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Small-RNA asymmetry is directly driven by mammalian Argonautes

Nature Structural & Molecular Biology volume 22, pages 512–521 (2015)Cite this article

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Abstract

Asymmetric selection of single-stranded guide RNAs from double-stranded RNA precursors is crucial in RNA silencing–mediated gene regulation. However, the precise mechanisms of small-RNA asymmetry are unclear, especially because asymmetric selection can still occur when the putative asymmetry sensors Drosophila R2D2 and mammalian Dicer are depleted. Here we report a direct contribution of mammalian Argonaute 2 (Ago2) to microRNA (miRNA) asymmetry. Ago2 selects strands with 5′-uridine or 5′-adenosine and thermodynamically unstable 5′ ends in parallel through its two sensor regions, which contact the 5′ nucleobases and 5′-phosphates of prospective guide strands. Hence, miRNA asymmetry shows superposed patterns reflecting 5′-end nucleotide identity ('digital' pattern) and thermodynamic stability ('analog' pattern). Furthermore, we demonstrate that cancer-associated miRNA variations reprogram asymmetric selection. Finally, our study presents a model of this universal principle, to aid in comprehensive understanding of miRNA function and development of new RNA-silencing therapies in precision medicine.

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Figure 1: General nature of microRNA asymmetry in mammals.
Figure 2: Asymmetry profiles for four mammalian Argonautes.
Figure 3: The PAZ domain of Ago2 is dispensable for asymmetry sensing.
Figure 4: Twin-drive model indicating the direct roles of the nucleotide-selection loop and phosphate-binding pocket in Ago2.
Figure 5: Effects of G-drive and N-drive mutations on TS and 5′-nt rules.
Figure 6: Global modeling of asymmetry kinetics.
Figure 7: Effects of MID-domain mutations on global miRNA asymmetry.
Figure 8: Cancer-associated variations in miRNA genes alter asymmetric miRNA biogenesis.

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Acknowledgements

We thank T. Yamazaki, H. Mihira, A. Mizutani, T. Kosaka, A. Chiu and members of the K.M. and P.A. Sharp laboratories for discussions and assistance; X. Wang (Northwestern University) and T.J. Kelly and J.R. Zamudio (Massachusetts Institute of Technology) for Ago-knockout ES cells; and H. Siomi and P.A. Sharp for critical reading. H.I.S. is supported by a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad. This work was supported by Grants-in-Aid for Young Scientists (A) (24689018, H.I.S.) and Grants-in-Aid for Scientific Research on Innovative Areas (23112702, H.I.S.; 22112002, K.M.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Strategic Basic Research Program from the Japan Science and Technology Agency (H.M. and K.M.); the Cell Science Research Foundation (H.I.S.); the Skoltech Institute (courtesy of P.A. Sharp). The results shown here are in part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/. Information about TCGA and the investigators and institutions that constitute the TCGA research network can be found at http://cancergenome.nih.gov/.

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  1. Hiroshi I Suzuki and Kohei Miyazono: These authors jointly supervised this work.

Authors and Affiliations

  1. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Hiroshi I Suzuki

  2. Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Hiroshi I Suzuki, Akihiro Katsura & Kohei Miyazono

  3. Department of Cellular Signaling, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Takahiko Yasuda, Toshihide Ueno & Hiroyuki Mano

  4. Department of Hematology and Oncology, JR Tokyo General Hospital, Tokyo, Japan

    Koichi Sugimoto

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Contributions

H.I.S. conceived and designed the hypothesis and research, performed the experiments and analyses and wrote the paper; A.K., T.Y., T.U. and H.M. performed small-RNA sequencing; K.S. and K.M. discussed the results and wrote the paper.

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Correspondence to Hiroshi I Suzuki or Kohei Miyazono.

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H.I.S. and K.M. have submitted a patent related to this work to the US Patent and Trademark Office under application no. 62/168051.

Integrated supplementary information

Supplementary Figure 1 Asymmetrical function of 5p and 3p arms of miR-181 and miR-30 families and miR-142 and miR-154.

(a) Control experiments using miRNA sensor vector and empty pri-miRNA overexpression vector in HEK 293T cells.

(b) Luciferase reporter assays showing activities of 5p and 3p arms of mouse miR-181, -30 families and miR-142, -154. Samples are from HEK 293T cells transfected with miRNA sensor vector and pri-miRNA expression vector of indicated miRNAs (48 hr after transfection). An inhibitory effect of each arm is summarized in Fig. 1b.

(c) Luciferase reporter assays in HepG2 and HCT116 cells showing activities of 5p and 3p arms of mouse miR-181, -30 families and miR-142, -154.

(d) Luciferase reporter assays in HEK 293T cells showing activities of 5p and 3p arms of human miR-30 family.

(e) Effects of mutation in a catalytic residue of Ago2 on 5p/3p arm ratios. miRNA reads of 5p and 3p arms in wild-type and Ago2 D597A mutant fetal livers (Cheloufi, S. et al., Nature. 465, 584-9, 2010) are plotted. This analysis suggests that 5p arm bias is not associated with the slicer activity of Ago2.

