Evolutionary Features of Mirtrons Support Their Status as Regulatory RNAs
Pre-miRNA hairpins collectively yield functional RNAs from both 5′ and 3′ hairpin arms (). In contrast, mirtron hairpins yield predominant small RNAs from only their 3′ arms (Ruby et al., 2007
). The 5′ splice consensus may intrinsically bias mirtron processing, since miRNAs and mirtron-derived small RNAs exhibit strong preferences to begin with U residues, whereas 5′ mirtron-derived RNAs must begin with a G residue (Ruby et al., 2007
). In addition, mirtrons typically exhibit extensive pairing between the 5′ and 3′ splice sequences (), a layout that may bias the selection of their 3′ arms as regulatory species (Khvorova et al., 2003
; Schwarz et al., 2003
). The asymmetry of mirtron processing toward 3′ products is rational from a biological perspective, since the regulatory potential of their 5′ products is perhaps undesirably constrained by the splice donor sequences within their prospective seed regions.
Further support for the regulatory status of mirtrons came from the observation that several mirtronic introns are well conserved among the sequenced Drosophilids (Figures and S1
; Ruby et al., 2007
). On the other hand, most mirtrons are preserved only within species of the melanogaster subgroup (D. melanogaster
, D. simulans/sechellia
, and/or D. yakuba/erecta
), suggesting that they were born within the last 5–10 million years (Figures and S1
; Ruby et al., 2007
). Nevertheless, detailed inspection revealed that the evolution of both “old” and “young” mirtrons parallels that of miRNAs in two aspects (reviewed by Lai et al. 
). First, as is the case for miRNA hairpins, both young and old mirtrons exhibit accelerated divergence in loop regions relative to the hairpin stems (Figures and S1
). Second, mirtron loci display preferential conservation of the 5′ seed region, a key determinant for miRNA target recognition (e.g., ). In fact, small RNAs generated by mirtrons resident in CG6695 and CG31772 (miR-1003 and miR-1004, respectively) have the same seed (Figures , , and S2
), indicative of a functional subfamily.
Mirtrons Repress Both Perfect-Match and Seed-Match Targets
These observations suggested that mirtrons are RNA genes related to miRNAs. Since experimental evidence presented in this study (see below) and a contemporary study (Ruby et al., 2007
) supported their identity as a functional subclass of miRNA genes, we refer to the hairpin introns as mirtrons and their small-RNA products as miRNAs.
Mirtrons Display Distinct Temporal and Spatial Patterns of Expression
We began our functional studies by using northern analysis to ask whether processed mirtrons could be detected across development or in cultured cells. We probed total RNA from 0–24 hr embryos, third-instar larvae/pupae, adults, and S2 cells to northern analysis with γ-32P-labeled locked nucleic acid (LNA) oligos antisense to the terminal 22–24 nt of mirtron hairpins for mir-1003, mir-1010, and mir-1008. These probes detected mature ~21–24 nt RNAs and rarer ~55–70 nt precursors (). As with miRNAs, such discretely hybridizing bands reflected precision in mirtron processing and argued against the possibility that the cloned sequences merely represent metabolic intermediates of spliced introns. We also note that mirtrons exhibited variety in their developmental and spatial expression profiles, similar to miRNAs. For example, the small-RNA products of mir-1003 and mir-1008, but not of mir-1010, were detected in S2 cells. mir-1010 also differed in that its expression was much reduced in adults relative to earlier stages ().
Distinct Temporal and Spatial Expression of Endogenous Mirtrons
Introns Can Autonomously Dictate Their Entry into the Mirtron Pathway
We next investigated whether mirtrons could be expressed exogenously. To do so, we cloned ~400 nt primirtron genomic fragments, whose termini lie within the exons flanking the mirtron, and inserted them into the 3′ UTR of a UAS-DsRed
vector (, construct A). This generic strategy successfully generates mature Drosophila
miRNAs from similarly sized pri-miRNA fragments (Lai et al., 2005
; Stark et al., 2003
). For these studies, we selected both highly conserved (mir-1003
) and newly born (mir-1008
) mirtron loci. When transfected into S2 cells with ub-Gal4
, such constructs directed the expression of all four mirtrons and their mature small-RNA products (, lanes 2 and 7, and data not shown).
