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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Curr Biol. Author manuscript; available in PMC Apr 13, 2010.
Published in final edited form as:
PMCID: PMC2854039
NIHMSID: NIHMS184415

Argonaute loading contributes to the precision of the 5′ ends of both microRNAs and their miRNA* strands in flies

Summary

MicroRNAs (miRNAs) are short (~21–23 nt) regulatory RNAs that direct repression of their mRNA targets. The miRNA “seed”—nucleotides 2 through 7—establishes miRNA target specificity, because small silencing RNAs bind their targets through this region [1-5]. Accurate processing of the miRNA 5′ end is thought to be under strong selective pressure [6, 7], as a shift by just one nucleotide in the 5′ end of a miRNA would alter its seed sequence, redefining its repertoire of targets (Figure 1). Animal miRNAs are produced by the sequential cleavage of partially double-stranded precursor RNAs by the RNase III endonucleases Drosha, which acts in the nucleus to convert primary miRNA transcripts into pre-miRNAs, and Dicer, which cleaves the pre-miRNA in the cytoplasm to generate a transitory double-stranded intermediate comprising the mature miRNA paired to its partially complementary miRNA* strand [8, 9]. Here, we report that in flies, the 5′ end of a miRNA and of its miRNA* strand is typically more precisely defined than the 3′ ends of either the miRNA or its miRNA*. Surprisingly, the 5′ ends of both miRNA and miRNA* sequences present in mature Argonaute2 (Ago2) complexes are more precisely defined than in the total small RNA population. Our data imply that either many miRNA* sequences are under evolutionary pressure to maintain their seed sequences—that is, they have cellular or exogenous RNA targets—or that secondary constraints such as the sequence requirements for loading small RNAs into functional Argonaute protein complexes narrow the range of miRNA and miRNA* 5′ ends that accumulate in flies.

Figure 1
Inaccurate processing of the 5′ end of a miRNA alters its seed sequence

Results and Discussion

We used high-throughput pyrosequencing of 18–30 nt RNAs to identify miRNAs expressed in Drosophila melanogaster heads and in cultured Drosophila S2 cells. Among the 120,896 miRNA reads (66,377 from fly heads; 54,519 from S2 cells), we observed two sources of heterogeneity for the ends of fly miRNAs: the addition of nucleotides not present in the gene from which the miRNA is transcribed (non-templated nucleotides) and inaccurate or alternative cleavage by Drosha or Dicer. About 5% of the reads for a typical miRNA contained non-templated nucleotides on at least one end (Figure 2A and S1), most frequently the addition of a single uridine or adenosine to the 3′ end, but longer extensions were also observed, both on the 5′ and the 3′ ends (Table S1). Interestingly, longer extensions were also U- and A-rich at the 3′ end, while at the 5′ end, the 3′-most non-templated nucleotide was frequently a cytidine, and other added nucleotides were typically uridines. This observation could prove useful for the identification of the 5′-elongating enzymatic activity. The non-templated addition of nucleotides, especially uridines, to the 3′ ends of miRNAs has been reported previously in wild-type Caenorhabditis elegans [6] and hen1 mutant Arabidopsis thaliana [10]. Overall, the addition of non-templated nucleotides to the 5′ end of miRNAs was more rare (~1%; Figure 2A and Table S1).

Figure 2
Cleavage inaccuracies are more frequent than non-templated additions

We also observed a second, more frequent type of heterogeneity: variability in the position of the miRNA 5′ and 3′ ends within the sequence of the miRNA precursors (Figure 2B). Non-templated nucleotides fortuitously matching the templated sequence are predicted to occur much less often than the heterogeneity we observe (Table S2). Similar terminal heterogeneity has been noted for the 3′ ends of C. elegans [6] and the 5′ and 3′ ends of mouse [11] miRNAs. The aberrant miRNA termini we observe likely reflect imprecision in precursor cleavage by Drosha and Dicer. They are unlikely to correspond to degradation products, because we recorded nearly as many miRNA reads that were longer than the dominant species as were shorter Figure S1), and because 93% (S2 cells) and 99% (fly heads) of sequences of the fly-specific 30 nt 2S ribosomal RNA (rRNA)—whose termini are expected to be single-stranded—were full-length (Supplemental Discussion). 3′ degradation was slightly more common than 5′ degradation: we detected 3′ degradation for 1,010 reads versus 5′ degradation for 201 reads among the 33,505 total 2S rRNA reads from S2 cells and fly heads combined; 5 reads corresponded to 2S rRNA trimmed from both ends.