(f) Luciferase reporter assays showing examples of 3p arm-dominant miRNAs. Samples are from HEK 293T cells transfected with miRNA sensor vector and pri-miRNA expression vector of indicated miRNAs (48 hr after transfection).

Error bars, s.d. (n = 3 technical replicates).

Supplementary Figure 2 Validation of asymmetry codes by a lattice of pri-miRNAs.

(a) Control experiments using miRNA sensor vector and empty pri-miRNA overexpression vector in HEK 293T cells.

(b) Luciferase reporter assays showing activities of 5p and 3p arms of a lattice of pri-miRNAs. Samples are from HEK 293T cells transfected with miRNA sensor vector and pri-miRNA expression vector of indicated miRNAs (48 hr after transfection). A relative inhibitory effect of each arm is summarized in Fig. 1f.

Error bars, s.d. (n = 3 technical replicates).

Supplementary Figure 3 The PAZ domain is dispensable for asymmetric selection.

(a) Overall structure of Ago2 with single mature miRNA strand (4F3T) (Elkayam, E. et al., Cell. 150, 100-10, 2012).

(b) A stochastic mode of initial recognition of RNA duplexes by either MID or PAZ domain.

(c) Sequences and thermodynamic stability profiles of symmetrical RNA duplexes used in Fig. 3 and Fig. 4. The terminal sequences of these duplexes were designed to show similar thermodynamic stability profiles. 5′-nt identity and thermodynamic stability profiles of these symmetrical 2nt overhang RNA duplexes covered four nucleotides and 0-2 nt mismatches (MM 0-2). Three end nucleotides and mutated nucleotides are highlighted in red and blue, respectively.

(d) Preparation of GST-PAZ and GST-MID protein fragments.

(e) Competitor experiments. Radiolabelled RNA duplexes, GST-PAZ and unlabeled competitors were incubated, UV photo-crosslinked and analyzed by PAGE and autoradiography. Quantification result is shown in the bottom.

(f) Immunoblot analysis showing expression of Ago2 mutants (F294A, L339A, and R277A-R280A-K335A) in HEK 293T cells.

Supplementary Figure 4 Asymmetric selection is not affected by mutation in the RTTPQT motif.

(a) Asymmetry sensing by MID domain. UV photo-crosslinking experiments showing binding properties of MID domain for symmetrical RNA duplexes. Radiolabelled RNA duplexes, GST-MID and unlabeled competitors were incubated, UV photo-crosslinked and analyzed by PAGE and autoradiography. Quantification result is shown in right.

(b) Prediction of RNA-binding motif in Ago2 MID domain by BindN program (Wang, L. et al., Nucleic Acids Res. 34, W243-8, 2006). The predicted RNA-binding residues are labeled with ‘+’.

(c) Structural comparison of human Ago2 with miR-20a (4F3T) and T. thermophilus Ago with guide DNA and target RNA duplex (3F73) (Elkayam, E. et al., Cell. 150, 100-10, 2012; Wang, Y. et al., Nature. 456, 921-6, 2008). Nucleotide selection loop and 554-RTTPQT-559 motif are highlighted in blue and orange, respectively.

(d) UV photo-crosslinking experiments showing binding properties of RTTPQT motif mutant for symmetrical RNA duplexes.

(e) Immunoblot analysis showing expression of Ago2 RTTPQT mutant in HEK 293T cells.

(f) RIP-qRT-PCR analysis showing effects of mutation in RTTPQT motif of Ago2 on asymmetry sensing for type 1 miR-154 (both arm type). Samples are from HEK 293T cells transfected with Ago2 mutant expression vector and pri-miRNA expression vector. Error bars, s.d. (n = 3 technical replicates).

Supplementary Figure 5 Recognition of TS and 5′-nt rules by conserved amino acid residues in the MID domain.

(a) Proposed mechanisms for detection of thermodynamic stability of RNA duplex ends by phosphate-binding pocket (tract).

(b) Sequences of MID and PIWI domains including G-drive (phosphate-binding pocket (tract), pink) and N-drive (nucleotide selection loop, blue) in Ago1-4. N, P, and S indicate nucleobase, phosphate, and ribose, respectively.

(c) Immunoblot analysis showing expression of G-drive and N-drive mutants of Ago2 in HEK 293T cells.

(d) RIP-qRT-PCR analysis showing effects of mutations in PAZ domain and MID domain on asymmetry sensing for type 1 miR-216a (5p arm type). Samples are from HEK 293T cells transfected with Ago2 mutant expression vector and pri-miRNA expression vector. 5p arm of type 1 miR-216a is predominantly selected by both TS rule and 5′-nt rule. Two G-drive mutants (K566A and R792A C793A) and two N-drive mutants (524-GKT-526 to GKGT and 523-PGKTP-527 to AAAAA) reduced 5p/3p ratios. Error bars, s.d. (n = 3 technical replicates).

(e) Preparation of GST-MID mutants.

Supplementary Figure 6 Role of 5′-nt identity in RISC stabilization.

(a) Design of siRNA duplexes used in Fig. 5a. Sequences are shown in Supplementary Table 4.

