain why some trimmed miRNAs do not accumulate iso- mutation is not homozygous viable. To test whether loss of forms longer than 22 nt even after Nibbler was depleted by Nibbler affects fly development, we used RNAi to deplete RNAi (e.g., miR-11; Figure S4A), suggesting that miRNA length Nibbler in vivo [40]. When driven by an Actin5C-Gal4 driver, alone does not define a Nibbler substrate. Perhaps additional a UAS-hairpin RNA (hpRNA) transgene on the second chromo- proteins help recruit Nibbler to Ago1-RISC for some miRNAs. some (UAS-hpRNAv52550) reduced the fraction of miR-34 that A requirement for Nibbler cofactors could also explain why was trimmed to 43% of all miR-34 isoforms, compared to Nibbler trims miR-7 in flies but not in S2 cells (Figure 4; 68% in flies expressing the Act5C-Gal4 driver alone or to Figure S5A). 66% in the flies carrying only the UAS-hpRNA transgene Is miRNA trimming conserved in other organisms? Small (Figures 5C and 5D). An insertion of the same hpRNA construct RNAs in the human cervical carcinoma cell line HeLa exhibit on the third chromosome (hpRNAv52612) reduced the fraction an overall miRNA 30 heterogeneity similar to that observed of miR-34 that was trimmed to 13% of all miR-34 isoforms, for fly miRNAs (Figures S5E and S5F). Several human miRNAs compared to 60% in flies carrying only the Actin5C-Gal4 driver with high 30 heterogeneity show a length distribution in HeLa or 53% in flies bearing only the UAS-hpRNA transgene (Figures cells reminiscent of Nibbler-substrates in flies (Figure S5G). 5E and 5F). Notably, 29% (69 of 239) of the flies expressing Perhaps a human homolog of fly Nibbler processes these UAS-hpRNAv52612, the RNAi transgene with the stronger effect miRNAs. The C. elegans homolog of Nibbler, Mut-7, is required on miR-34 trimming, failed to eclose from their puparia. Only for the accumulation of the 22G RNAs that direct worm Piwi 5% (16 of 327) of the Actin5C-Gal4/CyO; Dr/TM3,Sb control proteins to represses transposon expression [26, 53]. We do flies and only 2% (5 of 298) of the +; UAS-hpRNAiv52612 control not yet know whether Nibbler functions in the analogous flies died as pupae. Although miRNAs regulate fly develop- piRNA pathway in flies or whether Mut-7 has a yet undiscov- ment and Nibbler acts on miRNAs, we currently cannot ered role in miRNA maturation in worms. Nibbler Trims MicroRNAs 1885

C DEEF

Figure 5. miRNA Trimming Requires Nibbler In Vivo (A, C, and E) High resolution northern hybridization of miR-34 from 3–5 day-old male ﬂies. 2S rRNA served as a loading control. (B, D, and F) Abundance of miR-34 isoforms in ﬂies carrying a nibbler mutant allele (B) or in which Nibbler was depleted by RNAi (D and F). miR-34 isoform abundance was measured relative to the indicated controls. (G) A model for miRNA trimming by Nibbler. See also Figure S5.

Experimental Procedures For miRNA trimming, 50 32P-radiolabeled RNAs (2 nM) were incubated with 0–2 hr embryo lysate as described [54], except that RNase inhibitor Pre-miRNA Processing and Trimming Assays was omitted. Products were resolved by electrophoresis through a 15% Pre-miR-34 was transcribed with T7 RNA polymerase using a double- denaturing polyacrylamide sequencing gel. Gels were dried, exposed to stranded DNA oligonucleotide template (see Supplemental Experimental storage phosphor screens (Fuji), and quantiﬁed using ImageGauge 4.22 Procedures), dephosphorylated with Calf Intestinal Phosphatase (New (Science Lab 2003, Fuji). England Biolabs) and 50 32P-radiolabeled with T4 Polynucleotide Kinase To analyze miRNA trimming for synthetic 50 32P-radiolabeled 24 nt (New England Biolabs). Pre-miR-34 (2 nM) was incubated with recombinant miR-34, we considered all isoforms shorter than 24 nt to be trimmed. Dicer-1/Loquacious PB (5 nM) or S2 cell or 0–2 hr embryo lysate for 15 min at When pre-miR-34 was used as a substrate, we considered only isoforms 25C in a typical RNAi reaction [54]. Ago1 immunodepletion was as shorter than 23 nt to be trimmed, because Dicer-1 produces 23, 24, and described [20]. 25 nt miR-34 isoforms from pre-miR-34, so only isoforms shorter than Current Biology Vol 21 No 22 1886

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