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Sequence determinant of small RNA production by DICER

Nature volume 615, pages 323–330 (2023)Cite this article

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Abstract

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.

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Fig. 1: Massively parallel assay identifies the GYM motif.
Fig. 2: The GYM motif recognized by the dsRBD enhances pre-miRNA processing and determines the cleavage site.
Fig. 3: Endogenous miRNAs are regulated by the dsRBD in a GYM-motif-dependent fashion.
Fig. 4: The GYM motif enhances gene silencing potency.

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Data availability

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).

Code availability

Custom analysis codes are available at https://github.com/haedongkim615/dicer_gym_motif.

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Acknowledgements

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.).

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Author notes

  1. These authors contributed equally: Young-Yoon Lee, Haedong Kim

Authors and Affiliations

  1. Center for RNA Research, Institute for Basic Science (IBS), Seoul, Republic of Korea

    Young-Yoon Lee, Haedong Kim & V. Narry Kim

  2. School of Biological Sciences, Seoul National University, Seoul, Republic of Korea

    Young-Yoon Lee, Haedong Kim & V. Narry Kim

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Contributions

Y.-Y.L. carried out structural modelling and protein purification. Y.-Y.L. and H.K. carried out biochemical and cell biological experiments. H.K. carried out bioinformatic analyses. Y.-Y.L., H.K. and V.N.K. designed the study and wrote the paper.

Corresponding author

Correspondence to V. Narry Kim.

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Competing interests

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.

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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 information

Supplementary Fig. 1

Uncropped gels.

Reporting Summary

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.

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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

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