RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs)1,2. However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3′ overhang and a terminal loop3,4,5,6,7,8,9,10,11. Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis-acting element, termed the ‘GYM motif’ (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified ‘ruler’-like counting mechanisms from the 5′ and 3′ ends of pre-miRNA3,4,5,6. Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics.
This is a preview of subscription content, access via your institution
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 per month
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
The massively parallel assay data and rescue data were deposited to the GEO repository (accession numbers GSE202535 and GSE215866). Other structural models cited in this study for analysis (5ZAL and 2EZ6) are also accessible on PDB. The Cancer Genome Atlas data for the DICER gene was accessed at cBioPortal (https://www.cbioportal.org).
Custom analysis codes are available at https://github.com/haedongkim615/dicer_gym_motif.
Elbashir, S. M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).
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).
Macrae, I. J. et al. Structural basis for double-stranded RNA processing by Dicer. Science 311, 195–198 (2006).
Macrae, I. J., Li, F., Zhou, K., Cande, W. Z. & Doudna, J. A. Structure of Dicer and mechanistic implications for RNAi. Cold Spring Harb. Symp. Quant. Biol. 71, 73–80 (2006).
Park, J. E. et al. Dicer recognizes the 5′ end of RNA for efficient and accurate processing. Nature 475, 201–205 (2011).
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).
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, 58755885 (2002).
Gu, S. et al. The loop position of shRNAs and pre-miRNAs is critical for the accuracy of Dicer processing in vivo. Cell 151, 900–911 (2012).
Tsutsumi, A., Kawamata, T., Izumi, N., Seitz, H. & Tomari, Y. Recognition of the pre-miRNA structure by Drosophila Dicer-1. Nat. Struct. Mol. Biol. 18, 1153–1158 (2011).
Feng, Y., Zhang, X., Graves, P. & Zeng, Y. A comprehensive analysis of precursor microRNA cleavage by human Dicer. RNA 18, 2083–2092 (2012).
Liu, Z., Wang, J., Li, G. & Wang, H. W. Structure of precursor microRNA’s terminal loop regulates human Dicer’s dicing activity by switching DExH/D domain. Protein Cell 6, 185–193 (2015).
Lee, Y., Jeon, K., Lee, J. T., Kim, S. & Kim, V. N. MicroRNA maturation: stepwise processing and subcellular localization. EMBO J. 21, 4663–4670 (2002).
Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003).
Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F. & Hannon, G. J. Processing of primary microRNAs by the Microprocessor complex. Nature 432, 231–235 (2004).
Gregory, R. I. et al. The Microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240 (2004).
Han, J. et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027 (2004).
Khvorova, A., Reynolds, A. & Jayasena, S. D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).
Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).
Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).
Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P. & Burge, C. B. Prediction of mammalian microRNA targets. Cell 115, 787–798 (2003).
Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).
Tian, Y. et al. A phosphate-binding pocket within the platform-PAZ-connector helix cassette of human Dicer. Mol. Cell 53, 606–616 (2014).
Liu, Z. et al. Cryo-EM structure of human Dicer and its complexes with a pre-miRNA substrate. Cell 173, 1191–1203 (2018).
Heo, I. et al. Mono-uridylation of pre-microRNA as a key step in the biogenesis of group II let-7 microRNAs. Cell 151, 521–532 (2012).
Kim, H. et al. Bias-minimized quantification of microRNA reveals widespread alternative processing and 3′ end modification. Nucleic Acids Res. 47, 2630–2640 (2019).
Chiang, H. R. et al. Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. Genes Dev. 24, 992–1009 (2010).
Kim, H. et al. A mechanism for microRNA arm switching regulated by uridylation. Mol. Cell 78, 1224–1236 (2020).
Kim K. et al. A quantitative map of human primary microRNA processing sites. Mol. Cell 81, P3422–3439.E11 (2021).
Fang, W. & Bartel, D. P. The menu of features that define primary microRNAs and enable de novo design of microRNA genes. Mol. Cell 60, 131–145 (2015).
Han, C. et al. The RNA-binding protein DDX1 promotes primary microRNA maturation and inhibits ovarian tumor progression. Cell Rep. 8, 1447–1460 (2014).
Cerami, E. et al. The cBio Cancer Genomics Portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 (2012).
Gao, J. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 6, pl1 (2013).
Gao, Z., Herrera-Carrillo, E. & Berkhout, B. Delineation of the exact transcription termination signal for type 3 polymerase III. Mol. Ther. Nucleic Acids 10, 36–44 (2018).