The 5′ ends of miRNAs were more precisely defined than their 3′ ends, irrespective of whether the miRNA originated from the 5′ or 3′ arm of the premiRNA (Figure 3A). Thus, the difference in cleavage accuracy between the 5′ and 3′ ends cannot be attributed to an intrinsic difference in fidelity between Drosha and Dcr-1. We expected that the 3′ ends of miRNA* strands would be precisely defined, because they are created by the pair of cuts that generates the 5′ ends of miRNA, and that the 5′ ends of miRNA* strands would be imprecisely determined, because they are created by the pair of cleavages that generates the highly heterogeneous 3′ ends of miRNA. Instead, we found that the 5′ end of a strand (for example, the miRNA) was more accurate than the 3′ end of the adjacent strand (in this example, the miRNA*; Figure 3B); these two extremities are produced by a pair of cuts catalyzed by the same enzyme.

Figure 3
miRNA and miRNA* 5′ ends are more precisely defined than their 3′ ends

Current dogma holds that the local sequence or structure of miRNA precursors is under strong selective pressure to generate accurate 5′ ends, because a precise miRNA 5′ end directly establishes the seed sequence and hence the targets of the miRNA. Since we observe that, in flies, the 5′ ends of both the miRNA and the miRNA* are more precisely determined than the 3′ ends of either strand, this explanation implies that miRNA* sequences are under selective pressure to establish a unique seed sequence, implying that they, too, have regulatory targets.

It is also possible that both Drosha and Dcr-1—whose active sites are homologous—may also be intrinsically more precise in 5′ cleavage than in 3′ cutting. A third alternative is that 5′ and 3′ ends might be generated with similar, imperfect accuracy, but subsequent constraints in RISC loading or stability select for those small RNAs that begin with a particular nucleotide or sequence. The subsequent destruction of miRNAs without these 5′ features would increase the apparent accuracy of miRNA 5′ ends while retaining miRNA 3′ heterogeneity. To test this idea, we separately sequenced small RNAs containing modified 3′ termini (Table S3). In flies, the 3′ termini of small RNAs that are loaded into Ago2 [12], but not those bound to Argonaute1 [13], are 2′-O-methylated by Drosophila Hen1 as the last step in Ago2-RISC maturation [14]. To sequence small RNAs bearing 2′-O-methylated 3′ ends, we treated the total small RNA with NaIO4 followed by β-elimination; this method blocks ligation of adapters to small RNAs bearing 2′,3′ hydroxy termini, preventing them from being sequenced.

To determine if the greater accuracy of miRNA and miRNA* 5′ versus 3′ ends reflects the constraints of RISC assembly or stability, rather than more accurate 5′ versus 3′ cleavage by Drosha and Dicer, we compared the terminal heterogeneity of miRNA and miRNA* reads from the 3′ modified population to the heterogeneity of the total miRNA and miRNA* population. As a control, we compared the 3′ heterogeneity between the two populations. For both analyses, we only considered miRNA or miRNA* strands displaying some heterogeneity in the total population. For both fly heads and S2 cells, we observed a dramatic increase in the precision of the 5′—but not the 3′—ends of miRNAs and miRNA* strands upon loading into Ago2 (Figure 4). We also performed the analysis for those small RNAs that both had heterogeneous termini and were specifically enriched in the b-eliminated sequences relative to the non-b-eliminated set. For the 13 small RNAs (4 miRNAs and 9 miRNA*s) meeting these criteria, the 5′ ends in the sub-population of miRNA and miRNA* sequences loaded into Ago2—i.e., those that were 2′-O-methylated—were again more precisely defined than the 5′ ends of the same small RNA sequences in the total small RNA population (Figure S3). We conclude that loading or stabilization of miRNAs in Ago2, and, perhaps Argonaute proteins in general, imposes a purifying selection on their 5′ ends.

Figure 4
Ago2-loading, as evidenced by 3′ terminal 2′-O-methylation, refines miRNA and miRNA* 5′ ends

The mechanism responsible for the homogenization of 5′ ends remains to be determined. We can imagine that the efficiency of Argonaute loading is affected by the nature of the 5′ end of a small RNA, much as the stability of its pairing to the other strand influences this process [21]. The 5′ sequence itself may also play a role in RISC assembly, with some miRNA variants loaded more efficiently than others, according to the identity of their 5′ nucleotide(s). Alternatively, some Argonaute complexes might be selectively stabilized after their assembly, for example, by the presence of a target RNA whose binding stabilizes those RISCs containing miRNA isoforms with a complementary seed sequence.

Experimental Procedures

Biological Sources

Fly heads were isolated by vigorously shaking liquid nitrogen-frozen flies expressing a long double-stranded hairpin RNA corresponding to white [15, 16] in nested, pre-chilled sieves (U.S.A. standard sieve, Humboldt MFG Co., Chicago, IL, USA), allowing the heads to pass through the top sieve (No. 25) and collecting them on the bottom sieve (No. 40). S2 cell RNA was prepared from a clonal line containing the stably-integrated GFP transgene (pKF63) and transiently transfected with a double-stranded RNA against GFP [17].

