12. Siolas, D. et al. Synthetic shRNAs as highly potent RNAi triggers. Nature Biotechnol. (submitted).

13. Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

14. Llave, C., Xie, Z., Kasschau, K. D. & Carrington, J. C. Cleavage of Scarecrow-like mRNA targets

directed by a class of Arabidopsis miRNA. Science 297, 2053–2056 (2002).

15. Yekta, S., Shih, I. H. & Bartel, D. P. MicroRNA-directed cleavage of HOXB8 mRNA. Science 304,

594–596 (2004).

16. Olsen, P. H. & Ambros, V. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis

elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev. Biol. 216, 671–680

(1999).

17. Zeng, Y., Wagner, E. J. & Cullen, B. R. Both natural and designed micro RNAs can inhibit the

expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327–1333 (2002).

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(2003).

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preribosomal RNA processing. J. Biol. Chem. 275, 36957–36965 (2000).

20. Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. Role for a bidentate ribonuclease in the

initiation step of RNA interference. Nature 409, 363–366 (2001).

21. Giot, L. et al. A protein interaction map of Drosophila melanogaster. Science 302, 1727–1736 (2003).

22. Shiohama, A., Sasaki, T., Noda, S., Minoshima, S. & Shimizu, N. Molecular cloning and expression

analysis of a novel gene DGCR8 located in the DiGeorge syndrome chromosomal region. Biochem.

Biophys. Res. Commun. 304, 184–190 (2003).

23. Reinhart, B. J. et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis

elegans. Nature 403, 901–906 (2000).

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411–414 (2003).

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doi:10.1038/nature03120 (this issue).

26. Han, M. H., Goud, S., Song, L. & Fedoroff, N. The Arabidopsis double-stranded RNA-binding protein

HYL1 plays a role in microRNA-mediated gene regulation. Proc. Natl Acad. Sci. USA 101, 1093–1098

(2004).

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1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109, 861–871 (2002).

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associate with the RNA interference machinery. Genes Dev. 16, 2491–2496 (2002).

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Supplementary Information accompanies the paper on www.nature.com/nature.

Acknowledgements We thank F. Rivas for critical reading of the manuscript. Strain BC3825 was

obtained from the C. elegans Genetics Center, and drsh-1(tm0654) was obtained from the NBP in

Japan (Mitani laboratory). We thank E. Cuppen and his group for help in target-selected

mutagenesis. A.M.D. is a David Koch Fellow of the Watson School of Biological Sciences. G.J.H. is

supported by an Innovator Award from the US Army Breast Cancer Research Program. This work

was also supported by a grant from the NIH (G.J.H.) and by a VENI fellowship from the

Netherlands Organisation for Scientific Research (R.F.K.).

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests for materials should be addressed to G.H. (hannon@cshl.org).

..............................................................

The Microprocessor complexmediates the genesis of microRNAsRichard I. Gregory*, Kai-ping Yan*, Govindasamy Amuthan,Thimmaiah Chendrimada, Behzad Doratotaj, Neil Cooch& Ramin Shiekhattar

The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA

* These authors contributed equally to this work

.............................................................................................................................................................................

MicroRNAs (miRNAs) are a growing family of small non-pro-tein-coding regulatory genes that regulate the expression ofhomologous target-gene transcripts. They have been implicatedin the control of cell death and proliferation in flies1,2, haemato-poietic lineage differentiation in mammals3, neuronal patterningin nematodes4 and leaf and flower development in plants5–8.miRNAs are processed by the RNA-mediated interferencemachinery. Drosha is an RNase III enzyme that was recently

implicated in miRNA processing. Here we show that humanDrosha is a component of two multi-protein complexes. Thelarger complex contains multiple classes of RNA-associatedproteins including RNA helicases, proteins that bind double-stranded RNA, novel heterogeneous nuclear ribonucleoproteinsand the Ewing’s sarcoma family of proteins. The smaller complexis composed of Drosha and the double-stranded-RNA-bindingprotein, DGCR8, the product of a gene deleted in DiGeorgesyndrome. In vivo knock-down and in vitro reconstitutionstudies revealed that both components of this smaller complex,termedMicroprocessor, are necessary and sufficient inmediatingthe genesis of miRNAs from the primary miRNA transcript.

