Small-RNA asymmetry is directly driven by mammalian Argonautes
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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The A>G substitution is predicted to destabilize the 5′-end of the 5p arm and thus to increase the 5p/3p ratio for both 5p/3p.1 and 5p/3p.2 duplexes.
“The A>G substitution is predicted to destabilize the 5′-end of the 5p arm and thus to increase the 5p/3p ratio for both 5p/3p.1 and 5p/3p.2 duplexes”
From Gain-of-function mutation of microRNA-140 in human skeletal dysplasia (2019) · cites this paper as [20]
Selection of a single mature miRNA strand from a miRNA duplex is determined by a combination of 5′-end nucleotide identity and thermodynamic asymmetry of miRNA duplex termini.
“Selection of a single mature miRNA strand from miRNA duplex is determined by combination of 5′-end nucleotide identity and thermodynamic asymmetry of miRNA duplex termini20.”
From Gain-of-function mutation of microRNA-140 in human skeletal dysplasia (2019) · cites this paper as [20]
Ago2 prefers miRNA strands with 5′-uridine or 5′-adenosine and thermodynamically unstable 5′-ends.
“Ago2 prefers strands with 5′-uridine or 5′-adenosine and thermodynamically unstable 5′-ends.”
From Gain-of-function mutation of microRNA-140 in human skeletal dysplasia (2019) · cites this paper as [20]
Residues within the Argonaute PIWI domain contribute to a phosphate binding tract along the MID/PIWI interface that is proposed to sense thermodynamic stability, while a nucleotide selection loop within the MID domain senses the identity of the 5' nucleotide.
“Residues within the Argonaute PIWI domain contribute to a phosphate binding tract that is formed along the interface of the MID and PIWI domains that is proposed to sense TS while a nucleotide selection loop within the MID domain senses the identity of the 5′ nucleotide (Suzuki et al., 2015; Figure 2a).”
From microRNA strand selection: Unwinding the rules (2021) · cites this paper as Suzuki et al., 2015
The presence and positions of central mismatches do not appear to play a substantial role in miRNA strand selection in human cells.
“miRNA duplexes also contain mismatches; however, the presence and positions of central mismatches do not appear to play a substantial role in miRNA strand selection in human cells (Suzuki et al., 2015).”
From microRNA strand selection: Unwinding the rules (2021) · cites this paper as Suzuki et al., 2015
Argonaute protein appears to play a direct role in miRNA strand selection, as recombinant Argonaute MID domain is sufficient to recognize the preferred 5' nucleotide and to favor thermodynamically unstable ends.
“Perhaps not surprisingly, Argonaute protein appears to play a direct role in miRNA strand selection, as recombinant Argonaute MID domain is sufficient to recognize the preferred 5′ nucleotide and to favor thermodynamically unstable ends (Suzuki et al., 2015).”
From microRNA strand selection: Unwinding the rules (2021) · cites this paper as Suzuki et al., 2015
Deletion of the PAZ domain did not alter miRNA loading, suggesting the PAZ domain is not required for sensing TS.
“however, deletion of the PAZ domain did not alter miRNA loading, suggesting PAZ domain is not required for sensing TS (Suzuki et al., 2015).”
From microRNA strand selection: Unwinding the rules (2021) · cites this paper as Suzuki et al., 2015
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BackgroundInfluentialSystems and Synthetic microRNA Biology: From Biogenesis to Disease Pathogenesis
Matsuyama, H., & Suzuki, H. I. (2019) · International Journal of Molecular Sciences · doi:10.3390/ijms21010132 · Cited by 246 · see citing works
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“Moreover, recent studies indicate that Dicer is not necessary for asymmetric duplex loading in mammals.(45,46) These results clearly show that fly Ago2 possesses a different property from that of fly Ago1 and mammalian AGO proteins.”
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MicroRNA Control of TGF-β Signaling
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Nakanishi, K. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac519 · Cited by 113 · see citing works
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Komatsu, S., Kitai, H., & Suzuki, H. I. (2023) · Cells · doi:10.3390/cells12020306 · Cited by 123 · see citing works
Self-citationGain-of-function mutation of microRNA-140 in human skeletal dysplasia
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“We analyzed miRNA arm ratio as previously described (16) with the following model (Figure 3C): ln (5p/3p) = k G5p−3p + ( N5p − N3p ) , where k and N5p(3p) represent the constant for the relative thermodynamic stability and the constant corresponding to the 5′ end identity, respectively.”
