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  1. Author Response

    Reviewer #1 (Public Review):

    Canonical miRNA-targeting involves pairing between the miRNA seed region (nucleotides 2-7, counting from the miRNA 5' end) and a target mRNA. Pairing downstream of the seed can also influence target recognition, and in some cases 3' pairing can compensate for imperfect seed complementarity. In this study, McGeary et al. investigated the features of such miRNA 3' compensatory sites in a high-throughput manner by adapting the RNA bind-n-seq (RBNS) method used previously to characterize binding of purified Argonaute2-miRNA complexes to a random pool of target RNAs.

    Strengths To focus on 3'-compensatory sites, which are rare in random libraries, the authors designed libraries of RNAs containing imperfect seed complementarity followed by 25 nucleotides of random sequence. This approach allowed investigation of a range of 3' pairing possibilities far more extensive than any previous work. Results provide several unexpected findings. Contrary to the prevailing model that miRNA nucleotides 13-16 are most efficacious for 3' pairing, the authors found the optimal position varies between miRNA sequences and is often shifted to include G nucleotides in the miRNA. The number of unpaired nucleotides bridging seed and 3'-paired regions is also a factor-certain let-7 sites preferring an offset of +4 target nucleotides, indicating a high affinity target-binding mode previously unknown. Additionally, the contribution of miRNA 3' pairing correlates poorly with predictions from nearest-neighbor parameters. Overall findings greatly expand insights into miRNA 3' pairing and provide metrics for improving target prediction.

    Weaknesses Conclusions are drawn entirely from RBNS data sets, leading to a few limitations. Affinity measurements are limited to relative KD values, making comparison to other work in the field indirect and potentially problematic. For example, let-7 target sites in lin-41 have 11-19 3' compensatory pairing, +1nt offset, which (based on Fig. 2B and 2C) has a greater relative KD than the let-7 8mer canonical site. However, a recent result showed an in vivo lin-41 reporter with two 8mer sites is less repressed than same reporter bearing the wild type 3'-compensatory sites (1). In the absence of KD values and/or cellular repression data for these specific sites the noted differences are difficult to reconcile. Additionally, analyses assume miRNA-target complementarity directly correlates with physical pairing between miRNA and target. However, because physical pairing occurs within the Argonaute2-miRNA complex, this may not always be the case

    1. Duan, Ye, Isana Veksler-Lublinsky, and Victor Ambros. "Critical contribution of 3'non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA." bioRxiv (2021).

    Although conclusions from our affinity measurements agree with those of the Duan et al. (2021) bioRxiv submission with respect to the relative importance of pairing to let-7a nucleotides 11 and 12 compared to that of pairing to nucleotides 18 and 19 (Figure 7—figure supplement 1D), some of the more detailed conclusions do indeed differ. These differences might be due to our measurement of site affinity rather than repression (as mentioned by the referee). Alternatively, they might be due to either differences between flanking sequences of the sites or differences between human and C. elegans systems. With respect to whether two 8mer sites without 3′ pairing are as effective as the endogenous lin-41 sites, the results of Duan et al. (2021) are another step removed from ours because they measure mutant phenotypes rather than lin-41 repression. Nonetheless, as the reviewer suggests, the strain in which lin-41 seed pairing is restored and 3′ pairing is disrupted has phenotypes consistent with insufficient repression by let-7, which would not be expected if relative affinity of 8mer sites matched the affinities of the endogenous lin-41 sites.

    To investigate the relationship between 3′-pairing affinity in vitro and repression in cells, we performed a massively parallel reporter assay and have included these new results in our revision (Figure 3). Overall, we found that affinity in vitro corresponded well to repression in cells (r2 = 0.71). With respect to the example mentioned by the referee, we observed that dual 8mer sites imparted repression similar to that of the lin-41 sites (Figure 3B, bottom). We also observed some benefit of the endogenous flanking-sequence context of the lin-41 sites, but this benefit extended to the other sites as well, including to the 8mer sites (Figure 3—figure supplement 2). Thus, differences between our results an those of Duan et al. (2021) appear to be primarily attributable to differences between our two systems—perhaps a difference between human and C. elegans.

    With respect to the referee’s last point, we use “pairs” and “pairing” as synonymous with “Watson–Crick matches” and “complementarity.” In our revision, we have clarified that our use of “pairing” refers to potential pairing, not physical pairing.

    Reviewer #3 (Public Review):

    The Bartel Lab tackles the elusive role of the 3' part of miRNAs to contribute to the binding of target RNAs. In short, the presented data lead to the following conclusions:

    1- The positions most important for 3′ pairing differed between different miRNAs;

    2- Compared to Grimson et al. 2007, the authors show that preferred pairing often does not correspond precisely to positions 13-16, but it does always at least partially overlap such stretch of nucleotides;

    3- Two distinct 3′-binding modes seem to exist. Yet, arriving to that conclusion (that is at the core of the title) is not easy for the reader (see below);

    4- Increasing miRNA length can sometimes improve 3′ binding affinity, but it cannot substitute for other features required for high affinity to the miRNA 3′ region.