Structure-Function Analysis of Mirtron Biogenesis
We then tested the ability of mirtrons to be processed when resident in the coding context of a designed vector. To exclude the potential contribution of specific exonic sequences to mirtron maturation, we designed a host vector in which the mirtronic intron alone is inserted into the coding region of a DsRed-myc transcript (, construct B). We found that mature miR-1003, miR-1008, and their corresponding mirtrons were produced from such constructs at levels comparable to their expression from endogenous exonic contexts (, lanes 3 and 8; compare with lanes 2 and 7). Therefore, the sequence of a short intron can autonomously dictate its ability to enter the mirtron pathway.
Mirtron Biogenesis Exhibits Little Dependence on Drosha
The concordance between mirtron ends and splice sites (Figures and ) suggested that their biogenesis might bypass the essential miRNA-producing enzyme Drosha. We tested this by treating S2 cells with dsRNA against Drosha
. Western blot analysis confirmed robust knockdown of Drosha protein after 4 days (Figure S2A
). As a control, we tested the behavior of a UAS-DsRed-mir-1
construct; S2 cells do not normally express miR-1. As shown in (lanes 1 and 2), the levels of pre-miR-1 hairpin and mature miR-1 were significantly reduced in Drosha
dsRNA-treated cells relative to green fluorescent protein
) dsRNA-treated cells. In contrast, endogenous miR-1003 and its mirtron were not demonstrably changed by these treatments (, lanes 6 and 7), and the accumulation of exogenous miR-1003, miR-1010, and their associated mirtrons was also similar between GFP
-knockdown cells (, lanes 11 and 12 and 19 and 20). While these data do not exclude a contribution of Drosha to mirtron processing, they indicate that mirtron biogenesis does not exhibit the strong dependence on Drosha that is characteristic of canonical miRNA pathway substrates.
Mirtron Biogenesis Involves a Hybrid Pathway that Couples Splicing and Dicing
Mirtron Biogenesis Requires Intron Splicing and Lariat Debranching
We next tested the alternative hypothesis that splicing might directly initiate mirtron biogenesis. To do so, we made single G -> C substitutions in the 5′ splice sites of otherwise functional mir-1003 and mir-1008 mirtron-expression constructs (, construct C). Such mutations completely abolished the accumulation of mirtron hairpins and mature ~22 mers (, lanes 4 and 9), indicating that both forms are obligate splicing products.
Since spliced introns adopt a lariat structure in which the 5′ splice junction is covalently linked to the 3′ branch point, we hypothesized that Drosophila
lariat-debranching enzyme (Ldbr) is needed for spliced mirtron lariats to adopt hairpin folds required for their subsequent cleavage by Dicer. We treated cells with Ldbr
dsRNA and analyzed their ability to process endogenous mirtrons and products of UAS-DsRed-
small-RNA plasmids. Quantitative RT-PCR analysis indicated successful knockdown of Ldbr
function, as evidenced by a 15-fold accumulation of the actin
intron relative to cells that received GFP
dsRNA (Figure S2B
). These cells exhibited only a minor decline in pre-miR-1 hairpin and mature miR-1 (, lane 3). In contrast, the accumulation of hairpin mirtrons and their mature products was strongly decreased or abolished by this treatment, including those of endogenous mir-1003
(, lane 8) and ectopic mir-1003
(, lanes 13 and 21).
We sought to confirm a positively acting role for Ldbr in mirtron production in the animal using a UAS-LdbrRNAi
transgene (Conklin et al., 2005
). When activated with da-Gal4
, individuals survived well to late larval stages but died during pupation. We therefore selected late third-instar larvae as a compromise time point that was suboptimal for Ldbr
knockdown but obviated secondary concerns surrounding the analysis of sickly animals. As with the S2 experiments, RNA from Ldbr
knockdown larvae showed increased levels of actin
intron (Figure S2B
) but decreased levels of endogenous mirtron hairpin and mature product for mir-1010
(). When the same blots were stripped and reprobed for miR-1, we observed little change in the accumulation of this miRNA. We quantified a 67% reduction in miR-1010 as normalized to miR-1 under these conditions (Figure S3
Finally, to rule out the possibility that specific sequences in mature mirtrons per se influence their choice of nuclear processing pathway, we created a hybrid miRNA/mirtron construct in which the mature miR-1003 sequence was programmed into a pri-mir-6-1 precursor structure (, construct D). Such a construct still produced mature miR-1003 (, lane 5). However, the hybrid mir-6-1/mir-1003 construct now exhibited behaviors characteristic of a canonical miRNA precursor, in that its processing displayed strong dependence on Drosha but was little affected by Ldbr depletion (, lanes 17 and 18). Thus, we are able to control the choice of RNA substrates to enter the nuclear mirtron or miRNA pathways by manipulating sequence and structural features defined by our biogenesis experiments.
