Báo cáo khoa học: Treatment with small interfering RNA affects the microRNA pathway and causes unspecific defects in zebrafish embryos

Treatment with small interfering RNA affects the microRNA pathway and causes unspecific defects in zebrafish embryos Xiao-Feng Zhao, Anders Fjose, Natalia Larsen, Jon V. Helvik and Øyvind Drivenes

Department of Molecular Biology, University of Bergen, Norway

Keywords dicer; maternal mRNA; microRNA; RNA interference (RNAi); zebrafish development

Correspondence A. Fjose, Department of Molecular Biology, University of Bergen, PO Box 7803, N-5020 Bergen, Norway Fax: +47 555 89683 Tel: +47 555 84331 E-mail: anders.fjose@mbi.uib.no Ø. Drivenes, Department of Molecular Biology, University of Bergen, PO Box 7803, N-5020 Bergen, Norway Fax: +47 555 89683 Tel: +47 555 84325 E-mail: oyvind.drivenes@mbi.uib.no

MicroRNAs (miRNAs) are generated from primary transcripts through sequential processing by two RNase III enzymes, Drosha and Dicer, in association with other proteins. This maturation is essential for their func- tion as post-transcriptional regulators. Notably, Dicer is also a component of RNA-induced silencing complexes, which incorporate either miRNA or small interfering RNA (siRNA) as guides to target specific mRNAs. In ze- brafish, processed miRNAs belonging to the miR-430 family have previ- ously been shown to promote deadenylation and degradation of maternal mRNAs during early embryogenesis. We show that injection of one-cell- stage zebrafish embryos with siRNA causes a significant reduction in the endogenous levels of processed miR-430 and other miRNAs, leading to unspecific developmental defects. Coinjection of siRNA with preprocessed miR-430 efficiently rescued development. This indicates that the abnormali- ties generally observed in siRNA-treated zebrafish embryos could be due to inhibition of miR-430 processing and ⁄ or activity. Our results also suggest that the miRNA pathway in mammals, under some experimental or thera- peutic conditions, may be affected by siRNA.

(Received 7 December 2007, revised 18 February 2008, accepted 3 March 2008)

doi:10.1111/j.1742-4658.2008.06371.x

cytoplasm by a nuclear transport receptor complex, exportin-5 ⁄ RanGTP [7,8]. In the cytoplasm, a second RNase III, Dicer, cleaves the pre-miRNA to generate the mature miRNA [3,9,10].

MicroRNAs (miRNAs) are small RNA molecules of (cid:2) 21 nucleotides in metazoan animals and plants that influence mRNA stability and translation [1–3]. These mature miRNAs are generated from longer primary transcripts (pri-miRNA) in two processing steps cata- lyzed by two related RNase III endonucleases. In ani- mals, a nuclear microprocessor complex, containing the RNase III enzyme Drosha and the dsRNA-binding protein DGCR8, cleaves the pri-miRNA and exises a stem loop of (cid:2) 70 nucleotides [3–6]. This precursor then exported to the is miRNA (pre-miRNA)

The function of miRNAs and the ability to knock down expression of specific genes by RNA interfer- ence (RNAi) methods depend to a large extent on the same cellular machinery [3]. One important example is Dicer, which is required for miRNA processing as interfering well as cleavage of dsRNA into small (siRNAs). Moreover, mature miRNA and RNAs

Abbreviations GFP, green fluorescent protein; hpf, hours postfertilization; miRNA, microRNA; MMB, mesencephalic–metencephalic boundary; MZdicer, maternal–zygotic dicer; pre-miRNA, precursor miRNA; pri-miRNA, primary transcript of microRNA; RISC, RNA-induced silencing complex; RNAi, RNA interference; siGFP, siRNA specific for green fluorescent protein coding sequence; siRNA, small interfering RNA; TRBP, transactivating response RNA-binding protein.

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Results

Different siRNAs cause similar unspecific defects

siRNA are assembled into the RNA-induced silencing complexes (RISCs) miRISC and siRISC, respectively [3]. Both of these effector complexes contain, in addi- tion to similar Argonaute proteins, Dicer and other factors such as the transactivating response RNA- binding protein (TRBP) [11,12]. Interestingly, it has also been demonstrated that TRBP associates with Dicer to facilitate generation of siRNAs and mature miRNAs [13,14].

