Uncovering the mechanisms that recognise a microRNA (miRNA) target is 1 of the biggest
challenges because the Ago–miRNA complex is able to overcome different derogations of
complementarity when binding targets. However, the recently solved crystallographic structure
of Argonaute2 (Ago2) and a high-throughput analysis that used repurposed sequencing
techniques has brought us closer to achieving this goal

Marzec, Marek

PLoS Genetics

Repozytorium Uniwersytetu Śląskiego RE-BUŚ

             Title: New insights into the function of mammalian Argonaute2  Author: Marek Marzec  Citation style: Marzec Marek. (2020). New insights into the function of mammalian Argonaute2. "PLOS Genetics" iss. 11 (2020), art. no. e1009058, doi 10.1371/journal.pgen.1009058 REVIEWNew insights into the function of mammalianArgonaute2Marek MarzecID*University of Silesia in Katowice, Faculty of Natural Sciences, Institute of Biology, Biotechnology andEnvironmental Protection, Katowice, Poland* marek.marzec@us.edu.plAbstractUncovering the mechanisms that recognise a microRNA (miRNA) target is 1 of the biggestchallenges because the Ago–miRNA complex is able to overcome different derogations ofcomplementarity when binding targets. However, the recently solved crystallographic struc-ture of Argonaute2 (Ago2) and a high-throughput analysis that used repurposed sequencingtechniques has brought us closer to achieving this goal.MicroRNAs (miRNAs) are a group of short (approximately 22 nucleotides) noncoding RNAsthat are used by the Argonaute (Ago) proteins as guides for identifying complementary sites inthe messenger RNAs (mRNAs) that are targeted for repression [1]. It has been estimated thatthe expression of more than 50% of the protein-coding genes in humans are under miRNAcontrol [2]. miRNAs are categorised into families that are based on the sequence similarity,and each miRNA is able to target multiple mRNAs. The most conserved part of miRNA islocated at 50-end and is called the seed region. Guide (g) nucleotides from the seed region (g2–g7 or g2–g8) play a primary role in target recognition because more than 80% of the interac-tions between miRNAs and targets occurs via seed pairing [3]. However, for 20% of themiRNA–target interactions, other regions such as the supplementary region (g13–g16) ofmiRNA might be involved in targeting [4,5]. Although the miRNAs that belong to the samefamily share the same seed sequence, they contain divergent 30 regions. A functional redun-dancy has been described for representatives of the same family; however, there are also someexamples in which a single miRNA recognises a specific gene [6].Argonaute2 (Ago2) contains 3 RNA-binding domains: a P-element-induced whimpy testes(PIWI) domain, a middle (MID) domain, and a PIWI/Argonaute/Zwille (PAZ) domain. TheseRNA-binding domains are responsible for distinct tasks in binding of mature miRNAs: MIDdomain is responsible for anchoring the mono-phosphorylated 50-end, and PAZ domainbinds the hydroxylated 30-end [7,8]. Current structural insights into Ago-mediated miRNAtargeting in animals indicate that Ago creates a “seed chamber” for efficient and stable associa-tion with seed-paired target RNAs [8]. In contrast to the previous work which focused on thecrystal structure of seed chamber, Sheu-Gruttadauria and colleagues described the crystalstructure of the human Ago2 protein, which is where the base pairing of both the seed and thesupplementary regions between miRNA and the target occurs [9]. In that situation, Ago2PLOS GENETICSPLOS Genetics | https://doi.org/10.1371/journal.pgen.1009058 November 12, 2020 1 / 4a1111111111a1111111111a1111111111a1111111111a1111111111OPEN ACCESSCitation: Marzec M (2020) New insights into thefunction of mammalian Argonaute2. PLoS Genet16(11): e1009058. https://doi.org/10.1371/journal.pgen.1009058Editor: Jonathan Flint, University of California LosAngeles, UNITED STATESPublished: November 12, 2020Copyright: © 2020 Marek Marzec. This is an openaccess article distributed under the terms of theCreative Commons Attribution License, whichpermits unrestricted use, distribution, andreproduction in any medium, provided the originalauthor and source are credited.Funding: The author received no specific fundingfor this work.Competing interests: The author has declared thatno competing interests exist.opens an additional chamber (supplementary chamber) for up to 4 additional base pairs of themiRNA–target duplex (Fig 1). It was proven that the seed- and supplementary-paired regionsin an mRNA sequence might be separated by 1 to 15 nucleotides, which increased the targetaffinity by more than 10-fold (when a guanine/cytosine (GC)-rich supplementary sequencewas used) compared to seed-only pairing. The largest increase in affinity (more than 20-fold)was observed when 2 to 4 nucleotides created a bridge between a seed and the supplementarysequence in