Figure 1 Isolation of Drosha-containing complexes. a, Fractions of the immunoaffinity

eluate from M2 anti-Flag beads resolved by SDS–PAGE (4–12%). Flag-Drosha was

revealed by silver staining and western blotting with anti-Flag antibodies. Molecular

masses of marker proteins (left) and the polypeptide masses of associated subunits (right)

are indicated. b, Silver staining of Superose 6 gel-filtration fractions. Top, fraction

number; bottom, molecular mass markers. DAP, Drosha-associated proteins having

different molecular masses; asterisks, contaminating polypeptides; IP,

immunoprecipitation.

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It was recently suggested that most miRNA genes originate fromindependent transcription units9–11. However, about a quarter ofhuman miRNA genes are located in introns of pre-mRNAs. Becausethese miRNAs have the same orientation as pre-mRNAs, it is likelythat they are not transcribed from their own promoters but areprocessed from the introns12–14. The remaining miRNAs areclustered in the genome, predicting a multi-cistronic transcript9,10.Regardless of how different miRNAs originate, the primary miRNAtranscript (pri-miRNA)15 must be processed to yield a mature22-nucleotide (nt) miRNA. The present model for the pathwayby which mammalian miRNAs undergo maturation begins withcleavage of the pri-miRNA in the nucleus to release a ,60–70-ntstem loop intermediate, known as the pre-miRNA15,16. This proces-sing event is mediated by an RNase III endonuclease, Drosha, whichcleaves both strands of the stem at sites near the base of the primarystem loop17. Whether Drosha in itself is sufficient for this processingor whether other components associated with Drosha contribute tothe cleavage of pri-miRNAs has not previously been addressed.

To gain insight into the components of the miRNA-processingmachinery, we isolated a Drosha-containing complex from humancells. This was accomplished by developing HEK-293-derived stablecell lines expressing Flag-tagged Drosha. Flag-Drosha was isolatedby immunoaffinity chromatography. As Fig. 1a demonstrates,

the Flag affinity eluate contains a rich harvest of polypeptides.Drosha was represented in two forms differing in molecular mass by,10–15 kDa (Drosha a, ,160 kDa, and Drosha b, ,145 kDa; seeFig. 1a). It is currently not clear whether these two forms reflectpost-translational modification of Drosha or result from proteolyticcleavage sustained during preparation of the nuclear extract.

To characterize the elution profiles of the two forms of Droshaand to demonstrate that the Drosha-associated polypeptides(DAPs) constitute a multi-protein complex, the Flag-affinity eluatewas fractionated on a gel-filtration column (Fig. 1b). This analysisrevealed the presence of the larger form of Drosha (Drosha a) in twodistinct complexes: a high-molecular-mass complex containingmost Drosha-associated polypeptides (fractions 16–18) and alower-molecular-mass complex (fractions 22–26, ,600 kDa). Thesmaller form of Drosha (Drosha b) displayed an elution profileconsistent with a smaller complex with a molecular mass of,400 kDa (fractions 30–32).

We next determined the identity of the Drosha–associatedpolypeptides. The Flag-affinity eluate was separated on a polyacryl-amide gel, bands were stained with colloidal blue, and individualpolypeptides were excised from the gel and subjected to massspectrometric sequencing. Nineteen specific Drosha-associatedpolypeptides were identified in two independent sequencing

Figure 2 Composition and miRNA processing activity of Superose 6 gel-filtration

fractions. a, Western blotting with the antibodies shown at the left. IP,

immunoprecipitation. b, Flag-affinity eluates and an enriched Dicer fraction were used for

miRNA processing. ‘1 £ ’ corresponds to 10 ng of Drosha and 5 ml of enriched Dicer

fraction. c, miRNA processing with a pri-miRNA miR-(17,18,19a,20,19b-1) fragment.

Flag-Drosha eluate (Input) corresponded to 5, 10, 20 and 40 ng of Drosha, determined as

described in Methods.