BackgroundMethodThe landscape of miRNA editing in animals and its impact on miRNA biogenesis and targetingPreprint
Li, L., Song, Y., Shi, X., Liu, J. F., Xiong, S., Chen, W., Fu, Q., Huang, Z., Gu, N., & Zhang, R. (2017) · Genome Research · doi:10.1101/gr.224386.117 · Cited by 111 · see citing works
“The selection of the guide strand is an asymmetric, nonrandom process; in many cases, both strands function as mature miRNAs (Yang et al. 2011; Suzuki et al. 2015).”
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Wilson, S. E. (2021) · Experimental Eye Research · doi:10.1016/j.exer.2021.108594 · Cited by 108 · see citing works
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“Strand selection preference is given based on the strand possessing the least stable paired 5′-end [36,37], as well as a 5′-terminal A or U residue [38,39].”
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“This pocket makes specific interactions with a 5′-terminal U or A 396,397 , thereby affecting strand selection: nematode, fly and human miRNAs show a strong bias for a U or an A at the 5′ position of the miRNA guide strand 396,398–401 .”
Structural basis of siRNA recognition by TRBP double‐stranded RNA binding domains
Masliah, G., Maris, C., König, S. L. B., Yulikov, M., Aeschimann, F., Malinowska, A., Mabille, J., Weiler, J., Holla, A., Hunziker, J., et al. (2018) · EMBO Journal · doi:10.15252/embj.201797089 · Cited by 55
“The protein Ago has also been shown to play a role in guide strand selection, and structural studies have unveiled a direct readout of the 5 0 nucleobase by Ago MID domain (Frank et al , 2010; Suzuki et al , 2015).”
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“Pri-miRNA expression vectors were generated according to our previous reports (Suzuki et al. 2011, 2015).”
MethodSelf-citationRoles of MicroRNAs in Disease Biology
Suzuki, H. I. (2023) · JMA Journal · doi:10.31662/jmaj.2023-0009 · Cited by 58 · see citing works
“AGO proteins prefer strands with 5′-uridine or 5′-adenosine and thermodynamically unstable 5′-ends, thereby directly contributing to small RNA asymmetry (7), (8), ((9)).”
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“Also the secondary structure of the miRNA [59] and the identity of its first nucleotide [58, 60, 61] have been described to impact strand selection.”
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“Here, dsRNA forms a complex with Ago2 by direct loading: during this process, the 5’-phosphate-binding pocket formed by the MID and PIWI domains of Ago selectively binds the 5’-terminal phosphate of the nucleotide located at the less stable end of the siRNA duplex via a Dicer-independent pathway [16] (reviewed in [17,…”
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Chen, L., Sun, H., Wang, C., Yang, Y., Zhang, M., & Wong, G. (2018) · eBioMedicine · doi:10.1016/j.ebiom.2018.11.003 · Cited by 47 · see citing works
“For example, AGO1 tends to select the strand with U at the 5′ end as a guide strand [9–11]; while the pocket within AGO2 prefers a pA or pU 5′ terminus [12].”
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Pollutri, D., Gramantieri, L., Bolondi, L., & Fornari, F. (2016) · International Journal of Molecular Sciences · doi:10.3390/ijms17122029 · Cited by 39 · see citing works
“Notably, Suzuki and coworkers demonstrated a cancer-associated variation in asymmetric miRNA biogenesis [50], altering the 5p/3p ratios and functions of certain miRNAs and thus contributing to disease modulation and progression.”
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“Indeed, a very recent report has suggested a direct role for mAGO2 in miR strand choice (30).”
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Naruse, K., Matsuura-Suzuki, E., Watanabe, M., Iwasaki, S., & Tomari, Y. (2018) · RNA: A publication of the RNA Society · doi:10.1261/rna.063891.117 · Cited by 32 · see citing works
“…have previously reported that Dicer and its partner protein TRBP/PACT depletions in mammals do not hinder siRNA-initiated RISC assembly of human Ago proteins (Martinez et al. 2002; Kanellopoulou et al. 2005; Murchison et al. 2005; Betancur and Tomari 2012; Kim et al. 2014; Suzuki et al. 2015).”
Kelly, T. J., Suzuki, H. I., Zamudio, J. R., Suzuki, M., & Sharp, P. (2019) · RNA: A publication of the RNA Society · doi:10.1261/rna.071621.119 · Cited by 31 · see citing works
“Resultant miRNA duplexes are loaded into Argonaute (Ago) proteins (Ago1 – Ago4 in mammals), and one strand is retained in Ago and serves as the mature miRNA strand (Suzuki et al. 2015).”