    5- Central to the paper and underlying several analyses, the authors show that parameters derived from interactions of purified RNAs in solution are not directly relevant to miRNAs associated with AGO2;

    6- GG/GC/CG dinucleotides in positions 13-16 most likely participate in productive 3' pairing, and extra Gs beyond this stretch also favor.

    7- Importantly, there is a functional difference between 3′-supplementary and 3′- compensatory pairing in regard to the presence of mismatches in the seed.

    8- By using chimeric miRNAs, the authors separate effects of seed-mismatches, to those effects derived from the length, position, offset, and nucleotide-identity preferences of the 3′ region;

    9- Finally, the two different 3' binding modes presented in this manuscript help rationalizing some aspects of target-dependent miRNA degradation (TDMD).

    The title: Should the term "seed mismatch" be included to highlight one of the most important aspects of the paper?

    We have changed the title to “MicroRNA 3′-compensatory pairing occurs through two binding modes, with affinity shaped by nucleotide identity and position,” to indicate that the conclusions of the title were derived using seed-mismatch sites.

    The Introduction: Well-written and informative, but perhaps too long. The authors should explain why they have chosen Ago2 for all their experiments, when they continuously refer to "AGOs" in the Introduction.

    The Introduction has been shortened to enhance readability. We have also added justification for using AGO2, pointing out that this paralog is typically the most highly expressed and is the one most frequently used by others for biochemical and structural studies of AGO–miRNA complexes.

    The results: Specific comments: The authors jump from Fig. 1A to Fig. 1C. Fig. 1B is mentioned at the Introduction. Should Fig. 1B be moved to the supplement?

    Although we jump from Figure 1A to Figure 1C in the Results section, Figure 1A and Figure 1B are both cited in the Introduction section. Because of the importance of Figure 1B for the Introduction, we have opted to keep this panel in the main text.

    The authors mainly focus on let-7a and two well-known miRNAs: miR-1 and miR-155. The RNA bind-n-seq analysis reveals different binding behaviors. Are those miRNAs representatives? In how much the analysis provided by the authors get close to a (nearly) full picture of 3' miRNA binding modes?

    We initially observed evidence for the positive-offset binding mode in our analysis of let-7a (Figures 2 and 3) but not in our analyses of miR-1 and miR-155 (Figure 4). Nonetheless, we also observed evidence of this second binding mode in our analyses of miR-124, lsy-6, and miR-7, the three other miRNAs with AGO2-RBNS datasets (Figure 5—figure supplement 4). With respect to the question of whether there are more than two binding modes, we have no evidence for additional binding modes but of course cannot rule out this possibility.

    The (many) figures displaying color-gradient squares to calculate Kds are elegant but I would argue that replacing some of them by tables and numbers would be more informative and less demanding for the eye of the reader.

    Although replacing each of the color-gradient squares with a number might be more informative when comparing values for just a few sites, we wonder if digesting the entire table of numbers would be less demanding for the reader. Because replacing the heat maps with tables of numbers would also take much more space, we have opted to keep the heat maps.

    I would also suggest to bring back TargetScan at the Discussion (as in the previous paper by Mc Geary et al. 2019), to highlight the benefits of the biochemical approach on top of the powerful and universally used TargetScan.

    Ideally, the insights gained in this type of study could be used to improve the ability of TargetScan to predict the efficacy of sites with 3′ pairing. However, even with machine learning, we will need binding information for 3′ pairing of more miRNAs before we can build a model that generalizes to miRNAs of any sequence and thereby improve TargetScan.

    A general comment goes towards the presentation of the data. In contrast to other manuscripts, the authors rely on a unique type of data, that emerges from binding assays on nitrocellulose membranes, and their quantification. For a better visualization, I would encourage the authors to include examples of such bindings and quantifications.

    As the reviewer points out, most data of this paper come from AGO-RBNS experiments, which include a filter-binding step to separate library molecules that are bound from those that are unbound. However, because these data are derived from sequencing of amplicons prepared from RNA extracted from the nitrocellulose membranes, they cannot be visualized in the same manner as data from classical filter-binding experiments.