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analyses (Supplementary Fig. S1). The Drosha-associated proteinscomprised specific classes of RNA-associated proteins displayingcommon structural domains. These included the DEAD-box andDEAH-box family of RNA helicases, proteins with domains thatbind double-stranded RNA, heterogeneous nuclear ribonucleo-proteins (hnRNPs) and the Ewing’s sarcoma family of proteinscontaining an RNA recognition motif (RRM) and a zinc-fingerdomain.

To address the association of the aforementioned different classesof polypeptides with Drosha, we analysed alternate fractions ofSuperose 6 gel-filtration chromatography by western blot analysis.This analysis revealed that Drosha coelutes with the protein productof Ewing’s sarcoma gene (EWS), the RNA helicase, DDX17/P72and hnRNPM4 in a large complex peaking in fraction 17 (Fig. 2a).In contrast with these polypeptides, a single polypeptide of120 kDa corresponding to DGCR8 coelutes with the smaller

complex peaking in fractions 25–27 (Figs 1b and 2a).We next analysed the Flag-affinity eluate for pri-miRNA proces-

sing. A 790-base-pair fragment corresponding to a cluster ofpri-miRNAs miR-(17,18,19a,20,19b-1) was used as a substratefor analysis of pri-miRNA processing. It was shown recently thatpri-miRNA processing catalysed by Drosha resulted in the for-mation of a ,60–70-base-pair pre-miRNA precursor17. We firstexamined the activity of the Flag-Drosha affinity eluate in proces-sing the pri-miRNA. Addition of increasing concentrations ofFlag-Drosha affinity eluate to the pri-miRNA processing reactionresulted in the appearance of a distinct 63-nt pre-miRNA fragment(Fig. 2b). Further addition of a fraction enriched for Dicer, whichcatalyses further processing of the pre-miRNA, converted thisfragment to a mature ,22-nt miRNA, confirming that this pre-miRNA is the correct processing product (Fig. 2b).

We next analysed the gel-filtration fractions for pri-miRNAprocessing activity and found it was present in two distinct peakscorresponding to the two Drosha-containing complexes (Fig. 2c).Although the larger complex (fractions 16–18) displayed somepri-miRNA processing activity, the bulk of pri-miRNA processingactivity co-eluted with the smaller Drosha complex containingDGCR8. Moreover, closer examination of the reaction revealedthat the fractions containing the larger Drosha complex containeda non-specific RNase activity, resulting in reduced probeconcentrations.

To characterize the polypeptide composition of the smallerDrosha-containing complex, fractions 24–27 were concentratedby trichloroacetic acid, treated as described above and subjectedto mass spectrometric sequencing. The three largest polypeptidescorresponded to Drosha a and b forms and the DGCR8 protein(Supplementary Fig. S2). (The smaller polypeptide corresponded toSKB1, a common contaminant of Flag-affinity purification.) Wethen analysed the specific pri-miRNA processing activity of the twoDrosha complexes by normalizing the amounts of Drosha in eachcomplex (Supplementary Fig. S2). These studies revealed thatthe Drosha–DGCR8 complex displays a nearly eightfold greaterpri-miRNA processing activity than the large Drosha complex(Supplementary Fig. S2).

We next developed HEK-293-derived stable cell lines expressingFlag-DGCR8. Flag-DGCR8 was isolated by affinity chroma-tography. As shown in Fig. 3a, the DGCR8 affinity eluate containeda single polypeptide corresponding to Drosha in addition toDGCR8 itself. The polypeptide below DGCR8 corresponds to acarboxy-terminal truncation of the protein, because only theamino-terminal DGCR8 antibodies recognize this truncated pro-tein (Fig. 3a). The Flag-DGCR8–Drosha complex was then used toassess its activity for pri-miRNA processing. Consistent with theresults obtained with the smaller Drosha complex (SupplementaryFig. S2), Flag-DGCR8–Drosha exhibited robust pri-miRNA proces-sing activity (Fig. 3b). These results demonstrate the stable associ-ation of Drosha and DGCR8 in an active pri-miRNA processingcomplex. For the sake of consistency we have called thiscomplex Microprocessor18. A parallel study shows that a Drosophilahomologue of DGCR8 associates with Drosha and pri-miRNAprocessing activity in Drosophila, Caenorhabditis elegans andmammals for a role of this protein in miRNA biogenesis18.