mRNA. Adenine/uracil (AU)-rich sequences had a weaker effect on affinity; how-ever, for a 4-nucleotide bridge, there was a 10-fold increase. Moreover, an analysis of themiRNA isoforms that contain additional 30 nucleotides (30 isomiRs) revealed that longer23-nucleotide 30 isomiRs stabilise the miRNA–target interaction compared to the 21-nucleo-tide 30 isomiRs. Additionally, it was proven that Ago2 can use supplementary interactionswithout any central (g9–g12) pairing, and this situation is energetically favoured for miRNA–target pairing beyond the seed [9]. Moreover, it should be stressed that supplementary pairingplays various roles in increasing the target affinity for different miRNAs, which was alsoproven by Sheu-Gruttadauria and colleagues. Because the seed region can be separated by upto 15 nucleotides from supplementary region, and only 2 GC supplementary base pairs can sig-nificantly increase target affinity, it cannot be excluded that a lot of supplementary sites wereoverlooked in previous studies. According to the miRBase, about 25% of human miRNAs con-tain�3 GC nucleotides within positions g13–g16, which pointed out how important is thefindings of Sheu-Gruttadauria and colleagues [9]. Similar results were obtained by 2 otherresearch groups that used a high-throughput analysis to uncover the rules of miRNA targetrecognition. Becker and colleagues described the methods to measure the binding affinity andFig 1. Overview of the miRNA–target interactions in the human Ago2. Ago, Argonaute2; miRNA, microRNA.https://doi.org/10.1371/journal.pgen.1009058.g001PLOS GENETICSPLOS Genetics | https://doi.org/10.1371/journal.pgen.1009058 November 12, 2020 2 / 4cleavage rates that was examined for mouse Ago2 that had been loaded with 2 different miR-NAs (let-7a (seed-only pairing) and miR-21 (seed and supplementary pairing)) [10]. An Ilu-mina MiSeq platform was used to create a library of approximately 20,000 distinct RNAtargets per miRNA including targets with various mismatches or indels as well as targets thathad been predicted by different algorithms. Binding affinity was measured using a catalyticallyinactive Ago2 mutant (D669A) that had been loaded with fluorescent-labelled miRNA, andthe wild-type Ago2 was used to measure the cleavage rates. The results confirmed that amiRNA–target complementarity in the seed region plays a crucial role in binding affinity. Sim-ilarly, when indels were present in the target sequence complementary to the miRNA seed, thebinding affinity of miRNA to the target RNA was reduced. However, deletions had a strongereffect than insertions at the same target positions. For some miRNAs with a low GC content inthe seed region (such as miR-21), mismatches in the 30 supplementary region have a similareffect in reducing affinity as those in the seed region. In that case, pairing in the supplementaryregion is necessary for a high-affinity binding. It was also proven that insertions in the targetsequence, which is opposite the central region of the miRNA, do not affect binding affinity[10]. This is in agreement with the findings of Sheu-Gruttadauria and colleagues [9], accordingto which the seed- and supplementary-paired regions in an mRNA sequence might be sepa-rated by up to 15 nucleotides. Additionally, binding to the noncanonical target sequences (thatcontain less than 6 contiguous nucleotide matches to the seed region of a miRNA) was ana-lysed. For the majority of the analysed noncanonical targets that had been predicted by differ-ent algorithms, no interactions with miRNA were detected (95% for miR-21 and 89% for let-7a) [10]. However, it might be that the predictions of noncanonical sites by different algo-rithms were incorrect. This explanation is supported by the work of McGeary and colleagues[11]. It was possible to analyse the binding affinity of miRNAs to RNA random sequencesusing an RNA Bind-n-Seq (RBNS). This high-throughput approach enables all known canoni-cal sites to be analysed and new noncanonical sites for 6 different miRNAs (miR-1, let-7a,miR-7, miR-124, miR-155, and lsy-6) to be described. For example, after an analysis of1,398,016 motifs up to 10 nucleotides long, 8 previously unknown sites with a binding affinitysimilar to 6 nucleotide seed sequences were identified for miR-1. Moreover, for other miRNAs,the new noncanonical sites were also characterised. The results not only clearly indicated dif-ferences in the noncanonical site types observed for the analysed miRNAs but also miRNA-specific differences in the affinities of the canonical sites. However, 1 observation was highlyconsistent between the different miRNAs and miRNA site types: the dinucleotides that flankedthe canonical sequence had a great impact (up to 100-fold) on the binding affinity. An analysisof 6 miRNAs revealed that A/U enhanced affinity, G reduced affinity, and C did not affectaffinity. This effect was highly