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Kotani, S., Yoda, A., Kon, A., Kataoka, K., Ochi, Y., Shiozawa, Y., Hirsch, C., Takeda, J., Ueno, H., Yoshizato, T., et al. (2018) · Leukemia · doi:10.1038/s41375-018-0253-3 · Cited by 31 · see citing works
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“The 50 terminal UMP of the 5p strand is known to have an Ago binding affinity approximately 20 times greater than that of the CMP nucleotide on the 50 terminus of the 3p strand (figure 2c) [33].”
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“…parameters of the miRNA duplex: the identity of the 5 ′ -nucleotide and the thermodynamic stability of the two ends of the miRNA duplex. is thermodynamically The strand is favored if its 5 ′ -nucleotide is adenine or uracil and its 5 ′ -end is thermodynamically unstable [34,35]. unstable [34,35].”
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“” Together with AGO, the mature miRNA forms the “RNA-induced silencing complex” (RISC) [51,53].”
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“The miRNAs, in their mature form (20 – 24 nucleotides in length), create ribonucleoprotein complexes (miRNPs) by interacting with a member of the Argonaute family of proteins (Suzuki et al., 2015).”
Systematic characterization of seed overlap microRNA cotargeting associated with lupus pathogenesis
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“2, derived from the 3'- strand of premiR-140, after cleavage of the primary transcript by Drosha and subsequent cleavage of the loop region by the endonuclease, Dicer (Gebert and MacRae 2019; Suzuki et al. 2015).”
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Pinhal, D., Gonçalves, L. D. B., Campos, V. F., & Patton, J. G. (2025) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-025-05663-3 · Cited by 13 · see citing works
“Suzuki et al. [41] proposed the “Twin-drive model” to explain how the terminal thermodynamic asymmetry rule and 5 ′ nucleotide identity rule can coexist and how they superposition.”
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“Ther-modynamic stabilities of miRNA duplex were analyzed according to a previous report [30].”
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“Duplexes are loaded into Argonautes after which one strand is discarded according to the strand selection rule forming mature miRNA-Ago complexes containing just one strand of the small RNA duplex (12,13).”
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Conformational Plasticity in dsRNA-Binding Domains Drives Functional Divergence in RNA Recognition.
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Structural Insights into the Advances and Mechanistic Understanding of Human Dicer
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Labeling microRNA precursors for Dicer assays.
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Sharma, D., Rödelsperger, C., Röseler, W., Witte, H., Werner, M. S., & Sommer, R. J. (2026) · bioRxiv · doi:10.1101/2024.07.19.604269 · Cited by 6
Orbán, T. I. (2023) · Biologia Futura · doi:10.1007/s42977-023-00154-7 · Cited by 10 · see citing works
Diagnostic potential of miR-483 family for IGF-II producing non-islet cell tumor hypoglycemia.
Nagao, M., Fukuda, I., Asai, A., Esguerra, J., Hizuka, N., Eliasson, L., & Sugihara, H. (2020) · European Journal of Endocrinology · doi:10.1530/eje-20-0706 · Cited by 6
Schamberger, A., Várady, G., Fóthi, Á., & Orbán, T. (2021) · Genes · doi:10.3390/genes12071068 · Cited by 5
“Up to our present knowledge, strand selection is mainly determined by thermodynamic characteristics (strands with low thermodynamic stability at their 5′-end are favorable) and the 5′ nucleotide identity (A and U are favorable [28]).”
BackgroundAngart, P. A., Carlson, R. J., Adu-Berchie, K., & Walton, S. (2016) · Nucleic Acid Therapeutics · doi:10.1089/nat.2016.0612 · Cited by 5
“Recently, sensing of siRNA strand thermodynamics was proposed to occur through Ago2 MID domain interactions with the phosphate backbone of the first four nucleotides of the siRNA guide strand [45], sensing the accessibility and single-strand character of weakly base paired termini [45].”
Structural basis for RISC assembly of human Argonaute2
Zhang, H.-Q., Adhav, V. A., Kehling, A. C., Savidge, A., Shen, Z.-F., Fu, T., & Nakanishi, K. (2026) · Molecules and Cells · doi:10.1016/j.molcel.2026.04.029 · Cited by 4 · see citing works
“8,18 However, the relationship between these two rules remains unclear.”
DICER1 hotspot mutation induces 3p microRNA gain of function via Argonaute strand switch
Malagobadan, S., Shi, C.-M., Yang, A., Mondal, I., Baldeh, H., Spain, K., Guiblet, W., & Gu, S. (2025) · Nature Structural & Molecular Biology · doi:10.1038/s41594-025-01671-w · Cited by 4
“The first step is guided by the MID domain of AGO, which enforces strand selection based on its affinity for one strand of the miRNA duplex 16 .”