We next analysed the activity of Microprocessor purified by eitherFlag-Drosha or Flag-DGCR8 affinity chromatography for proces-sing of two other pri-miRNAs. miR-(15,16) and miR-(23,27,24-2)were processed to yield expected miRNA precursors as was shownpreviously15 (Fig. 3c). Moreover, analysis of the processed fragmentsby northern blot analysis and further processing by Dicer confirmedthe specific processing by Microprocessor (Supplementary Fig. S3).

To demonstrate rigorously that pri-miRNA processing requiredDGCR8 in addition to the RNase III Drosha, we reconstitutedpri-miRNA processing activity by using recombinant Droshaproduced in insect cells and recombinant DGCR8 generated in

Figure 3 Microprocessor is the Drosha–DGCR8 complex mediating miRNA processing.

a, Isolation of Flag-DGCR8 complex. SDS–PAGE followed by silver staining and western

blot analysis with antibodies shown on the figure are displayed. b, Analysis of pri-miRNA

processing activity of Flag-DGCR8–Drosha complex. c, Analysis of miRNA processing

activity of Microprocessor purified through Flag-Drosha (fraction 25; Fxn 25) or Flag-

DGCR8 with the use of the miRNAs shown.

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bacteria (Fig. 4a). Both recombinant proteins were purified tonear homogeneity and were used in near-stoichiometric amountsin pri-miRNA processing assays (Fig. 4b, c). Although nativeMicroprocessor displayed robust pri-miRNA processing activity,neither recombinant Drosha nor recombinant DGCR8 showed anysignificant pri-miRNA processing activity (Fig. 4c, lanes 4–7).However, addition of the two recombinant proteins reconstitutedthe pri-miRNA processing activity to levels similar to those seenwith native complex (Fig. 4c, lanes 8 and 9). Whereas the addition ofincreasing concentrations of DGCR8 inhibited the processing reac-tion perhaps through a squelching mechanism, a further increase inDrosha stimulated the processing of pri-miRNA (Fig. 4c, comparelanes 10, 11 and 12, 13). Interestingly, whereas Drosha alonedisplayed non-specific RNase activity on the substrate, the additionof DGCR8 inhibited these non-specific effects and promotedDrosha’s pri-miRNA processing activity (Fig. 4c, compare lanes 5and 9). These results show the requirement for DGCR8 in directingthe specific processing of pri-miRNAs by Drosha.

To assess the role of Microprocessor in the initiation of miRNAprocessing in vivo, we used RNA interference to deplete DGCR8 and

Drosha. For these experiments short interfering RNA (siRNA)against Drosha was used as a positive control, whereas siRNAagainst transcription factor TFII-I was used as negative control(Fig. 5a). Knock-down of DGCR8 caused a similar effect to Droshadepletion for all miRNAs tested, resulting in a pronounced decreasein mature miRNA levels (Fig. 5b). Depletion of both Droshaand DGCR8, however, resulted in a substantial accumulation ofpri-miRNA, showing the requirement for Microprocessor inmiRNA processing in vivo (Fig. 5c). These results show the obliga-tory role for Drosha and DGCR8 in microRNA processing in vivoand in vitro.

We have isolated two multi-protein complexes that containDrosha as their catalytic engine. We show that the smaller Micro-processor complex containing Drosha and DGCR8 is necessary andsufficient for the processing of pri-miRNA to pre-miRNA. Thiscontention is based on the following observations. First, Micro-processor purified by Flag-Drosha affinity purification displaysspecific and robust activity in pri-miRNA processing. Second,isolation of Microprocessor through Flag-DGCR8 using stablecell lines revealed its close association with Drosha and miRNA

Figure 4 Reconstitution of Microprocessor by using recombinant Drosha and DGCR8.

a, Analysis of recombinant Drosha and DGCR8 with colloidal blue staining; 100 ng of each

protein was analysed. b, As in a except that ‘1 £ ’ corresponds to 50 ng of BSA, used to

determine Drosha and DGCR8 concentrations. c, Reconstitution of miRNA processing by

using recombinant Drosha and DGCR8. ‘1 £ ’ corresponds to 10 ng of Drosha in fraction

26 (Fxn 26) of Superose 6 and 20 ng of each of the recombinant (r) proteins assayed for

pri-miRNA processing activity as described in Methods.