We have previously characterized the structure and embryonic expression of zebrafish six3a (originally named six3 [25]), and more recently we have identified genomic and cDNA sequences representing eri1, a zebrafish homolog of the enhanced RNA interference-1 (eri-1) gene in C. elegans (see Experimental proce- dures). Using specific siRNAs (siEri1 and siSix3a) to target the mRNAs expressed from these two genes, we observed similar but not identical embryonic defects (Fig. 1C–F). Following injection of sufficient amounts of siRNA (see Experimental procedures), most embryos at 28 h postfertilization (hpf) displayed tail truncations and loss of distinct morphological features at the mesencephalic–metencephalic boundary (MMB; Table 1). In addition, enlarged heart cavities occurred at lower frequencies in these embryos (not shown). Notably, we also observed tail truncations and MMB defects in embryos treated with siRNA specific for green fluorescent protein (GFP) coding sequences (siG- FP; Fig. 1G,H; Table 1), suggesting that these malfor- mations are general consequences of siRNA treatment.

A

B

WT

C

D

*

RNAi has been employed extensively as a research tool in several animal models, including Caenorhabd- itis elegans, Drosophila melanogaster and mice, as well as mammalian cells [15–18]. RNAi targeting of specific also been demonstrated in zebrafish genes has (Danio rerio) cell lines [19]. However, experimental studies on zebrafish embryos have revealed substantial unspecific defects of RNAi that have prevented further use of this technology for gene function analyses [19– 22]. By circumventing these problems, studies on zebrafish maternal–zygotic dicer (MZdicer) mutants, which do not process pre-miRNA, have uncovered extensive embryonic abnormalities reflecting the impor- tance of miRNAs in developmental processes [23]. Remarkably, the brain morphogenesis defects in MZ dicer mutants could be suppressed by injection of preprocessed miR-430 family miRNAs, which are the only abundant miRNAs during the first hours after the transition from maternal to zygotic gene expression [23]. Further studies of the developmental regulatory these miRNAs function of miR-430 revealed that accelerate deadenylation and degradation of several hundred different types of maternal mRNAs, leading to a sharpening of the maternal-to-zygotic transition [24].

siEri1

We have

E

F

*

siSix3a

G

H

*

siGFP

Fig. 1. Injection of different siRNAs causes brain and tail defects. Light micrographs are shown for wild-type (WT) and siRNA-injected zebrafish embryos. At the 28 hpf stage, the zebrafish embryos injected with siRNAs targeting eri1 (siEri1), six3a (siSix3a) and GFP (siGFP) show tail and brain defects. The MMB is easily visible in wild-type embryos (arrowhead), whereas this morphological con- striction is missing in embryos injected with the different siRNAs [stars in (D), (F) and (H)].

connection investigated the possible between the miRNA pathway and the unspecific defects caused by RNAi in zebrafish embryos. Accom- panying the induction of unspecific defects, the treat- ment with different siRNAs was also shown to significantly reduce the levels of processed miR-430 and other miRNAs. Moreover, we demonstrated, by coinjecting siRNA with preprocessed miR-430, that most of the morphological abnormalities could be pre- vented. Hence, the unspecific defects generally caused by RNAi in zebrafish embryos are mainly due to inhi- bition of miR-430, which has been shown previously to have an essential role in the clearance of maternal mRNAs [24]. These observations may have implica- tions for the development of new RNAi techniques in zebrafish. In addition, our results suggest a need for investigating whether treatment of mammalian cells with larger amounts of siRNA may also cause some inhibition of the miRNA pathway.

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A

the efficiencies of different siRNAs and Table 1. Analysis of related RNA molecules in inducing MMB defects. asGFP, antisense strand of GFP; dsGFP, double-stranded GFP without 3¢-overhangs; sGFP, sense strand of GFP.