Amarzguioui, M. et al. Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nat. Protoc. 1, 508–517 (2006).
Kim, D. H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, 222–226 (2005).
Snead, N. M. et al. Molecular basis for improved gene silencing by Dicer substrate interfering RNA compared with other siRNA variants. Nucleic Acids Res. 41, 6209–6221 (2013).
Masliah, G. et al. Structural basis of siRNA recognition by TRBP double-stranded RNA binding domains. EMBO J. 37, e97089 (2018).
Ma, E., Zhou, K., Kidwell, M. A. & Doudna, J. A. Coordinated activities of human Dicer domains in regulatory RNA processing. J. Mol. Biol. 422, 466–476 (2012).
Gan, J. et al. Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III. Cell 124, 355–366 (2006).
Kwon, S. C. et al. Molecular basis for the single-nucleotide precision of primary microRNA processing. Mol. Cell 73, 505–518 (2019).
Kim, B., Jeong, K. & Kim, V. N. Genome-wide mapping of DROSHA cleavage sites on primary microRNAs and noncanonical substrates. Mol. Cell 66, 258–269 (2017).
Bofill-De Ros, X. et al. Structural differences between pri-miRNA paralogs promote alternative Drosha cleavage and expand target repertoires. Cell Rep. 26, 447–459 (2019).
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).
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).
Lee, Y.-Y., Lee, H., Kim, H., Kim, V. N. & Roh S.-H. Structure of the human DICER–pre-miRNA complex in a dicing state. Nature https://doi.org/10.1038/s41586-023-05723-3 (2023).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).
Heo, I. et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through premicroRNA uridylation. Cell 138, 696–708 (2009).
Kim, Y. K., Kim, B. & Kim, V. N. Re-evaluation of the roles of DROSHA, Export in 5, and DICER in microRNA biogenesis. Proc. Natl Acad Sci. USA 113, E1881–E1889 (2016).
Bogerd, H. P., Whisnant, A. W., Kennedy, E. M., Flores, O. & Cullen, B. R. Derivation and characterization of Dicer- and microRNA-deficient human cells. RNA 20, 923–937 (2014).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Kozomara, A. & Griffiths-Jones, S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res. 42, D68–D73 (2014).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Fromm, B. et al. MirGeneDB 2.0: the metazoan microRNA complement. Nucleic Acids Res. 48, D1172 (2020).
Auyeung, V. C., Ulitsky, I., McGeary, S. E. & Bartel, D. P. Beyond secondary structure: primary-sequence determinants license pri-miRNA hairpins for processing. Cell 152, 844–858 (2013).
Bellaousov, S., Reuter, J. S., Seetin, M. G. & Mathews, D. H. RNAstructure: web servers for RNA secondary structure prediction and analysis. Nucleic Acids Res. 41, W471–W474 (2013).
Bofill-De Ros, X. & Gu, S. Guidelines for the optimal design of miRNA-based shRNAs. Methods 103, 157–166 (2016).
We thank J.-S. Woo for the mammalian cell transfection protocol; B. Cullen for HEK293T DICER-knockout cell lines; M. Lee for Drosophila cDNA; B. Um, H. Jang, K. Kim, M. Kim, S. Son and Y. Park for valuable discussions; and Y.-G. Choi, S.-M. Ji, J. Yang, D.-E. Choi, S. Bang and E. Kim for technical assistance. This research was supported by Institute for Basic Science funding from the Ministry of Science and ICT of Korea (IBS-R008-D1 to Y.-Y.L., H.K. and V.N.K.), BK21 research fellowships from the Ministry of Education of Korea (to Y.-Y.L. and H.K.) and a National Research Foundation of Korea grant funded by the Korean government (NRF-2018-Global PhD Fellowship Program to Y.-Y.L. and NRF-2015-Global PhD Fellowship Program to H.K.).
Y.-Y.L., H.K. and V.N.K. are coinventors on pending patent application (KR 10-2022-0059227), submitted by Institute for Basic Science and Seoul National University, which covers the use of the GYM motif for RNA interference.
Peer review information
Nature thanks Haruhiko Siomi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 A yet-unknown mechanism of DICER processing mediated by the upper stem region.
a, Illustration of the mechanism of cleavage site choice by DICER. b, Cleavage site decision of pre-let-7a-1 and pre-miR-324. c, In vitro processing of a pre-miR-324 variant by DICER. “No-bulge pre-miR-324” was used for this assay to avoid the influence of the bulge27. A mismatch near the cleavage was replaced with a base-pair marked in pink. Cleavage sites and their corresponding products are marked with arrowheads. For gel source data, see Supplementary Fig. 1. *, radiolabeled 5′ phosphate.