InfluentialStructural basis for chaperone-guided assembly of RNA-induced silencing complex
Lee, Y.-Y., Jeong, M., Lee, H., Lee, D., Lee, J., Park, J., Kim, V. N., & Roh, S. (2026) · Nature · doi:10.1038/s41586-026-10640-2 · Cited by 3 · see citing works
shRNAI: A deep neural network for the design of highly potent shRNAs
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Reduced glycolysis links resting zone chondrocyte proliferation in the growth platePreprint
Kobayashi, T., Young, C., Zhou, W., & Rhee, E. P. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.01.18.524550 · Cited by 3 · see citing works
Ooki, A., Onodera, S., Saito, A., Oguchi, A., Murakawa, Y., Sakamoto, T., Sueishi, K., Nishii, Y., & Azuma, T. (2020) · Bone · doi:10.1016/j.bone.2020.115582 · Cited by 3
How miRNA Structure of Animals Influences Their Biogenesis
Vorozheykin, P. S., & Titov, I. I. (2020) · Russian Journal of Genetics · doi:10.1134/s1022795420010135 · Cited by 3 · see citing works
AQ-seq: Accurate quantification of microRNAs and their variants
Kim, H., Kim, J., Kim, K., Chang, H., You, K., & Kim, V. (2018) · bioRxiv · doi:10.1101/339606 · Cited by 3
Xiao, W., Zheng, Y., Zhang, H., Xu, B., Zhang, R., & Wu, L. (2026) · Nature Communications · doi:10.1038/s41467-026-68812-7 · Cited by 2 · see citing works
“Subsequent application of a strand selection model to all miRNA, as previously described 67 , and compared with the strand ratio data obtained by 4NBoost and TruSeq showed that 4NBoost data shared a stronger correlation with model predictions (Pearson ’ s coef fi cient = 0.67) than TruSeq (Pearson ’…”
InfluentialMedley, J. C., & Zinovyeva, A. (2026) · RNA Biology · doi:10.1080/15476286.2026.2649359 · Cited by 3 · see citing works
“To investigate whether different methods of calculating the thermodynamic stability of miRNA duplex end affected predictions of miRNA strand selection, we used the twin-drive model [50] to calculate the predicted strand choice of C. elegans miRNAs.”
InfluentialLi, B.-X., Pan, M., Wu, D.-Z., Zheng, Z.-M., & Pei, D. (2025) · Frontiers in Plant Science · doi:10.3389/fpls.2025.1629166 · Cited by 2 · see citing works
“Among these, the MID domain preferentially selected miRNA guide strands that initiated with uridine (U) or adenine (A) at the 5 ′ end (Khvorova et al., 2003; Suzuki et al., 2015).”
The role of noncoding RNAs in pancreatic birth defects
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“…termed RNA-induced silencing complex (RISC), in which the Argonaute family (e.g., Ago2) protein expels one strand of the miRNA duplex, while the remaining strand remains in RISC to form the mature RISC complex (Liu et al., 2004; Rivas et al., 2005; Schwarz et al., 2003; Suzuki et al., 2015).”
BackgroundRNAi and miRNA pathways in mammals I – molecular mechanisms
Svoboda, P. (2020) · Karolinum Press eBooks · doi:10.14712/9788024643724.3 · Cited by 2 · see citing works
“Selection of the guide strand involves multiple sensors – this includes AGO2 strand selection capability (Noland and Doudna, 2013; Suzuki et al., 2015), which is enhanced in complex with Dicer and TARBP2 or PACT.”
BackgroundMicroRNA arm switching regulated by uridylation
Kim, H., Kim, J., Yu, S., Lee, Y.-Y., Park, J., Choi, R., Yoon, S., Kang, S., & Kim, V. (2020) · bioRxiv · doi:10.1101/2020.04.06.027813 · Cited by 2
“Thermodynamically unstable 5′ ends would be more readily accessible to the pocket, leading to the preference for unstable 5′ ends by AGO (Khvorova et al., 2003; Schwarz et al., 2003).”
BackgroundInfluentialGoh, E., & Okamura, K. (2018) · Methods in molecular biology · doi:10.1007/978-1-4939-7339-2_3 · Cited by 2
Roas, K., Kovalski, I., Mouhadeb, O., Aminov, T., Weinstein-Marom, H., & Nissim, L. (2026) · Nature Communications · doi:10.1038/s41467-026-74408-y · Cited by 1
“Furthermore, since long intronic dsRNA could be processed by the miRNA production machinery, it is critical to exclude basalUG, Drosha,and Dicer processing motifs 14,41 .”