Embryos analyzed

MMB defects (%)

250 pg of siGFP 250 pg of siEri1 400 pg of siSix3a 250 pg of dsGFP 125 pg of sGFP 125 pg of asGFP

134 87 122 114 109 126

129 (96.3) 76 (87.4) 100 (82.0) 11 (9.6) 2 (1.8) 15 (11.9)

B

Consistent with this assumption, equal numbers of molecules of the same GFP sequence caused very few abnormalities when injected as ssRNA (sense and anti- sense in separate experiments) or dsRNA without the 2 bp 3¢-overhangs (Table 1).

siRNAs affect the endogenous levels of miRNAs

C

The defects caused by siRNA injections showed a resemblance to the abnormalities reported previously for MZdicer mutants, which are due to failure in the processing of miR-430 [23]. Interestingly, the MZdicer phenotype also included tail and MMB defects [23]. Therefore, it seemed plausible that miRNA processing could be affected by the injection of siRNAs. To inves- tigate this possibility, we analyzed by northern blotting the levels of processed miR-430b and three additional miRNAs expressed in early embryos [26,27]. This anal- ysis showed that all three siRNAs caused reductions in the amounts of mature miRNAs at 12 hpf (Fig. 2A). Although the level of processed miR-430b seemed to be most strongly affected, we also detected significant reductions for the other three miRNAs tested. Further- more, we generally observed stronger reductions for the largest dosage of siRNA (500 pg). These results were reproducible, and for miR-430b the level was reduced by as much as 70% (supplementary Fig. S4). The effects of siRNAs on miRNA levels were less prominent at later embryonic stages, as revealed by analyses at 24 hpf, where a significant reduction in the amount of the mature form was detected only for miR-430b (supplementary Fig. S1).

Fig. 2. Injection of different siRNAs affects the levels of miRNAs. (A) The endogenous levels of the processed forms of four different miRNAs were analyzed at the 12 hpf stage by northern blotting. Treatment with each of the three different siRNAs caused reduc- tion of the levels of all four miRNAs as compared to the wild-type (WT). (B) Northern blot analysis of the endogenous level of mature miR-430b at 12 hpf following injection of different types of RNA molecules corresponding to the same GFP sequence. Single- stranded and double-stranded molecules without the characteristic features of siRNA did not affect the level of miR-430b, as com- pared to non-injected (WT) and buffer-injected embryos. (C) Endog- enous levels of mature miR-430b were reduced at 12 hpf following injection of miR-206. The different amounts (pg) injected are indi- cated for each type of RNA. asGFP, antisense strand of GFP; dsGFP, double-stranded GFP without 3¢-overhangs; sGFP, sense strand of GFP.

To examine whether the reduced amounts of pro- cessed miRNAs could be due to a unique feature of siRNAs, we analyzed the possible effects following microinjection of other types of RNA molecules con- taining the same GFP sequence. As expected from the minimal effects on embryonic development caused by injection of equal numbers of these GFP-specific RNA molecules (Table 1), we did not detect any reduction in

the level of processed miR-430b at 12 hpf (Fig. 2B). Consistent with the common MMB defects of the three siRNAs, expression of the pax2a gene, which is an

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B

A

important midbrain marker [28,29], was also clearly reduced in the affected region of the brain (supplemen- tary Fig. S2).

Coinjection with preprocessed miR-430b can prevent unspecific defects

D

C

F

E

H

G

indicating that

In support of

J

I

Microinjection of preprocessed miR-430 has previously been shown to rescue tail and brain defects of MZdicer mutants [23]. Similarly, we investigated whether coin- jection of miR-430b could rescue the effects of siRNA treatment. In these experiments, we also performed control injections with miR-206, and a mutated version of miR-430b (miR-430b-mis) that lacks the power to rescue MZdicer embryos [23]. Consistent with the results of Giraldez et al. [23], the dsRNA molecules of preprocessed miR-430b did not induce any embryonic defects (Fig. 3A,B; Table 2). However, preprocessed miR-206 duplexes clearly affected the morphologies injected embryos of both the tail and MMB of (Fig. 3C,D; Table 2), treatment of embryos with miRNA duplexes may zebrafish generally induce the same kind of unspecific defects as siRNAs. this assumption, we also observed reduced levels of mature miR-430b in miR-206-injected embryos (Fig. 2C).