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processing activity. Third, miRNA processing activity could bereconstituted with recombinant Drosha and DGCR8. Last, knock-down of Drosha and DGCR8 resulted in diminished mature miRNAand accumulation of pri-miRNA in vivo.

DGCR8 is an evolutionarily conserved protein that containstwo double-stranded RNA-binding domains and a WW domainknown to interact with proline-rich peptides. The WW domain ofDGCR8 is most probably the interacting surface with the proline-rich N-terminal domain of Drosha. DGCR8 is one of an estimated30 genes in the chromosomal region 22q11.2 whose heterozygousdeletion results in the most common human genetic deletionsyndrome, known as DiGeorge syndrome19,20. The clinical manifes-tations of the disease are highly variable, with 75% of patientsdisplaying congenital heart defects. Other common featuresinclude, among many others, characteristic facial appearance,immunodeficiency resulting from thymic hypoplasia, and develop-mental and behavioural problems. It will be important to explainthe role of DGCR8 in the genesis and development of DiGeorgesyndrome. Future experiments with knockout mouse models ofDGCR8 in homozygous and heterozygous animals will shed light onthe likely function of DGCR8 in developmental control and theexpression of DiGeorge syndrome-like phenotypes.

In the past we have used the 2-MDa chromatin remodellingcomplex, SWI–SNF, as a size maker for gel-filtration chroma-tography. Comparing the elution profiles of the large Droshacomplex with that of SWI–SNF (which peaks in fraction 20 on

Superose 6) we conclude that the large Drosha complex is largerthan SWI–SNF. We have identified nearly 20 polypeptides that arespecifically associated with Drosha in this complex. These includedRNA helicases containing a DEAD-box or DEAH-box, hnRNPs andproteins with either double-stranded RNA-binding or RRMdomains. We have also identified EWS as components of this largerDrosha complex. Ewing’s family of tumours result from tumour-associated chromosomal translocations between EWS genes andone of five different ETS transcription factors21.

We cannot exclude a role for the large Drosha complex in miRNAprocessing because it displayed a weak pre-miRNA processingactivity in vitro. Moreover, our analysis in vivo with siRNA knock-down of three components, p62/DDX5, p72/DDX17 andhnRNPU1-like, revealed a small decrease in mature miRNAs,although abrogation of these subunits never approached the effectsobserved after DGCR8 and Drosha depletions. It is therefore morelikely that the large Drosha-containing complex has a function inother RNA processing pathways. Because Drosha has also beenpreviously shown to participate in preribosomal RNA processing22,the large Drosha complex might mediate such preribosomal RNAprocessing activities. A

MethodsAffinity purification of Flag-Drosha and Flag-DGCR8Flag-Drosha and a selectable marker for puromycin resistance were cotransfected intoHEK-293 human embryonic kidney cells. Transfected cells were grown in the presence of2.5 mg ml21 puromycin, and individual colonies were isolated and analysed for Flag-Drosha expression. To purify the Drosha complex, nuclear extract generated from 20015-cm plates (4 £ 109 cells or ,150 mg of nuclear extract) was incubated with anti-FlagM2 affinity resin (Sigma). After two washes with buffer A (20 mM Tris-HCl pH 7.9, 0.5 MKCl, 10% glycerol, 1 mM EDTA, 5 mM dithiothreitol, 0.5% Nonidet P40, 0.2 mMphenylmethylsulphonyl fluoride), the affinity column was eluted with buffer A containingFlag peptide (400mg ml21) in accordance with the manufacturer’s instructions (Sigma).Analysis of Drosha by Superose 6 gel filtration was similar to that previously described23,24.Fractions from the gel-filtration chromatography were concentrated by precipitation withtrichloroacetic acid and analysed by SDS–PAGE followed by silver staining. Flag-DGCR8was isolated by using a similar protocol. Protein identification by liquid chromatography–tandem mass spectroscopy was performed as detailed23. Multiple sequencing analyses havedetermined SKB1, MEP50 and a–tubulin as common contaminants of Flag-affinitypurification. Anti-hnRNP M4 antibody was obtained from Santa Cruz Biotechnology.Anti-Drosha and anti-DGCR8 were generated against the last 20 amino acids in the aminoand carboxy termini of each protein (Open Biosystems). Recombinant Drosha andDGCR8 were purified by methods previously described for the purification ofrecombinant proteins from insect cells and bacteria23,24. In brief, each protein wasexpressed as a Flag-tagged protein and purified with anti-Flag M2 affinity resin similar inmanner to the protocol described for Flag-Drosha and Flag-DGCR8.

