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Dicer recognizes the 5′ end of RNA for efficient and accurate processing


A hallmark of RNA silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer is crucial for the functionality of microRNAs (miRNAs). The current model posits that Dicer selects cleavage sites by measuring a set distance from the 3′ overhang of the double-stranded RNA terminus. Here we report that human Dicer anchors not only the 3′ end but also the 5′ end, with the cleavage site determined mainly by the distance (22 nucleotides) from the 5′ end (5′ counting rule). This cleavage requires a 5′-terminal phosphate group. Further, we identify a novel basic motif (5′ pocket) in human Dicer that recognizes the 5′-phosphorylated end. The 5′ counting rule and the 5′ anchoring residues are conserved in Drosophila Dicer-1, but not in Giardia Dicer. Mutations in the 5′ pocket reduce processing efficiency and alter cleavage sites in vitro. Consistently, miRNA biogenesis is perturbed in vivo when Dicer-null embryonic stem cells are replenished with the 5′-pocket mutant. Thus, 5′-end recognition by Dicer is important for precise and effective biogenesis of miRNAs. Insights from this study should also afford practical benefits to the design of small hairpin RNAs.

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Figure 1: Human Dicer counts 22 nucleotides from the 5′ end of RNA to locate the cleavage site.
Figure 2: The 5′ counting rule is conserved in Drosophila Dicer-1 but not in Giardia Dicer.
Figure 3: Residues required for the recognition of dsRNA terminus.
Figure 4: The 5′ pocket is critical for efficient and accurate cleavage in vivo.

Accession codes

Primary accessions

Gene Expression Omnibus

Protein Data Bank

Data deposits

Small RNA sequencing data were deposited in the Gene Expression Omnibus ( under accession number GSE27903.


  1. Kim, V. N., Han, J. & Siomi, M. C. Biogenesis of small RNAs in animals. Nature Rev. Mol. Cell Biol. 10, 126–139 (2009)

    Article  CAS  Google Scholar 

  2. Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003)

    Article  ADS  CAS  Google Scholar 

  3. Ketting, R. F. et al. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans . Genes Dev. 15, 2654–2659 (2001)

    Article  CAS  Google Scholar 

  4. 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)

    Article  ADS  CAS  Google Scholar 

  5. Grishok, A. et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23–34 (2001)

    Article  CAS  Google Scholar 

  6. Hutvagner, G. et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834–838 (2001)

    Article  CAS  Google Scholar 

  7. Knight, S. W. & Bass, B. L. A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans . Science 293, 2269–2271 (2001)

    Article  ADS  CAS  Google Scholar 

  8. Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. & Hannon, G. J. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150 (2001)

    Article  CAS  Google Scholar 

  9. Tabara, H., Yigit, E., Siomi, H. & Mello, C. C. The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans . Cell 109, 861–871 (2002)

    Article  CAS  Google Scholar 

  10. Mourelatos, Z. et al. miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev. 16, 720–728 (2002)

    Article  CAS  Google Scholar 

  11. Han, J. et al. Molecular basis for the recognition of primary microRNAs by the Drosha–DGCR8 complex. Cell 125, 887–901 (2006)

    Article  CAS  Google Scholar 

  12. Vermeulen, A. et al. The contributions of dsRNA structure to Dicer specificity and efficiency. RNA 11, 674–682 (2005)

    Article  CAS  Google Scholar 

  13. Zhang, H., Kolb, F. A., Brondani, V., Billy, E. & Filipowicz, W. Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21, 5875–5885 (2002)

    Article  CAS  Google Scholar 

  14. Zhang, H., Kolb, F. A., Jaskiewicz, L., Westhof, E. & Filipowicz, W. Single processing center models for human Dicer and bacterial RNase III. Cell 118, 57–68 (2004)

    Article  CAS  Google Scholar 

  15. MacRae, I. J., Zhou, K. & Doudna, J. A. Structural determinants of RNA recognition and cleavage by Dicer. Nature Struct. Mol. Biol. 14, 934–940 (2007)

    Article  CAS  Google Scholar 

  16. MacRae, I. J. et al. Structural basis for double-stranded RNA processing by Dicer. Science 311, 195–198 (2006)

    Article  ADS  CAS  Google Scholar 

  17. Burroughs, A. M. et al. A comprehensive survey of 3′ animal miRNA modification events and a possible role for 3′ adenylation in modulating miRNA targeting effectiveness. Genome Res. 20, 1398–1410 (2010)

    Article  CAS  Google Scholar 

  18. Wu, H., Ye, C., Ramirez, D. & Manjunath, N. Alternative processing of primary microRNA transcripts by Drosha generates 5′ end variation of mature microRNA. PLoS ONE 4, e7566 (2009)

    Article  ADS  Google Scholar 

  19. Heo, I. et al. Lin28 mediates the terminal uridylation of let-7 precursor microRNA. Mol. Cell 32, 276–284 (2008)

    Article  CAS  Google Scholar 

  20. Chiang, H. R. et al. Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. Genes Dev. 24, 992–1009 (2010)

    Article  CAS  Google Scholar 

  21. Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009)

    Article  CAS  Google Scholar 

  22. Tomari, Y., Matranga, C., Haley, B., Martinez, N. & Zamore, P. D. A protein sensor for siRNA asymmetry. Science 306, 1377–1380 (2004)

    Article  ADS  CAS  Google Scholar 

  23. Serra, M. J. et al. Effects of magnesium ions on the stabilization of RNA oligomers of defined structures. RNA 8, 307–323 (2002)

