Recent methods for the analysis of miRNA pathways

Micro-RNAs (miRNAs) are small, single-stranded, non-coding ribonucleic acids (RNAs). First discovered in 1993 in C. elegans, they are now known to take part in regulating gene expression.

In recent years, scientists uncovered fundamental information on miRNA machinery's structural and molecular dynamics. For example, how the transcriptome selects miRNA substrates and targets and the regulation of miRNA biogenesis and turnover. Recent technological advances include massive parallel assays, cryogenic electron microscopy, single-molecule imaging, and CRISPR-Cas9 screening. These technological advances contributed heavily to the latest discoveries. Scientists now know that miRNAs regulate gene expression and control cell development and metabolism.

Scientists estimate that humans express more than 2,000 mature miRNAs; some appear to be associated with cancer, cardiovascular, inflammatory diseases, and a broad range of neurodevelopmental and autoimmune disorders. Furthermore, miRNAs are considered important biomarkers for disease assessment.

miRNAs are secreted to extracellular fluids, bound to specific proteins, or are part of extracellular vesicles. Through RNA sequencing, next-generation RNA sequencing enables their detection in liquid biopsies and biological fluids, including plasma and serum, saliva, cerebrospinal fluid, and breast milk.

Technologies recently applied for the analysis of miRNA pathways

High-throughput substrate screening

This screening method is similar to classical in vitro selection assays. The method often involves parallel processing of a large pool of endogenous substrates or a library of designed or randomized variants. Deep sequencing is used as a read-out, to infer functionally relevant features for processing. This approach allows characterization of pri-miRNA and pre-miRNA features (Auyeung et al. 2013; Fang et al. 2015; Li et al. 2020; Lee et al. 2023; Nguyen et al. 2022).


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). This study is the first to utilize massively parallel substrate assays to reveal motifs involved in pri-miRNA processing.

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).

Lee, Y. Y., Kim, H. & Kim, V. N. Sequence determinant of small RNA production by DICER. Nature 615, 323–330 (2023).

Li, S., Nguyen, T. D., Nguyen, T. L. & Nguyen, T. A. Mismatched and wobble base pairs govern primary microRNA processing by human microprocessor. Nat. Commun. 11, 1926 (2020).

Nguyen, T. D., Trinh, T. A., Bao, S. & Nguyen, T. A. Secondary structure RNA elements control the cleavage activity of DICER. Nat. Commun. 13, 2138 (2022).

Cryogenic electron microscopy (cryo-EM)

Cryo-EM uses an electron beam for imaging specimens under cryogenic conditions. Data processing of electron microscopy densities allows the assignment of atomic coordinates. Cryo-EM is suitable for studying proteins or complexes of large molecular weight molecules. Relatively small samples are needed, and the method can capture multiple conformational states in a single experiment without crystallization.

Cryo-EM allowed the study of the Mammalian Drosha/DGCR8 complex (Jin et al. 2020; Partin et al. 2020), Mammalian Dicer complexes (Liu et al 2018; Lee et al. 2023; Zapletal et al. 2022), Drosophila Dicer complexes Dicer-1 (Jouravleva et al. 2022) and Dicer-2 (Yamaguchi et al 2022; Su et al 2022), and Arabidopsis Dicer complexes: DCL1 (Wei et al 2021) and DCL3 (Wang et al. 2021).


Jin, W., Wang, J., Liu, C. P., Wang, H. W. & Xu, R. M. Structural basis for pri-miRNA recognition by Drosha. Mol. Cell 78, 423–433.e5 (2020).

Jouravleva, K. et al. Structural basis of microRNA biogenesis by Dicer-1 and its partner protein Loqs-PB. Mol. Cell 82, 4049–4063.e6 (2022).

Lee, Y. Y., Lee, H., Kim, H., Kim, V. N. & Roh, S. H. Structure of the human DICER–premiRNA complex in a dicing state. Nature 615, 331–338 (2023).

Liu, Z. et al. Cryo-EM structure of human dicer and its complexes with a pre-miRNA substrate. Cell 173, 1191–1203.e12 (2018).

Partin, A. C. et al. Cryo-EM structures of human Drosha and DGCR8 in complex with primary microRNA. Mol. Cell 78, 411–422 e414 (2020). Together with Jin et al. (2020), this work is the first cryo-EM study of Microprocessor structures.

Su, S. et al. Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD. Nature 607, 399–406 (2022).

Wang, Q. et al. Mechanism of siRNA production by a plant Dicer–RNA complex in dicing-competent conformation. Science 374, 1152–1157 (2021).

Wei, X. et al. Structural basis of microRNA processing by Dicer-like 1. Nat. Plants 7, 1389–1396 (2021).

Yamaguchi, S. et al. Structure of the Dicer-2–R2D2 heterodimer bound to a small RNA duplex. Nature 607, 393–398 (2022).

Zapletal, D. et al. Structural and functional basis of mammalian microRNA biogenesis by Dicer. Mol. Cell 82, 4064–4079.e13 (2022). Together with Lee et al. (Nature, 2023), this work reports new cryo-EM structures for active mammalian Dicer complex.

