A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA
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D. Yang et al. Curr. Biol. 10 1191 (2000).
RNA was isolated from in vitro processing reactions as described (21) and RNA from pupae and HeLa cells was isolated with TRIzol Reagent (Life Technologies) according to the manufacturer's instructions. RNAs were separated by electrophoresis on 15% denaturing polyacrylamide gels and electroblotted onto Hybond N+ (Amersham). DNA oligonucleotides were radioactively labeled with polynucleotide kinase and [γ- 32 P]ATP (ICN). Membranes were hybridized with the probe at 42°C in 50% formamide 5× saline sodium phosphate EDTA (pH 7.4) 5× Denhardt's reagent 0.5% SDS denatured herring sperm DNA (80 μg/ml) and washed at 42°C in 2× saline sodium citrate and 0.1% SDS. In Fig. 1A the deoxyoligonucleotide probe was 5′-AACTATACAACCTACTACCTCACCGGATCC-3′.
Primer extension was performed with Superscript II reverse transcriptase (Life Technologies) at 37°C for 40 min followed by incubation at 50°C for 30 min with 5 μg of RNA with 1 pmol of the [γ- 32 P]ATP-labeled deoxyoligonucleotide 5′-CTACTATACAACCTACTAC-3′. Extension products were separated by electrophoresis in a 15% denaturing polyacrylamide gel.
A. E. Pasquinelli G. Ruvkun unpublished data.
Pre- let-7 RNA was transcribed from a synthetic deoxynucleotide template with T7 RNA polymerase in the presence of α- 32 P–labeled nucleotide gel isolated and incubated at ≤50 nM in a standard RNAi reaction as described (21). Northern hybridization experiments and controls were performed as described (19). Synthetic RNAs ( let-7 in Fig. 1B) (5′-GGCAAAUUGAGGUAGUAGGUU-3′ and 5′-UAUACAAUGUGCUAGCUUUCU-3′; Dharmacon Research) were used to assess probe specificity in control hybridizations.
A. Nykänen P. D. Zamore unpublished data.
Supplementary Web data are available on Science Online at www.sciencemag.org/cgi/content/full/1062961/DC1.
RNAs were treated with 5 U of CIP (Promega) per 100 μg of RNA. Periodate treatment and β-elimination were as previously described (41).
G. Hutvágner et al. data not shown.
S. Matsuda et al. Biochim. Biophys. Acta 1490 163 (2000).
Synthetic RNAs (Dharmacon) were deprotected according to the manufacturer's directions. The siRNA duplex targeting Dicer mRNA was prepared by annealing 5′-UGCUUGAAGCAGCUCUGGAdTdT-3′ and 5′-UCCAGAGCUGCUUCAAGCAdTdT-3′; the control siRNA duplex was formed by annealing 5′-CUUUAAGCUCCCUGAGCGUUU-3′ with 5′-ACGCUCAGGGAGCUUAAAGUG-3′. siRNAs were annealed as described (25). siRNA-mediated gene silencing was performed as described (32). Briefly exponentially growing HeLa S3 cells were trypsinized and transferred to six-well plates. Cells (2 ml) were plated into each well (1 × 10 5 cells/ml). siRNAs were transfected at 18 nM concentration with LipofectAMINE 2000 reagent (Life Technologies) in media containing Dulbecco's modified Eagle's medium but lacking both serum and antibiotics. Untreated control cells received LipofectAMINE and buffer but no siRNA. Two hours after transfection the media were replaced with fresh media containing serum but no antibiotics. RNA was extracted 3 days after transfection. Transfection efficiency was determined by staining cells for β-galactosidase activity in a parallel transfection with a plasmid in which LacZ is driven by a cytomegalovirus promoter. Semiquantitative RT-PCR detection of human Dicer mRNA has been described previously (34). Actin was detected with the primers 5′-CGTGATGGTGGGCATGGGTCAG-3′ and 5′-CTTAATGTCACGCACGATTTCC-3′.
M. Zuker et al. in A Practical Guide in RNA Biochemistry and Biotechnology J. Barciszewski B. F. C. Clark Eds. (Kluwer New York 1999).
We thank A. Grishok C. Mello and G. Ruvkun for sharing data before publication; G. Stein for providing facilities for HeLa cell culture; and D. Bartel A. Grishok K. Knight C. Mello G. Ruvkun P. Sharp F. Slack and members of the Zamore laboratory for discussions and comments on the manuscript. We acknowledge A. Grishok and C. Mello for the suggestion that pre- let-7 hairpins might be engineered to generate non- let-7 products. P.D.Z. is a Pew Scholar in the Biomedical Sciences. Supported in part by a grant from the NIH (GM62862-01) to P.D.Z.