siSix3a

K

L

rescue

the MMB or

M

N

Coinjection of preprocessed miR-430b efficiently res- cued the siEri1-induced defects (Fig. 3E,F; Table 2), and a similar result was observed for coinjections of (supplementary Fig. S3; miR-430b with Table 2). Preprocessed miR-430b also rescued embryos from defects caused by siGFP, and the efficiency was clearly improved with a higher dosage (Fig. 3K,L; Table 2). By contrast, coinjection of miR-430b-mis, which has two point substitutions in the 5¢-seed region [23], did not tail defects caused by any of the three gene-specific siRNAs supplementary Fig. S3; Table 2). (Fig. 3G,H,M,N; Similarly, siEri1-induced defects were not rescued by coinjection with miR-206 duplexes (Fig. 3I,J; Table 2). These results show that unspecific defects induced by siRNA, which correlate with a significant reduction of the endogenous level of mature miR-430b, can be pre- vented by coinjection of this particular miRNA. If inhibition of miR-430 activity by siRNAs occurs also at the level of miRISC assembly and ⁄ or function, miR-430b coinjection would be expected to reduce this effect as well (see Discussion).

Discussion

Fig. 3. Rescue of siRNA-induced abnormalities by coinjection of miR-430b. The effects of injecting preprocessed duplexes of miRNAs alone and in combination with siRNAs were analyzed. Whereas embryos were not significantly affected by injection of miR-430b (A, B), injection of miR-206 caused similar tail and MMB (see Fig. 1).The tail ⁄ MMB defects as siRNA injections (C, D) defects caused by injections of the two different siRNAs, siEri1 and siGFP (see Fig. 1), were rescued by coinjection of miR-430b alone (E, F, K, L) but not by the mutated variant miR-430b-mis (G, H, M, N). Coinjection of miR-206 did not rescue the tail ⁄ MMB defects caused by siEri1 (I, J). Arrowheads and stars indicate the presence and absence of an MMB, respectively.

The rapidly growing knowledge on RNAi and miRNA has revealed many common factors and interconnec-

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Table 2. Analysis of the influence of siRNAs and miRNAs on MMB morphology. Coinjection of miR-430b efficiently rescued MMB defects caused by siRNA treatment. The MMB defects were not significantly rescued by miR-206 and the mutated variant miR-430b- mis.

Embryos analyzed

MMB defects (%)

miR-430 miRNAs [23]. Remarkably, further investiga- tions on the mRNA targets of miR-430, which are the only abundant miRNAs before gastrulation, demon- strated that miR-430 is essential for efficient removal of maternal mRNAs during the maternal-to-zygotic transition [24]. Hence, considering the common factors in the RNAi and miRNA pathways, and the impor- tance of miR-430 at early stages of zebrafish develop- ment, we assumed a possible involvement of miR-430 in the unspecific defects caused by siRNA treatment.

98 250 pg of miR-430b 91 250 pg of miR-206 111 250 pg of siEri1 + 250 pg of miR-430b 115 250 pg of siEri1 + 250 of pg miR-430b-mis 110 250 pg of siEri1 + 250 pg of miR-206 88 250 pg of siGFP + 250 pg of miR-430b 46 250 pg of siGFP + 450 pg of miR-430b 109 250 pg of siGFP + 250 pg of miR-430b-mis 400 pg of siSix3a + 250 pg of miR-430b 91 400 pg of siSix3a + 250 pg of miR-430b-mis 112

0 (0) 91 (100) 9 (8.1) 97 (84.3) 110 (100) 55 (62.5) 21 (45.7) 107 (98.2) 7 (7.7) 101 (90.2)

Using siRNAs corresponding to sequences in two endogenous genes (eri1 and six3a) and the exogenous reporter gene GFP, we investigated the possibility that miRNAs may in some way be influenced by the siR- NAs introduced into zebrafish embryos. By northern analysis of miR-430b and three additional miRNAs, we found a general reduction in the levels of processed miRNAs in embryos treated with siRNAs. Injection of other types of RNA molecules, such as ssRNAs and dsRNAs without the 3¢-overhangs, which contained the same sequence, did not cause any general abnor- malities, and the levels of mature miRNAs were not affected. These results suggest that the characteristic features of siRNAs are critical for reducing the levels of processed miRNAs, particularly miR-430, and this may lead to the unspecific defects observed in zebrafish embryos.

