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

Nature volume 475, pages 201–205 (2011)Cite this article

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Abstract

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.

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Primary accessions

Gene Expression Omnibus

Protein Data Bank

Data deposits

Small RNA sequencing data were deposited in the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) under accession number GSE27903.

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Acknowledgements

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.

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Author notes

  1. Jong-Eun Park and Inha Heo: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Biological Sciences, Seoul National University, Seoul 151-742, Korea

    Jong-Eun Park, Inha Heo, Hyeshik Chang, David Jee & V. Narry Kim

  2. Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, 10065, New York, USA

    Yuan Tian, Dhirendra K. Simanshu & Dinshaw J. Patel

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  1. Jong-Eun Park
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Contributions

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). https://doi.org/10.1038/nature10198

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