Erratum to: How Animal miRNAs Structure Influences Their Biogenesis
Vorozheykin, P. S., & Titov, I. I. (2020) · Russian Journal of Genetics · doi:10.1134/s1022795420220019 · Cited by 2 · see citing works
Yap1 mediates miR-143 regulation of ovarian estrogen synthesis in Larimichthys crocea
Min, Q.-W., Zhou, J.-Y., Yi, X., Li, R.-J., Huang, B., Lv, M.-Q., Gu, X.-J., Zhang, C.-Y., Li, Q., Zhao, N., et al. (2026) · Fish Physiology & Biochemistry · doi:10.1007/s10695-026-01822-w
“This process is precisely governed by two conserved mechanisms: digital sequence recognition based on 5 ′ -end nucleotide identity and analog thermodynamic selection based on the thermodynamic stability of the duplex ends (Frank et al. 2010; Seitz et al. 2011; Suzuki et al. 2015).”
Pérez‐Navarro, Y., López‐Camarillo, C., Flores-García, L. C., Álvarez-Sánchez, M. E., Ramírez, J. A. C., & Salinas-Vera, Y. M. (2026) · International Journal of Molecular Sciences · doi:10.3390/ijms27167363
“First, the intrinsic properties of the duplex, such as the thermodynamic stability of the 5 ′ end and the identity of the 5 ′ terminal nucleotide, influence the strand loading [53,76].”
InfluentialThe roles of nuclear miRNAs in cardiovascular disease
Han, L., Jin, K., & Chen, C. (2026) · Vessel Plus · doi:10.20517/2574-1209.2026.02
DICER1-related tumour predisposition mutations increase 3p-miRNA function and HERVH activity
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Gordon, K., Bellora, N., Witteveldt, J., Wojtus, J. K., Mueller, F., Kases, K. J., Marchante, P. G., Peris, G., Boyle, S., Heras, S. R., et al. (2025) · bioRxiv · doi:10.64898/2025.12.16.694338
3’ Nucleotide Asymmetry Directs miRNA Strand SelectionPreprint
Medley, J. C., Moriya, S. K., Ouyang, H., Crawshaw, H., Zhang, S. Y., Panzade, G., Sydzyik, W. J., Sydzyik, J. T., Bhandari, M., Hammell, C. M., et al. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.10.15.682714
“The twin-drive model postulates that miRNA strand selection can be predicted by modeling the relative effects of 5' nucleotide identity and thermodynamic asymmetry 17 .”
InfluentialIdentification and Functional Characterization of microRNAs in Ichthyosporea
Zhu, H., & Bråte, J. (2025) · F1000Research · doi:10.12688/f1000research.162501.1
Yoon, J. S. J., Baksh, A., Chamberlain, T. C., & Mui, A. (2021) · Preprints.org · doi:10.20944/preprints202111.0272.v1
“Thus, KSRP may be participating in strand selection by bringing specific proteins into the Dicer complex that enhances the thermodynamic stability [47] of the 5p strand binding to the Dicer protein.”
BackgroundZhao, B.-T., & Fan, C. (2019) · bioRxiv · doi:10.1101/817395
“The strand with lower stability base-pairing of the 2–4 nt at the 5’ end preferentially associates with RISC and thus becomes the active miRNA [Frank et al., 2010; Khvorova et al., 2003; Schwarz et al., 2003; Suzuki et al., 2015].”
Background
- Authors
- Suzuki, H. I., Katsura, A., Yasuda, T., Ueno, T., Mano, H., Sugimoto, K., & Miyazono, K.
- Affiliations
- Koch Institute for Integrative Cancer Research At MIT (US)
- The University of Tokyo (JP)
- JR Tokyo General Hospital (JP)
- Corresponding author
- Suzuki, Hiroshi I
- Miyazono, Kohei
- Venue
- Nature Structural & Molecular Biology
- Published
- 2015-06-22
- Volume
- 22
- Issue
- 7
- Pages
- 512-521
- ISSN
- 1545-9985 (ISSN-L)
- 1545-9993
- OpenAlex
- W1542414309
- Cited by
- 108
- References
- 0
- Citations by year
- 2026: 10 · 2025: 13 · 2024: 6 · 2023: 10 · 2022: 13 · 2021: 6 · 2020: 14 · 2019: 9 · 2018: 9 · 2017: 9 · 2016: 5 · 2015: 4
- FWCI
- 4.05
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Structural & Molecular Biology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Japan Society for the Promotion of Science (22112002)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- Argonaute 2, microRNA, RNA silencing, Dicer, R2D2
- MeSH terms
- Animals, Base Sequence, Humans, Models, Molecular, Neoplasms, Nucleic Acid Conformation, Protein Structure, Tertiary, MicroRNAs, Argonaute Proteins
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