endogenous

tions between these two pathways. Recently, it has also been shown that modulation of the processing of miR- NAs is an important feature of their regulatory func- tion and may be directly connected to cell signaling [30–32]. However, in relation to the extensive use of RNAi as a tool to knock down the expression of spe- cific genes, the possible influence on miRNA process- ing, which may cause various side effects, has been analyzed only recently [33]. In this study, we have investigated these aspects in zebrafish, where RNAi experiments have previously been shown to result in high frequencies of unspecific defects [19–22]. Because of these problems, RNAi has not become a useful technique for studying gene function in zebrafish. As an alternative, morpholino antisense oligonucleotides have been extensively used for transient knockdown of gene expression in zebrafish embryos and larvae [34,35]. However, unspecific effects can be a problem with this method as well, and it cannot be further developed as a transgenic technique with the possibili- ties of achieving tissue-specific and ⁄ or long-term knockdown of the targeted genes.

increase of pre-miRNAs, we

If this interpretation is correct, it will be natural to ask how injection of siRNAs can possibly interfere levels of mature miRNAs. with the Although correctly sized siRNAs ((cid:2) 21 bp) are not cut by Dicer, which is the enzyme responsible for the last processing step of miRNAs, siRNAs are known to be assembled into effector complexes (siRISCs) containing Argonaute proteins as well as Dicer and other factors [3]. One of the additional factors is TRBP, which together with Dicer facilitates generation of siRNAs and mature miRNAs from dsRNAs and pre-miRNAs, respectively [13,14]. Accordingly, the injection of large amounts of siRNAs would affect the availability of these factors for processing of pre-miRNAs. Thus, the most plausible explanation is that the observed reduc- tion of mature miRNAs is due to inhibition of pre- miRNA processing by siRNAs competing for binding to Dicer, TRBP, and ⁄ or other limiting factors. How- ever, since our northern blot analysis did not reveal a cannot concomitant exclude other possibilities, such as enhanced degrada- tion of mature miRNAs.

In Drosophila and mouse, transgenic RNAi tech- niques have been developed to facilitate tissue-specific or inducible knockdown [16,17]. In principle, this strat- egy can also be used in zebrafish, but it may not be feasible, due to the unspecific effects associated with RNAi. Although the reason why treatment with siR- NA causes a high frequency of general abnormalities in zebrafish has remained unclear, some clues regard- ing this issue have been obtained from studies of the MZdicer mutation [23]. The MZdicer mutant embryos, which display several defects similar to those caused by siRNAs, were rescued by injection of preprocessed

When discussing the relevance of miR-430 to the it should be unspecific defects caused by siRNAs, noted that these miRNAs are most abundant during early stages of zebrafish development [26,36,37]. Follow-

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the mRNA targets. For the same reason, coinjection of preprocessed miR-430b duplexes would be expected to reduce this inhibition.

that

The results

siRNAs reported here suggest injected into zebrafish embryos compete for limiting factors that are required in the miRNA pathway. By contrast, a recent study of systemic administration of synthetic siRNA in mouse and hamster did not reveal any effect on miRNA levels or activity in the liver [33]. However, a more complete investigation is required to analyze whether or not treatment of mammals with higher doses of siRNA can inhibit the endogenous miRNA pathway in particular tissues and ⁄ or during embryogenesis. Another issue, which is also relevant to therapeutic use of siRNA in humans, concerns the possible sensitivity to changes in miRNA levels or activity. Negative side effects reflecting such sensitivity have already been reported from experiments where short hairpin RNAs were expressed at high levels in the liver of mice [34]. This treatment was shown to sat- urate the nuclear exportin-5 transporter, leading to the levels of processed miRNAs and reduction of lethality.