Collectively, these data reveal that mirtron biogenesis, like that of certain snoRNAs (Ooi et al., 1998
), positively requires the action of lariat-debranching enzyme. These data do not exclude the possibility that Ldbr is required for the activity or processing of an intermediate factor that in turn mediates the resolution of mirtrons, but they are consistent with the parsimonious explanation that Ldbr acts directly upon mirtron lariats.
The Mirtron Pathway Merges with the Canonical miRNA Pathway during Hairpin Export
Consistent with previous results in mammalian and Drosophila
cells, we observed modest (~50%) reduction in endogenous mature miR-2b upon treatment with either of two nonoverlapping Exportin-5
dsRNAs (Figure S4
). However, mirtron hairpins proved to be more sensitive to manipulation of Exportin-5. We observed 60%–80% reduction in mirtron hairpins and mature products for endogenous mir-1003
in cells treated with either Exportin-5
dsRNA (). Similar, although slightly less robust, results were obtained using UAS-DsRed-mir-1003
mirtron-expression constructs (Figure S4
). It is conceivable that mirtron overexpression can partially overcome Exportin-5
knockdown or that there is an alternate mechanism for the nuclear export of pre-miRNAs and mirtrons. However, these data suggest that a considerable proportion of mirtron hairpins transit Drosophila
Exportin-5. In addition, the observation that mirtron hairpins decline following Exportin-5
knockdown is consistent with the previous suggestion that nuclear pre-miRNA hairpins are degraded when Exportin-5 is compromised (Yi et al., 2003
If the mirtron and canonical miRNA pathways merge during hairpin export, one might predict that their cytoplasmic processing should be similar. There are two Drosophila
Dicers, with Dicer-1 known to be genetically required for pre-miRNA maturation and Dicer-2 for processing of long dsRNA (Lee et al., 2004b
; Saito et al., 2005
). We tested their requirements by treating S2 cells with dsRNA against Dicer-1
). As shown previously (Okamura et al., 2004
), the maturation of miRNAs exhibited strong dependence on Dicer-1 but not Dicer-2 (, lanes 4 and 5). Dicer-1 was also strongly required for mirtron biogenesis, as its knockdown induced the accumulation of mirtron hairpins and depleted their small-RNA products (; lanes 9, 14, and 22). In contrast, no mirtron tested exhibited substantial sensitivity to Dicer-2
dsRNA (; lanes 10, 15, and 23).
We also analyzed the requirement of loquacious (loqs), a partner of Dicer-1 that is needed for efficient pre-miRNA cleavage (Forstemann et al., 2005
; Jiang et al., 2005
; Saito et al., 2005
). Treatment with loqs
dsRNA concomitantly increased the steady-state levels of endogenous mirtron hairpins for mir-1003
and decreased their mature products (, lanes 4 and 8). Therefore, loqs is also an important cofactor for mirtron cleavage by Dicer-1.
In summary, mirtron biogenesis differs from that of nuclear pre-miRNA biogenesis in that mirtron accumulation appears to bypass Drosha cleavage but, instead, exhibits strong dependence on splicing and intron lariat debranching. However, these pathways converge since both types of hairpins appear to transit Exportin-5 and require Dicer-1/loqs for cleavage into ~22 nt RNAs.
Mirtrons Generate Active Regulatory RNAs
We assayed the transregulatory activity of mirtrons using renilla luciferase “sensors” bearing sequences antisense to miR-1003, miR-1004, and miR-1010 in psiCHECK2; this vector contains a renilla luciferase “sensor” fused to test sequences and a firefly luciferase gene for normalization. When transfected into S2 cells along with ub-Gal4 and empty UAS-DsRed vector, miR-1003 sensor levels were much lower than those of the empty sensor or miR-1010 sensor (, lane 5; compare with lanes 1 and 19). Since miR-1003, but not miR-1010, is expressed by S2 cells (), this suggested that endogenous mirtron-derived miR-1003 directly repressed this sensor. To test this, we mutated the miR-1003 sensor to introduce noncomplementary bases at positions 2, 4, and 6 as measured from its 5′ end. In spite of 16/16 nucleotides of perfect match, the seed mutant miR-1003 sensor was no longer repressed in S2 cells, consistent with its failure to be recognized by endogenous miR-1003 (, lane 9; compare with lane 5).