RNA Preparation

400 μg total RNA was extracted using the mirVana kit (Ambion), then 18-to-30 nt long RNAs gel purified. 2S rRNA was depleted by hybridization to immobilized DNA oligonucleotide (5′-biotin-TCA ATG TCG ATA CAA CCC TCA ACC ATA TGT AGT CCA AGC A-3′). 1.6 nmol of the biotinylated oligonucleotide was bound to 32 mg M270 Streptabeads (Dynal, Norway) in 3.2 ml 0.5x SSC for 30 min on ice, then the beads were washed with ice-cold 0.5x SSC, resuspended in 8 ml 0.5x SSC, and incubated 5 min at 65°C. Gel-purified RNAs were diluted with 7 volumes 0.5x SSC to a final volume of 160 μl and denatured at 80°C for 5 min, then added to the bead suspension and incubated 1 h at 50°C. Beads were magnetically captured for 1 min at room temperature, then the 2S rRNA-depleted supernatant collected and precipitated with absolute ethanol. More than 99% of the 2S rRNA was routinely removed without measurably altering miRNA concentration; without the depletion step, nearly all the small RNA reads would correspond to 2S rRNA. Half the sample was then b-eliminated as described [18] and half was subject to the same treatment, except that sodium periodate was omitted.

Amplification and Pyrosequencing

Adapters were ligated to the small RNA sample, and the resulting library amplified by PCR as described [19], except that a truncation mutant of RNA ligase 2 [Rnl2(1-249); ref 20] was used for the 3′ ligation step; T4 RNA ligase (Ambion) was used for 5′ ligation. The 5′ adapter was 5′-dAdTdC dGdTrA rGrGrC rArCrC rUrGrA rArA-3′ (Dharmacon, Lafayette, CO, USA); 3′ ‘preadenylated’ adapters were 5′-rAppdCdA dCdTdC dGdGdG dCdAdC dCdAdA dGdGdA ddC-3′ for fly head and 5′-rAppdTdT dTdAdA dCdCdG dCdGdA dAdTdT dCdCdA dGddC-3′ for S2 cell RNA (IDT DNA, Coralville, IA, USA). After adapter addition, the RNA was amplified by PCR using DNA primers corresponding to the adapters. This PCR pool was gel purified (4% Metaphor Agarose, Cambrex, East Rutherford, NJ, USA) with Qiaex II (Qiagen, Valencia, CA, USA), then re-amplified by PCR (common 5′ primer, 5′-GCC TCC CTC GCG CCA TCA GAT CGT AGG CAC CTG AAA-3′; 3′ primer for fly heads, 5′-GCC TTG CCA GCC CGC TCA GTC CTT GGT GCC CGA GTG-3′; 3′-primer for S2 cells, 5′-GCC TTG CCA GCC CGC TCA GCT GGA ATT CGC GGT TAA A-3′). The PCR-amplified libraries were pyrosequenced by Roche Applied Science (Branford, CT, USA). Sequence and abundance data are available via the NCBI gene expression omnibus web site (http://www.ncbi.nlm.nih.gob/geo/) using accession number GSE9389.

Computational Methods

Eighteen-to-30-nt long reads were mapped to the Drosophila melanogaster genome (FlyBase assemblyR5.1; http://flybase.org/) and to the D. melanogaster “stem-loops” (which include the pre-miRNA sequences, usually extended by a few nucleotides) listed in miRBase (http://microrna.sanger.ac.uk/sequences/; version 10.0, August 2007). To identify non-templated microRNA additions, nongenome matching sequences were iteratively trimmed by 1 to 3 nucleotides on either the 5′ or the 3′ end and mapped to stem-loops.

Among stem-loop-matching reads, miRNA-matching and miRNA*-matching reads were identified, using either the experimentally detected miRNA* sequence (when it was available in the miRBase records) or the product of conceptual dicing of the hairpin [21]. To include reads that showed extremities different from those annotated in miRBase, a distance of as many as 9 nucleotides 5′ or 3′ from the annotated miRNA or miRNA* sequence was tolerated. Statistical calculations were made using the R statistical package. p-values were calculated using the Wilcoxon test.

Supplementary Material

Acknowledgments

We thank Stewart Shuman for providing the truncated Rnl 2 expression plasmid, Alicia Boucher for assistance with fly husbandry, Gwen Farley for preparing RNA ligase, and members of the Zamore lab for advice, suggestions, and critical comments on the text. We are especially grateful to Roche Applied Science for high-throughput sequencing. P.D.Z. is a W.M. Keck Foundation Young Scholar in Medical Research. This work was supported in part by grants from the National Institutes of Health to P.D.Z. (GM62862 and GM65236) and EMBO long-term (ALTF 910-2004) and HFSP (LT00575/2005-L) fellowships to HS.

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