Experimental procedures

ing siRNA injection, we detected a > 50% reduction of processed miR-430b, and this would be at least par- tially equivalent to the conditions in MZdicer embryos, in which processing of pre-miRNAs does not occur [23]. Similar to the rescue of MZdicer mutants by miR-430b [23], we observed efficient rescue when siRNAs were coinjected with the preprocessed duplex form of this particular miRNA. In contrast, coinjec- tion of another early embryonic miRNA (miR-206) or a mutated version of miR-430b (miR-430b-mis) did not give any rescue. These results are entirely consis- tent with the documented role of mature miR-430 in promoting deadenylation and degradation of maternal mRNAs, which is required for a normal maternal-to- zygotic transition [24]. Because of this crucial function of miR-430, development of zebrafish embryos is likely to be affected by treatment with siRNAs. Our results from experiments with three different siRNAs suggest that this effect is a general phenomenon in zebrafish. However, some variations with respect to the abilities of different siRNAs to cause unspecific defects and reduced levels of mature miRNAs suggest a certain degree of sequence dependence. This may simply reflect differences in the binding affinities of various siRNAs to one or more factors that are shared between the RNAi and miRNA pathways.

Isolation and analysis of genomic DNA and cDNA

Relevant to this issue, we also noted that single injections of miR-206 duplexes, in contrast to miR- 430b, caused a high frequency of brain and tail abnor- malities, as well as a reduction of the endogenous level of processed miR-430b. These observations indicated that the level of the mature form of miR-430 was affected by miRNA injection but was compensated by the introduction of preprocessed miR-430b. Therefore, we conclude that injected miRNA duplexes (with inter- nal mismatches) can probably affect the endogenous concentrations of mature miRNAs in the same way as siRNA duplexes.

endogenous

Injections of siRNA and miRNA duplexes

Two zebrafish eri1 expressed sequence tags (BQ285328 and reported previously [38], were subjected to BI888174), blast analysis against the zebrafish genomic database at ENSEMBL, and a genomic region containing the eri1 locus was identified (GenBank accession number BX511222). Using genscan [39], webgene [40] and eri1 expressed sequence tag alignments, we identified a putative eri1 cod- ing region composed of seven exons spanning a genomic region of 7542 bp. Using primers located in the putative 5¢-region and 3¢-region, ERI1F1 (5¢-AAA CCA GAT GTG AGT GTT TCT GA-3¢) and ERI1R1 (5¢-CAC AAC ATG GCA GGT TTT CA-3¢), we isolated the complete eri1 coding sequence by PCR using adult zebrafish cDNA as template.

The importance of miR-430 was confirmed by coin- jection of preprocessed miR-430b duplexes, which the unspecific defects apparently rescued most of caused by siRNAs. However, at lower doses of level of mature siRNAs, when the miR-430 was less affected, we also observed relatively high frequencies of unspecific defects. This could reflect a particularly high sensitivity to changes in the concen- tration of this important miRNA, but it seems more likely that siRNAs may cause an additional block of miRNA function at the level of the effector complex miRISC. Hence, excess amounts of siRNAs may effi- ciently compete with miR-430 (and other miRNAs) for binding to Dicer, Argonaute proteins and ⁄ or other factors of this complex, and prevent interaction with

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Adult fish were kept at 28.5 (cid:2)C on a natural 14 h light ⁄ 10 h dark cycle, and all embryos were obtained from natural mating. The siRNAs targeting eri1, six3a and GFP (see below) were designed using the Dharmacon siRNA design (http://www.dharmacon.com/sidesign/) center and pur- chased from MWG Biotech (Ebersberg, Germany). Embryos were injected in the yolk at the one-cell stage, with an average injection volume of 2 nL, which contained

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3 Rana TM (2007) Illuminating the silence: understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol 8, 23–36. 4 Denli AM, Tops BBJ, Plasterk RHA, Ketting RF & 250 pg of siRNA and ⁄ or miRNA. In the case of six3a, a larger amount of siRNA (400 pg) was required to achieve a high frequency of defects (Table 1). Following injection, embryos were incubated at 28.5 (cid:2)C in E3 medium.

Oligonucleotide sequences

Hannon GJ (2004) Processing of primary microRNAs by the Microprocessor complex. Nature 432, 231–235.