We next tested the response of these sensors to ectopic mirtrons expressed using ub-Gal4 and UAS-DsRed-mirtron plasmids. We observed that sensors for miR-1003, miR-1004, and miR-1010 were strongly inhibited (5- to 8-fold) in the presence of cognate mirtron-expression constructs (, lanes 6, 14, and 22; compare with lanes 5, 12, 19, respectively). On the other hand, the mir-1010 mirtron construct had little impact on the miR-1003 and miR-1004 sensors (, lanes 8 and 15), while the mir-1003 and mir-1004 mirtron constructs did not repress the miR-1010 sensor (, lanes 20 and 21). The consistent behavior of the different mirtron:sensor pairs demonstrates that mirtrons generate sequence-specific regulatory RNAs.
As is the case with miRNAs, few if any endogenous transcripts are perfectly complementary to mirtron-derived small RNAs. Therefore, we asked whether mirtron-derived small RNAs could recognize seed-matched sites, which constitute the bulk of endogenous miRNA target sites. Since miR-1003 and miR-1004 have the same seed, we performed this test by assaying their mirtron-expression constructs on reciprocal sensors. While weaker than its effect on a perfectly matched sensor, ectopic miR-1004 repressed the miR-1003 sensor by 2-fold (, lane 7); similar repression of the miR-1004 sensor by ectopic miR-1003 was also seen (, lane 13). To test whether the observed regulation was truly mediated by the proposed seed matches, we analyzed the response of seed mutant miR-1003 and miR-1004 sensors. Neither mir-1003 nor mir-1004 mirtron-expression construct could repress either mutant sensor (, lanes 9–11 and 16–18). These data demonstrate that mirtron products can repress targets via seed-matched sites, thereby acting as canonical miRNAs.
Mirtrons Require Ago1 to Repress Seed-Matched Targets
The biogenesis and regulatory properties of mirtrons strongly suggested that their products were incorporated into Ago complexes. We tested whether mirtron products could associate with Ago1, the primary effector of canonical miRNA-mediated regulation in Drosophila
(Okamura et al., 2004
). We immunoprecipitated (IP-ed) endogenous Ago1 from 0–10 hr embryos and subjected the associated RNAs to northern analysis. As shown in , endogenous mature miR-1003 and miR-1010 co-IPed with endogenous Ago1 protein. Specificity of these interactions was demonstrated by the failure of control T7 antibody to co-IP mirtron-derived small RNAs and the failure of Ago1 to coIP 30 nt 2S rRNA. Since the enrichment of mirtron-derived small RNAs in the IP fraction was less than the observed enrichment of Ago1, however, this left open the possibility that a population of these small RNAs might associate with other partners such as Ago2.
We then examined the functional consequences of Ago knockdown on the ability of mirtrons to regulate seed-matched targets. As a control, we examined the effect of GFP
, and Ago2
dsRNAs on the ability of miR-279 to regulate a luciferase-nerfin 3′ UTR sensor, which contains at least five miR-279-binding sites (Stark et al., 2003
). As seen in , lanes 1–3, knockdown of Ago1, but not Ago2, derepresses the nerfin sensor in the presence of ectopic miR-279. We then analyzed a target bearing four bulged sites for miR-1010 (“miR-1010mi sensor”). We observed that knockdown of Ago1, but not Ago2, also derepresses this sensor in the presence of the mir-1010
mirtron-expression construct (, lanes 4–6). Therefore, while we do not exclude that mirtrons might also function via Ago2, our data provide evidence that small RNAs derived from mirtron hairpins associate with Ago1 to regulate seed-matched targets.
Mirtrons Exhibit Negative Regulatory Activity in Transgenic Drosophila
With these tissue culture data in hand, we challenged mirtrons to regulate target genes in the animal. We used a transgenic assay in which the expression of a ubiquitously expressed GFP “sensor” is tested for modulation by ectopic miRNAs provided in a spatially restricted pattern (Stark et al., 2003
). For these tests, we used ptc-Gal4
, which is active in a stripe of cells at anterior-posterior compartment boundaries. Specific downregulation of GFP in the ptc > miRNA
“stripe” reflects an in vivo miRNA:target relationship.
We created transgenic strains carrying tub-GFP-miR-1003
sensors and a UAS-DsRed-mir-1004
mirtron-expression construct. Ectopic mir-1004
had no effect on a functional GFP sensor for miR-7 (Lai et al., 2005
; Stark et al., 2003
), demonstrating specificity of the assay (). On the other hand, miR-1004 strongly suppressed its perfect sensor () and weakly suppressed the imperfect, seed-matched, miR-1003 sensor (). These data constitute stringent evidence that mirtrons are processed into functional species that can inhibit both perfectly matched and seed-matched targets in vivo.