(b) A lattice of pri-miRNAs used for RIP-qRT-PCR analysis of Ago2 mutants in Fig. 5b.

(c) Predicted pattern of altered miRNA asymmetry caused by mutations in G-drive and N-drive.

(d) Luciferase reporter assays showing role of 5′-nt identity in initial RNA loading and RISC dissociation. Samples are from HEK 293T cells were transfected with miRNA sensor vector and pri-miRNA expression vector (mouse pri-miR-216a (5p arm type) with 5p arms with different 5′-nts). To evaluate the role of 5′-nt identity in RISC stabilization and/or dissociation, 24 hr after transfection, cells were treated with Actinomycin D (1 mg/mL) and subjected to luciferase reporter assay.

(e) RIP-qRT-PCR analysis showing role of 5′-nt identity in RISC stabilization. Samples were from HEK 293T cells transfected with pcDNA3-Flag-Ago2 and pri-miRNA expression vector (pri-miR-216a (5p arm type) with 5p arms with different 5′-nts) and treated with/without Actinomycin D (1 mg/mL, 24 hr).

(f) Prediction of inhibitory activities of both arms of diverse miRNAs (46 pri-miRNAs, 92 strands) shown in Fig. 6b by equation (iii). FR indicates fold repression. Exponential curves were fitted.

Error bars, s.d. (n = 3 technical replicates).

Supplementary Figure 7 Effects of MID-domain mutation on asymmetry pattern.

(a) Immunoblot analysis showing showing expression of Flag-tagged WT or mutant Ago2 in mouse Ago-knockout ES cells.　

(b) Comparison of qRT-PCR analysis and small RNA sequencing for small RNAs interacting with Flag-tagged WT Ago2 or Ago2 mutants in mouse Ago-knockout ES cells shown in (a). The results for miR-140 (both arm type), in which TS and 5′-nt rules favor 5p and 3p arms, respectively, are shown.

(c) Small RNA sequencing analysis showing asymmetry patterns of small RNAs interacting with Flag-tagged WT Ago2 or Ago2 mutants (G-drive mutants (Q548A and R792A C793A) and N-drive mutants (524-GKT-526 to GKGT and 523-PGKTP-527 to AAAAA)) in mouse Ago-knockout ES cells. MiRNAs were classified into 8 groups according to 5′-nt identity, as in Fig. 6a. y axis indicates log2 strand ratio.

(d) Univariate linear regression analysis using ΔΔG for miRNA asymmetry patterns. Correlation coefficients between strand ratio and ΔΔG were summarized. Mutation of K566 in the phosphate-binding pocket strongly attenuated global dependence on TS rule, in consistent with the most remarkable effect of this mutation on TS rule (Fig. 4 and Fig. 5). As for other phosphate-binding pocket mutants, R792A C793A mutant showed a weak but similar trend to reduce dependence on TS rule, and Q548A mutant showed a complex pattern, but reduced dependence on TS rule in several groups.

Supplementary Figure 8 Effects of cancer-associated variations on asymmetric activities of miR-142 and miR-146a.

(a) Details of miR-142 asymmetry. According to genome-wide high-throughput miRNA profiling (Chiang, H.R. et al., Genes Dev. 24, 992-1009, 2010; Griffiths-Jones, S. et al., Nucleic Acids Res. 36, D154-8, 2008; Park, J.E. et al., Nature. 475, 201-5, 2011), processing of pre-miR-142 shows considerable heterogeneity. Application of our model to each miRNA duplex can explain potent activities of both arms of miR-142 precursors. In this figure, we adopted conventional 22nt length processing to infer identities of each miRNA duplex from the results of deep sequencing.

(b) Proposed roles of variations in miR-142 and miR-146a duplexes on the asymmetric biogenesis.

(c) Effects of miR-142 mutation on expression levels of miR-142 and miR-142 targets in TCGA AML data. In AML database, two 3p variants and two 5p variants dominate miR-142 pool, suggesting that the processing of miR-142 shows a complex pattern of miRNA duplexes in this cell type. *P < 0.05 by two-tailed Mann-Whitney test.

(d and e) Impacts of genotypes (GG, CG, and CC) of miR-146a SNP (rs2910164) on patient survival in liver hepatocellular carcinoma (d) and skin cutaneous melanoma (e) in the TCGA database. The P values were calculated with two-tailed log-rank test.

(f and g) Effects of miR-146a genotypes (GG, CG, and CC) on miR-146a 5p/3p ratios in liver hepatocellular carcinoma (f) and skin cutaneous melanoma (g) in the TCGA database. Boxes represent the median and interquartile range (IQR). Error bars, 1.5X IQR. **P < 0.01; ***P < 0.001 by two-tailed Mann-Whitney test.

(h) GSEA showing upregulation of miR-146a-5p targets in the HCC patients with miR-146a CC genotype. In melanoma, the significant alteration of miR-146a-5p targets by miR-146a genotype was not observed.

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Suzuki, H., Katsura, A., Yasuda, T. et al. Small-RNA asymmetry is directly driven by mammalian Argonautes. Nat Struct Mol Biol 22, 512–521 (2015). https://doi.org/10.1038/nsmb.3050

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