5 Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N & Shiekhattar R (2004) The microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240.

sGFP, 6 Han JJ, Lee Y, Yeom KH, Kim YK, Jin H & Kim VN (2004) The Drosha–DGCR8 complex in primary micr- oRNA processing. Genes Dev 18, 3016–3027. The sequences are given in the 5¢- to 3¢-direction: siEri1, UCAGUGAUCCGGUGUAUAA(TT); siSix3a, CUAUCA GGAGGCCGAGAAA(TT); siGFP, AAGCUGACCCU GAAGUUCA(TT); dsGFP, AAGCUGACCCUGAAGU UCA; AAGCUGACCCUGAAGUUCA(TT); asGFP, UGAACUUCAGGGUCAGCUU(TT). blot Northern

7 Yi R, Qin Y, Macara IG & Cullen BR (2003) Exportin- 5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17, 3011–3016.

8 Lund E, Guttinger S, Calado A, Dahlberg JE & Kutay U (2004) Nuclear export of microRNA precursors. Science 303, 95–98. probes: miR-430b, BIO-GUACCC CAACUUGAUAGCACUUU; miR-206, BIO-CCACATG CTTCCTTATATTCCATA; miR-17a-1, BIO-ACTACCTG CACTGTAAGCACTTTG; miR-19b, BIO-TCAGTTTT GCATGGATTTGCACA. miR-206 duplex: AAUGUAA GGAAGUGUGUGGGU; CCACACACUUCCUUACAA UUU. miR-430b

Northern blot analysis

duplex: AAAGUGCUAUCAAGUUGGG GU; CCCAACUUGAUAGCACUAUUU. miR-430b-mis duplex, as described in [23]: AAAGACCUAUCAAG UUGGGGT; CCCAACUUGAUAGGUCUAUTT. 9 Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G & Mello CC (2001) Genes and mechanisms related to RNA interfer- ence regulate expression of the small temporal RNAs that control C-elegans developmental timing. Cell 106, 23–34.

10 Hutvagner G & Zamore PD (2002) A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060.

11 Chendrimada TP, Gregory RI, Kumaraswamy E, Nor- man J, Cooch N, Nishikura K & Shiekhattar R (2005) TRBP recruits the Dicer complex to Ago2 for micro- RNA processing and gene silencing. Nature 436, 740– 744.

12 Haase AD, Jaskiewicz L, Zhang HD, Laine S, Sack R, Gatignol A & Filipowicz W (2005) TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep 6, 961–967. 13 Saito K, Ishizuka A, Siomi H & Siomi MC (2005) Pro- Total RNA was isolated from wild-type and injected embryos at 12 hpf and 24 hpf using Trizol (Invitrogen, Carlsbad, CA, USA). Five micrograms of total RNA was separated on a 15% denaturing polyacrylamide gel contain- ing 8 m urea, and was blotted according to standard proce- dures. Biotin-labeled probes were purchased from MWG Biotech. Prehybridization and hybridization were carried out in 0.25 m sodium phosphate (pH 7.2), 7% SDS, and 0.5% sodium pyrophosphate. After hybridization, the membrane was washed in 2 · SSC and 1% SDS at 37 (cid:2)C. The biotin signal was detected using the Chemiluminescent Nucleic Acid Detection Module kit (Pierce, Rockford, IL, USA).

Acknowledgements

We thank Dr Hee-Chan Seo for technical advice and the Faculty of Mathematics and Natural Sciences at the University of Bergen for special support.

cessing of pre-microRNAs by the Dicer-1–Loquacious complex in Drosophila cells. PLoS Biol 3, 1202–1212. 14 Kok KH, Ng MHJ, Ching YP & Jin DY (2007) Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA. J Biol Chem 282, 17649–17657. 15 Grishok A (2005) RNAi mechanisms in Caenorhabditis elegans. FEBS Lett 579, 5932–5939. 16 Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y,

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siSix3a-induced MMB and tail

The following supplementary material online: Fig. S1. Northern blot analysis of the effects of siRNA injections on endogenous levels of different miRNAs. Fig. S2. Expression of the pax2a gene at the MMB is affected by siRNA injection. Fig. S3. Rescue of defects by coinjection of miR-430b. Fig. S4. Changes in the endogenous level of miR-430b following injection of different amounts of siGFP. Each column represents the average level of the mature form of miR-430 (relative to the wild-type), obtained from three different experiments.

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30 Obernosterer G, Leuschner PJF, Alenius M & Martinez J (2006) Post-transcriptional regulation of microRNA expression. RNA 12, 1161–1167.

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