miRNA processingIn vitro transcription was performed with the Promega Riboprobe system, using linearizedpGEM-7Z vector containing miR-(17,18,19a,20,19b-1), miR-(15,16) and miR-(23,27,24-2) as described15. In brief, the miRNA probes were amplified from HEK-293 RNA byreverse transcriptase-mediated polymerase chain reaction (RT–PCR) with 5

0-

tgctgaatttgtatggtttatagttgtta-3 0 as 5 0 primer and 5 0 -tacttttctacagacttttcactaccaca-3 0 as 3 0

primer for miR-(17,18,19a,20,19b-1), 50-CGCCCGGTGCCCCCCTCACCCCTGTGC

CAC-3 0 as 5 0 primer and 5 0 -CCCTGTTCCTGCTGAACTGAGCCAGTGTAC-3 0 as 3 0

primer for miR-(23,27,24-2) and 5 0 -CCTTGGAGTAAAGTAGCAGCAACTAATG-3 0 as 5 0

primer and 50-CTTACTCTGAGTTAAATCTTGAATAC-3

0as 3

0primer for miR-

(15,16). The processing reaction, containing the indicated amounts of Droshacomplex, 3ml of solution containing 32 mM MgCl2, 10 mM ATP, 200 mM creatinephosphate, 1 U ml21 HPRI (Takara) and the labelled pri-miRNA (2 £ 105 c.p.m.) andbuffer (20 mM Tris-HCl pH 7.9, 0.1 M KCl, 10% glycerol, 5 mM dithiothreitol, 0.2 mMphenylmethylsulphonyl fluoride), was added to a final volume of 30 ml. The reactionmixture was incubated at 37 8C for 90 min and extracted with phenol:chloroformmixture, then with chloroform and precipitated with 300 mM sodium acetate andethanol. The precipitated RNA was loaded on 15% denaturing polyacrylamide gels.For the reconstitution experiments, the two recombinant proteins were incubated for1 h on ice in buffer A containing 100 mM KCl before the addition of the reaction mix.All Drosha quantitative analyses were performed by comparing the Flag-affinity eluateand recombinant Drosha by quantitative western blot analysis. The amounts ofrecombinant Drosha were then deduced by using colloidal blue staining in comparisonwith known amounts of BSA.

siRNAs and transfectionThe siRNAs were synthesized by Dharmacon. The sequence of Drosha siRNA was 5 0 -AAGGACCAAGUAUUCAGCAAG-3

0, the DGCR8 siRNA was 5

0-AUCCGUUGAUCUC

GAGGAATT-3 0 , and the control siRNA against TFIII was 5 0 -UGUGGGGAAGCUCUU

Figure 5 A role for Microprocessor in vivo in miRNA processing. a, Analysis of transcript

levels by using RT–PCR for Drosha and DGCR8 after treatment of HeLa cells with siRNA

against each protein. siRNA against TFII-I was used as control. b, Northern blot analysis of

miRNA-21, miRNA-16, miRNA-23, let-7a-1 and miRNA-20 after treatment of HeLa cells

with siRNA against Drosha, DGCR8 or control siRNA. c, Analysis of pri-miRNA processing

after depletion of Drosha and DGCR8. 2RT, absence of reverse transcriptase.