Extended Data Fig. 2 Design of the massively parallel assay.
a, A structural model of human DICER in a dicing state. The dsRNA was modeled into the cryo-EM structure of human DICER23, based on the crystal structure of dsRNA-bound Aa RNase III39. DICER dsRBD was then superimposed with that of Aa RNase III to predict its position in a dicing state. b, Pre-miRNAs used in the massively parallel assay. The 5-bp and 3-bp windows (positions –1 to 3, –1 to 1, 1 to 3 relative to the starting position of 3p miRNA) were targeted for randomization based on the structural model. Secondary structures of pre-miRNAs were obtained using RNAstructure52. c, SDS-PAGE of purified proteins. For gel source data, see Supplementary Fig. 1. d, Size-exclusion chromatography of purified proteins.
Extended Data Fig. 3 Massively parallel assays performed with substrates with −1-to-1 or 1-to-3 randomization.
a–b, Distribution of read counts of variants. c–d, Distribution of cleavage scores of variants. e–h, Correlation of cleavage scores of variants between different conditions of varying reaction time. i, Distribution of the cleavage scores measured from the 2nd screening with 1-to-3 randomization.
Extended Data Fig. 4 Massively parallel assay reveals structural and sequence preferences at position 1.
a–b, Structural impact on cleavage scores. G–U pair was considered as a mismatch only when it is in between mismatches. p, pair; m, mismatch. c–d, Impact of the base combinations at the 1 position on cleavage scores. Variants with base-pairs at all but position 1 were included in this analysis.
Extended Data Fig. 5 The GYM motif affects efficiency and accuracy of DICER processing independently of TRBP.
a–c, f, In vitro processing of pre-let-7a-1 variants by human DICER (a–b), human DICER and TRBP (c), or fly Dcr-1 (f). Substrates were radiolabeled at their 5′ ends. †, nicked products at the 3p positions. a, Lanes 6–10 are identical with those in Fig. 2a. b, Squares indicate mean (n = 2, independent experiments). c, Bars indicate mean ± SD (n = 3, independent experiments). ***p < 0.001 by two-sided Student’s t test compared to GCm. d, DROSHA processing assay and miRNA abundance measurement of pre-miR-A1 variants in HEK293T cells. Left: Schematic outline of this experiment. Right top: Luciferase assay. Firefly luciferase signals were normalized to Renilla luciferase (Rluc) signals. Right bottom: miRNA levels measured by qRT-PCR. The TaqMan probe was designed to target the common sequence of variants. Bars indicate mean ± SD (n = 3, biological replicates). **p < 0.01, ***p < 0.001 by two-sided Student’s t test compared to GCm. e,g, In vitro processing of duplex variants by human DICER. Cleavage products and their corresponding cleavage sites are marked with arrowheads. *, radiolabeled 5′ phosphate. g, The duplex had a base-pair at its terminus (marked in orange) so that the 5′ end cannot be incorporated into the 5′ pocket. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 6 R1855 and E1859 of the DICER dsRBD are important for recognition of the mismatch.
a, In vitro processing of pre-let-7a-1 variants by human DICER ΔdsRBD with the indicated reaction time. b, Amino acid sequence alignment of dsRBDs of metazoan DICERs. c, In vitro processing of duplex variants by human DICER point mutants at the indicated position. Cleavage products and their corresponding cleavage sites are marked with arrowheads. *, radiolabeled 5′ phosphate. d,e, In vitro processing of pre-let-7a-1 variants by either DICER R1855L (d) or R1855A/E1859A (AA) (e) with the indicated reaction time. Bars indicate mean (n = 2, independent experiments) (d) or mean ± SD (n = 3, independent experiments) (e). *p < 0.05, ***p < 0.001 by two-sided Student’s t test compared to GCm. †, nicked products at the 3p positions. f, In vitro processing of duplex variants by DICER AA mutant. The cleavage product and its corresponding cleavage site marked with the arrowhead are largely unaffected by the GYM motif variations, which contrasts the result from WT DICER shown in Fig. 2b, d. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 7 Mutating the DICER dsRBD reduces efficiency and accuracy of DICER processing.
a, c, Comparison of miRNA expressions in either HCT116 (a) or HEK293T (c). Spike-ins were used for normalization. RPM, reads per million. b, d, Comparison of cleavage accuracy in either HCT116 (b) or HEK293T (d). For a given miRNA, the most abundant 5′-isomiR was identified in the WT sample. Then the fold change of its proportions in each sample was measured as cleavage accuracy. Grey, unannotated strand. Bar graphs show the number of miRNAs whose major 5′-isomiR was significantly affected by the mutation (p < 0.01 by two-sided Student’s t test).