consistent between the different miRNAs and miRNA sitetypes. The results can be explained by the impact of the nucleotides on the stabilisation of theRNA secondary structure; a lower propensity to stabilise the secondary structure results inreduced changes to abolishes binding [10].Last year saw great progress in uncovering the mechanisms that are related to miRNA tar-geting. First of all, different conformations of the human Ago2 have been described, and sec-ondly, high-throughput analysis, which is used to measure the binding affinities of differentRNAs to miRNAs, enables the biochemical basis of miRNA targeting to be described. Whenthe results that had been obtained in only 1 of the described works were used to train a convo-lutional neural network, the prediction of the miRNA repression outperformed all of the previ-ously used algorithms. Using other results, i.e., information that 1 to 15 nucleotides canseparate the seed- and supplementary-pairing regions, will enable the further improvement oftarget prediction algorithms. Using of the methods that have already described and adaptingthe new high-throughput methods will undoubtedly provide further insights into miRNAPLOS GENETICSPLOS Genetics | https://doi.org/10.1371/journal.pgen.1009058 November 12, 2020 3 / 4targeting. Also, new classes of miRNA were recently described, i.e., class of miRNA target sitesthat lack both perfect seed pairing and 30-compensatory pairing and instead have 11 to 12 con-tiguous Watson–Crick pairs to the center of the miRNA [12] or class of miRNA recognitionelements that function exclusively in coding sequence (CDS) regions [13]. Now, 1 of the big-gest challenge will be to find a way for identification of supplementary sites that can signifi-cantly increase target affinity and include them in the target prediction algorithms.References1. Bartel DP. Metazoan MicroRNAs. Cell. 2018; 173:20–51. https://doi.org/10.1016/j.cell.2018.03.006PMID: 295709942. Friedman RC, Farh KK-H, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets ofmicroRNAs. Genome Res. 2009; 19:92–105. https://doi.org/10.1101/gr.082701.108 PMID: 189554343. Grosswendt S, Filipchyk A, Manzano M, Klironomos F, Schilling M, Herzog M, et al. Unambiguous iden-tification of miRNA:target site interactions by different types of ligation reactions. Mol Cell. 2014;54:1042–1054. https://doi.org/10.1016/j.molcel.2014.03.049 PMID: 248575504. Broughton JP, Lovci MT, Huang JL, Yeo GW, Pasquinelli AE. Pairing beyond the seed supports Micro-RNA targeting specificity. Mol Cell. 2016; 64:320–333. https://doi.org/10.1016/j.molcel.2016.09.004PMID: 277206465. Salomon WE, Jolly SM, Moore MJ, Zamore PD, Serebrov V. Single-molecule imaging reveals that argo-naute reshapes the binding properties of its nucleic acid guides. Cell. 2015; 162:84–95. https://doi.org/10.1016/j.cell.2015.06.029 PMID: 261405926. Wahlquist C, Jeong D, Rojas-Muñoz A, Kho C, Lee A, Mitsuyama S, et al. Inhibition of miR-25 improvescardiac contractility in the failing heart. Nature. 2014; 508:531–535. https://doi.org/10.1038/nature13073 PMID: 246706617. Schirle NT, MacRae IJ. The crystal structure of human Argonaute2. Science. 2012; 336:1037–1040.https://doi.org/10.1126/science.1221551 PMID: 225395518. Schirle NT, Sheu-Gruttadauria J, MacRae IJ. Structural basis for microRNA targeting. Science. 2014;346:608–613. https://doi.org/10.1126/science.1258040 PMID: 253599689. Sheu-Gruttadauria J, Xiao Y, Gebert LF, MacRae IJ. Beyond the seed: structural basis for supplemen-tary microRNA targeting by human Argonaute2. EMBO J. 2019; 38:e101153. https://doi.org/10.15252/embj.2018101153 PMID: 3126860810. Becker WR, Ober-Reynolds B, Jouravleva K, Jolly SM, Zamore PD, Greenleaf WJ. High-throughputanalysis reveals rules for target RNA binding and cleavage by AGO2. Mol Cell. 2019; 75:741–755.e11.https://doi.org/10.1016/j.molcel.2019.06.012 PMID: 3132444911. McGeary SE, Lin KS, Shi CY, Pham TM, Bisaria N, Kelley GM, et al. The biochemical basis of micro-RNA targeting efficacy. Science. 2019; 366. https://doi.org/10.1126/science.aav1741 PMID: 3180669812. Shin C, Nam J-W, Farh KK-H, Chiang HR, Shkumatava A, Bartel DP. Expanding the microRNA target-ing code: functional sites with centered pairing. Mol Cell. 2010; 38:789–802. https://doi.org/10.1016/j.molcel.2010.06.005 PMID: 2062095213. Zhang K, Zhang X, Cai Z, Zhou J, Cao R, Zhao Y, et al. A novel class of microRNA-recognition elementsthat function only within open reading frames. Nat Struct Mol Biol. 2018; 25:1019–1027. https://doi.org/10.1038/s41594-018-0136-3 PMID: 30297778PLOS GENETICSPLOS Genetics | https://doi.org/10.1371/journal.pgen.1009058 November 12, 2020 4 / 4

New insights into the function of mammalian Argonaute2

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New insights into the function of mammalian Argonaute2

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