    Article  CAS  Google Scholar 

  24. Gunther, T. Concentration, compartmentation and metabolic function of intracellular free Mg2+ . Magnes. Res. 19, 225–236 (2006)

    CAS  PubMed  Google Scholar 

  25. Jiang, F. et al. Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila . Genes Dev. 19, 1674–1679 (2005)

    Article  CAS  Google Scholar 

  26. Saito, K., Ishizuka, A., Siomi, H. & Siomi, M. C. Processing of pre-microRNAs by the Dicer-1–Loquacious complex in Drosophila cells. PLoS Biol. 3, e235 (2005)

    Article  Google Scholar 

  27. Miyoshi, K., Miyoshi, T., Hartig, J. V., Siomi, H. & Siomi, M. C. Molecular mechanisms that funnel RNA precursors into endogenous small-interfering RNA and microRNA biogenesis pathways in Drosophila . RNA 16, 506–515 (2010)

    Article  Google Scholar 

  28. Förstemann, K. et al. Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol. 3, e236 (2005)

    Article  Google Scholar 

  29. Lee, Y. S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69–81 (2004)

    Article  CAS  Google Scholar 

  30. Roy, A., Kucukural, A. & Zhang, Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols 5, 725–738 (2010)

    Article  CAS  Google Scholar 

  31. Murchison, E. P., Partridge, J. F., Tam, O. H., Cheloufi, S. & Hannon, G. J. Characterization of Dicer-deficient murine embryonic stem cells. Proc. Natl Acad. Sci. USA 102, 12135–12140 (2005)

    Article  ADS  CAS  Google Scholar 

  32. Heo, I. et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 138, 696–708 (2009)

    Article  CAS  Google Scholar 

  33. Seitz, H., Ghildiyal, M. & Zamore, P. D. Argonaute loading improves the 5′ precision of both microRNAs and their miRNA* strands in flies. Curr. Biol. 18, 147–151 (2008)

    Article  CAS  Google Scholar 

  34. Chang, K., Elledge, S. J. & Hannon, G. J. Lessons from Nature: microRNA-based shRNA libraries. Nature Methods 3, 707–714 (2006)

    Article  CAS  Google Scholar 

  35. Silva, J., Chang, K., Hannon, G. J. & Rivas, F. V. RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age. Oncogene 23, 8401–8409 (2004)

    Article  CAS  Google Scholar 

  36. Kim, D. H. & Rossi, J. J. Strategies for silencing human disease using RNA interference. Nature Rev. Genet. 8, 173–184 (2007)

    Article  CAS  Google Scholar 

  37. Han, K. An efficient DDAB-mediated transfection of Drosophila S2 cells. Nucleic Acids Res. 24, 4362–4363 (1996)

    Article  CAS  Google Scholar 

  38. Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010)

    Article  CAS  Google Scholar 

  39. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009)

    Article  CAS  Google Scholar 

  40. Babiarz, J. E., Ruby, J. G., Wang, Y., Bartel, D. P. & Blelloch, R. Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. Genes Dev. 22, 2773–2785 (2008)

    Article  CAS  Google Scholar 

  41. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate—a practical and powerful approach to multiple testing. J. R. Stat. Soc., B 57, 289–300 (1995)

    MathSciNet  MATH  Google Scholar 

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We are grateful to G. Hannon for Dicer-null mouse ES cells; J. Doudna for Giardia Dicer cDNA; and M. Siomi for Drosophila Dicer-1, Dicer-2, Loqs-PB and R2D2 constructs. We also thank the members of the V.N.K. laboratory, particularly C. Joo, M.-J. Yoon, K.-H. Yeom and A. Cho for discussions and technical help. The V.N.K. laboratory was supported by the Creative Research Initiatives Program (2010000021) and National Honor Scientist Program (20100020415) through the National Research Foundation and the BK21 Fellowships (J.-E.P. and H.C.) from the Ministry of Education, Science and Technology. Research in the D.J.P. laboratory was supported by the National Institutes of Health.

Author information

Authors and Affiliations



J.-E.P., I.H. and D.J. performed biochemical and cell biological experiments. H.C. carried out bioinformatic analyses. Y.T., D.K.S. and D.J.P. performed structural studies. J.-E.P., I.H. and V.N.K. designed the study and wrote the paper.

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Correspondence to V. Narry Kim.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

The file contains Supplementary Figures 1-10 with legends. (PDF 2010 kb)

Supplementary Table 1

This table shows the numbers of sequence reads in steps of data processing and basic information for each library. (PDF 42 kb)

Supplementary Table 2

This table shows the sequencing data which include the sequences and counts of each read, and the statistical confidence of Drosha/Dicer cleavage site change. (HTML 938 kb)

Supplementary Table 3

This table shows the list of mature miRNAs with tendency of seed sequence changes by 5'-mutation. (XLS 192 kb)

Supplementary Table 4

This table shows the primer sequences used for mutagenesis experiments. (DOC 40 kb)

Supplementary Table 5

This table shows the RNA sequences used for generation of pre-miRNAs. (DOC 42 kb)

Supplementary Table 6

This table shows the sequences of pre-miRNAs used for in vitro assay. (DOC 36 kb)

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Park, JE., Heo, I., Tian, Y. et al. Dicer recognizes the 5′ end of RNA for efficient and accurate processing. Nature 475, 201–205 (2011).

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