Single-molecule assay

Single-molecule assays allow the analysis of real-time dynamics of biological reactions. In-vitro assays usually need purified materials. Observation times depend on reaction kinetics, photostability, and the lifetime of fluorophores. However, some reaction times can be as short as microseconds. The detection of single molecules is generally limited by diffraction (~200–300 nm spatial resolution). For example, the single-molecule Förster resonance energy transfer (smFRET) operates at 1 to 10 nm. smFRET allows resolving intermolecular and intramolecular motions. Fluorescently tagged molecules utilizing multimeric tags or scaffolds for enhanced detection enable single-molecule imaging in living cells.

Single-molecule assays allowed for studying the dynamic interplay of human Dicer and TRBP (Fareh et al. 2016), in-vitro target search and interrogation of human Ago2/RISC complexes (Solomon et al. 2015; Yao et al. 2015; Chandradoss et al. 2015; Cui et al. 2019; Willkomm et al. 2022), live cell imaging of targeting and regulation by human Ago2 (Ruijtenberg et al. 2020; Cialek et al., 2022; Kobayashi & Singer, 2022), and the assembly and dynamics of Drosophila AGO2/RISC complexes (Iwasaki et al. 2015; Tsuboyama et al. 2018).


Chandradoss, S. D., Schirle, N. T., Szczepaniak, M., MacRae, I. J. & Joo, C. A dynamic search process underlies microRNA targeting. Cell 162, 96–107 (2015). This study uses the distance sensing capability of smFRET to visualize strategies of target interrogation by RISC.

Cialek, C. A. et al. Imaging translational control by Argonaute with single-molecule resolution in live cells. Nat. Commun. 13, 3345 (2022).

Cui, T. J. et al. Argonaute bypasses cellular obstacles without hindrance during target search. Nat. Commun. 10, 4390 (2019).

Fareh, M. et al. TRBP ensures e%icient Dicer processing of precursor microRNA in RNA-crowded environments. Nat. Commun. 7, 13694 (2016).

Iwasaki, S. et al. Defining fundamental steps in the assembly of the Drosophila RNAi enzyme complex. Nature 521, 533–536 (2015).

Kobayashi, H. & Singer, R. H. Single-molecule imaging of microRNA-mediated gene silencing in cells. Nat. Commun. 13, 1435 (2022).

Ruijtenberg, S. et al. mRNA structural dynamics shape Argonaute–target interactions. Nat. Struct. Mol. Biol. 27, 790–801 (2020).

Salomon, W. E., Jolly, S. M., Moore, M. J., Zamore, P. D. & Serebrov, V. Single-molecule imaging reveals that argonaute reshapes the binding properties of its nucleic acid guides. Cell 162, 84–95 (2015).

Tsuboyama, K., Tadakuma, H. & Tomari, Y. Conformational activation of argonaute by distinct yet coordinated actions of the Hsp70 and Hsp90 chaperone systems. Mol. Cell 70, 722–729.e4 (2018).

Willkomm, S. et al. Single-molecule FRET uncovers hidden conformations and dynamics of human Argonaute 2. Nat. Commun. 13, 3825 (2022). This study uses single-molecule fluorescence to dissect internal motions within human Ago2 during transitions from guide RNA binding to target capture.

Yao, C., Sasaki, H. M., Ueda, T., Tomari, Y. & Tadakuma, H. Single-molecule analysis of the target cleavage reaction by the Drosophila RNAi enzyme complex. Mol. Cell 59, 125–132 (2015).

RNA bind-n-seq (RBNS)

This method yields relative quantitative binding affinities of an RNA-binding protein (RBP) across a library of target sites. Typically, a purified RBP is incubated with a randomized pool of RNAs. Co-purified RBP targets are then analyzed by deep sequencing to identify their features.

The method was recently applied to study Ago2–miRNA complex binding affinity to target RNAs (McGeary et al. 2019 and 2022).


McGeary, S. E. et al. The biochemical basis of microRNA targeting efficacy. Science https://doi.org/10.1126/science.aav1741 (2019). This paper reveals broad features of miRNA–target interactions using RBNS.

 McGeary, S. E., Bisaria, N., Pham, T. M., Wang, P. Y. & Bartel, D. P. microRNA 3′-compensatory pairing occurs through two binding modes, with affinity shaped by nucleotide identity and position. eLife https://doi.org/10.7554/eLife.69803 (2022).

CRISPR–Cas9 screening

Genetic mutagenesis screens enable the identification of factors involved in a cellular process of interest. miRNA screening often incorporates a specific reporter as a functional read-out, allowing cell sorting before deep sequencing enriched or depleted guide RNAs.

ERH and SAFB2 in miRNA cluster assistance ZSWIM8 in target-directed miRNA degradation (TDMD) (Shie et al. 2020; Han et al. 2020).


Shi, C. Y. et al. The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation. Science https://doi.org/10.1126/science.abc9359 (2020).

Han, J. et al. A ubiquitin ligase mediates target-directed microRNA decay independently of tailing and trimming. Science https://doi.org/10.1126/science.abc9546 (2020). Together with Shi et al. (2020), this work reveals the regulatory mechanism of TDMD, via ZSWIM8-mediated Argonaute protein degradation.

Legend: Ago, Argonaute; miRNA, microRNA; pre-miRNA, precursor miRNA; pri-miRNA, primary miRNA; RISC, RNA-induced silencing complex; TRBP, transactivation responsive RNA-binding protein.

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