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NATURE | VOL 432 | 11 NOVEMBER 2004 | www.nature.com/nature 239© 2004 Nature Publishing Group

GGCCTT-30. siRNA transfection in HeLa cells was performed with Lipofectamine

2000 (Invitrogen). In brief, cells were plated in 10-cm dishes to 40% confluence. Foreach dish, 1.6 nmol of siRNA was mixed with 24 ml of Lipofectamine 2000 in 3 ml ofOpti-MEM medium. The mixture was added to cells and incubated for 6 h. After 24 h asecond transfection was performed in the same way. Total RNA was prepared 3 days afterthe second transfection and was used for northern blot analysis or RT–PCR.

RNA isolation, RT–PCR and northern blot analysisTotal RNA from HeLa cells was prepared in TRIzol reagents (Invitrogen) in accordancewith the manufacturer’s instructions. To examine the effect of siRNA, RNA (2mg) wassubjected to complementary DNA synthesis with oligo(dT), using the SuperScript first-strand synthesis system for RT–PCR (Invitrogen). To examine pri-miRNAs, RNA (2 mg)was subjected to cDNA synthesis with random primers. As a control, RT–PCRs wereperformed in the absence of reverse transcriptase. Different PCR cycles were examined todetermine linear amplification.

Primer sequences for RT–PCRs in Fig. 5a were Drosha (5 0 -CATGCACCAGATTCTCCTGTA-3

0and 5

0-GTCTCCTGCATAACTCAACTG-3

0) and DGCR8 (5

0-TATCAGATCC

TCCACGAGTG-30

and 50-TCTTGGAGCTTGCTGAGGAT-3

0). Primer sequences

for RT–PCRs in Fig. 5c were pri-let-7a-1 (5 0 -GATTCCTTTTCACCATTCACCCTGGATGTT-3

0and 5

0-TTTCTATCAGACCGCCTGGATGCAGACTTT-3

0), pri-miR30a

(50-ATTGCTGTTTGAATGAGGCTTCAGTACTTT-3

0and 5

0-TTCAGCTTTGTAAAAA

TGTATCAAAGAGAT-3 0 ) and pri-miR17 (5 0 -TGCTGAATTTGTATGGTTTATAGTTGTTAG-3

0and 5

0-CACTACCACAGTCAGTTTTGCATGGATTTG-3

0). b-Actin was used

for internal control in Fig. 5a, c, and the primer sequences were 5 0 -AAAGACCTGTACGCCAACAC-3 0 and 5 0 -GTCATACTCCTGCTTGCTGAT-3 0 .

For northern blot analysis, total RNA (10 mg) was resolved on a 15% denaturingpolyacrylamide gel and electrotransferred to Hybond Nþ nylon membrane (Amersham).The membranes were crosslinked under ultraviolet and prewashed for 1 h at 65 8C in0.1 £ SSC/0.1% SDS. Prehybridization and hybridization were performed at 42 8C in10 £ Denharts solution, 6 £ SSC, 0.1% SDS. Oligonucleotides complementary tomiRNAs were end-labelled with [g-32P]ATP and used as probes for northern analysis. Thesequences of the oligonucleotides were 5 0 -gaaaatccctggcaatgtgat-3 0 (miR-23),5

0-actatacaacctactacctca-3

0(let-7a-1), 5

0-gccaatatttacgtgctgcta-3

0(miR-16),

50-tacctgcactataagcacttta-3

0(miR-20), 5

0-tcaacatcagtctgataagcta-3

0(miR-21) and

5 0 -caggcccgaccctgcttagcttccgagatcagacgagat-3 0 (5S rRNA). All of the probes were washedtwice for 10 min at 25 8C in 6 £ SSC/0.1% SDS.

Received 14 June; accepted 19 October 2004; doi:10.1038/nature03120.

Published online 7 November 2004.

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Supplementary Information accompanies the paper on www.nature.com/nature.

Acknowledgements We thank V. N. Kim for the Drosha cDNA; O. Delattre and A. I. Lamond for

the gift of EWS and DDX17 antibodies, respectively; K. Nishikura for providing recombinant

Drosha; and T. Beer (Wistar Proteomocs Facility) for expertise in the microcapillary HPLC/mass

spectrometry. R.S. was supported by grants from the NIH and the American Cancer Institute.

R.G. is a fellow of the Jane Coffin Child Memorial Fund for Medical Research.

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests for materials should be addressed to R.S.

(shiekhattar@wistar.upenn.edu).

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