Extended Data Fig. 8 Processing of pre-miRNAs are regulated by the GYM motit recognized by the DICER dsRBD.
a–c, In vitro processing of variants of pre-miR-27b (a), pre-miR-21 (b), and pre-let-7d/f-1/i (c) by either DICER WT or ΔdsRBD. Pre-miRNAs were radiolabeled at their 5′ end. Reactions were performed with different time points as indicated. For gel source data, see Supplementary Fig. 1. Bars indicate mean ± SD (n = 3, independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001 by two-sided Student’s t test compared to the WT substrate. †, nicked products at the 3p positions.
Extended Data Fig. 9 Examples of miRNAs whose DICER cleavage sites are affected by mutation of the DICER dsRBD.
a–b, The usage of 5′ ends of miRNAs in the DICER-knockout HCT116 cells rescued with indicated DICER. The annotation in miRBase release 21 was used as a reference. Cleavage sites and their corresponding positions are marked with arrowheads. RPM, reads per million.
Extended Data Fig. 10 The DICER dsRBD-GYM motif interaction plays a critical role in cleavage site decision of endogenous miRNAs.
a, c, The usage of 5′ ends of miR-34a-3p (a) or 3′ ends of let-7e-5p and 5′ ends of let-7e-3p (c) in the DICER-knockout HCT116 cells rescued with indicated DICER. The annotations in miRBase release 21 were used as references. Corresponding positions of the major cleavage sites are marked with arrowheads. RPM, reads per million. b, d, In vitro processing of pre-miR-34a variants (b) or pre-let-7e variants (d) by either DICER WT or ΔdsRBD. Pre-miRNAs were radiolabeled at their 5′ end. Major cleavage products and their corresponding cleavage sites are marked with arrowheads. Reactions were performed with different time points as indicated because DICER ΔdsRBD has reduced activity. e, The GYM scores at the position −1 of human pre-miRNAs. miRNAs registered in miRGeneDB (n = 383) were included in this analysis. The dashed line indicates the average of GYM scores of the surrounding positions (−2 and 0). f, Alternative processing of pre-miR-9. Cleavage sites were inferred from 5′ ends of miR-9-3p in the DICER-knockout HCT116 cells rescued with DICER WT. Average proportions are indicated at the corresponding cleavage sites marked with arrowheads. g, In vitro processing of pre-miR-9-1 by DICER. The GYM score for each window (grey and colored boxes) is shown. Pre-miRNAs were radiolabeled at their 5′ end. Major cleavage products and their corresponding cleavage sites are marked with arrowheads. For gel source data, see Supplementary Fig. 1.
Supplementary Fig. 1
Supplementary Table 1
Oligonucleotides used in the study. Oligonucleotides used to prepare pre-miRNA substrates by ligation or dsRNA substrates by annealing for in vitro processing assays, DsiRNA sequences for cellular transfection and shRNA sequences for cloning and plasmid transfection.
Supplementary Table 2
Massively parallel assays. Read counts obtained from individual variants in the input and uncleaved populations, calculated cleavage scores and GYM scores normalized to 0–100.
Supplementary Table 3
Exact P values calculated for in vitro assays and rescue experiments.
Supplementary Table 4
DICER rescue experiment in HCT116 cells. Read counts, spike-in-normalized abundances and the proportions of the main 5′-isomiR identified in the WT samples of rescued HCT116 cells.
Supplementary Table 5
DICER rescue experiment in HEK293T cells. Read counts, spike-in-normalized abundances and the proportions of the main 5′-isomiR identified in the WT samples of rescued HEK293T cells.
Supplementary Table 6
GYM motifs and corresponding GYM scores of human miRNAs.
Supplementary Table 7
Representative pre-miRNAs. Curated lists of representative animal miRNAs from diverse species.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Lee, YY., Kim, H. & Kim, V.N. Sequence determinant of small RNA production by DICER. Nature 615, 323–330 (2023). https://doi.org/10.1038/s41586-023-05722-4
This article is cited by
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.