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  2. Evaluation Summary:

    This manuscript will be of interest to readers in the field of microRNA (miRNA) biology, particularly those interested in miRNA targeting. The authors interrogated non-canonical miRNA target recognition to a depth vastly exceeding any study to date. The results revealed unexpected, sequence-specific diversity in miRNA-targeting modes, providing new insights relevant for improved target prediction.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Canonical miRNA-targeting involves pairing between the miRNA seed region (nucleotides 2-7, counting from the miRNA 5' end) and a target mRNA. Pairing downstream of the seed can also influence target recognition, and in some cases 3' pairing can compensate for imperfect seed complementarity. In this study, McGeary et al. investigated the features of such miRNA 3' compensatory sites in a high-throughput manner by adapting the RNA bind-n-seq (RBNS) method used previously to characterize binding of purified Argonaute2-miRNA complexes to a random pool of target RNAs.

    Strengths
    To focus on 3'-compensatory sites, which are rare in random libraries, the authors designed libraries of RNAs containing imperfect seed complementarity followed by 25 nucleotides of random sequence. This approach allowed investigation of a range of 3' pairing possibilities far more extensive than any previous work. Results provide several unexpected findings. Contrary to the prevailing model that miRNA nucleotides 13-16 are most efficacious for 3' pairing, the authors found the optimal position varies between miRNA sequences and is often shifted to include G nucleotides in the miRNA. The number of unpaired nucleotides bridging seed and 3'-paired regions is also a factor-certain let-7 sites preferring an offset of +4 target nucleotides, indicating a high affinity target-binding mode previously unknown. Additionally, the contribution of miRNA 3' pairing correlates poorly with predictions from nearest-neighbor parameters. Overall findings greatly expand insights into miRNA 3' pairing and provide metrics for improving target prediction.

    Weaknesses
    Conclusions are drawn entirely from RBNS data sets, leading to a few limitations. Affinity measurements are limited to relative KD values, making comparison to other work in the field indirect and potentially problematic. For example, let-7 target sites in lin-41 have 11-19 3' compensatory pairing, +1nt offset, which (based on Fig. 2B and 2C) has a greater relative KD than the let-7 8mer canonical site. However, a recent result showed an in vivo lin-41 reporter with two 8mer sites is less repressed than same reporter bearing the wild type 3'-compensatory sites (1). In the absence of KD values and/or cellular repression data for these specific sites the noted differences are difficult to reconcile. Additionally, analyses assume miRNA-target complementarity directly correlates with physical pairing between miRNA and target. However, because physical pairing occurs within the Argonaute2-miRNA complex, this may not always be the case

    1. Duan, Ye, Isana Veksler-Lublinsky, and Victor Ambros. "Critical contribution of 3'non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA." bioRxiv (2021).

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  4. Reviewer #2 (Public Review):

    In this manuscript, McGeary, Bisaria, and Bartel provide key additional insights into how 'compensatory' 3' binding contributes to the affinity of Ago2-miRNA complexes for target sites with imperfect seed matches.

    At the core of this work is an impressive and elegant series of experiments using the Ago2-RNA bind'n'seq (AGO-RBNS) technique they recently developed. Here they focused on three 'programmed' libraries designed to have an imperfect seed match (edit distance 1) for either let-7, miR-155, or miR-1 preceding a 25 nt randomized sequence.

    A detailed analysis of the sequences enriched upon incubation with Ago2 loaded with the corresponding miRNA challenges previous assumptions regarding the role of critical residues within compensatory sites. This study uncovers marked variability between different miRNAs with respect to the ability of 3' matches to compensate for imperfect seed matches. In particular they show that although for some miRNAs extensive 3' pairing can lead to a binding affinity comparable to the binding affinity of a perfect 8-mer seed match, for others the effect is much more modest.

    Another important finding is the discovery of two 3'-pairing modes for some miRNAs (let-7 in particular), one requiring the presence of a more extensive 'bulge' in the target but resulting in higher affinity.

    These observation will have important implication in the design of improved prediction algorithms and for the interpretation of CLIP experiments. Some of the findings reported here, for example the interesting observation that the nature of the seed mismatch profoundly affects the impact of the compensatory 3'-pairing, will prompt follow-up structural studies to be fully understood at the mechanistic levels.

    The manuscript is impeccably written and the experiments are well controlled and beautifully illustrated. Their results are consistent with the authors's interpretation. The vast scientific literature existing on the topic is appropriately cited and the statistical analyses used are, as far as I can judge, appropriate.

    Overall, this is a strong and important manuscript that will be widely appreciated by the broad scientific community and by the non-coding RNA field.

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  5. Reviewer #3 (Public Review):

    The Bartel Lab tackles the elusive role of the 3' part of miRNAs to contribute to the binding of target RNAs. In short, the presented data lead to the following conclusions:

    1- The positions most important for 3′ pairing differed between different miRNAs;
    2- Compared to Grimson et al. 2007, the authors show that preferred pairing often does not correspond precisely to positions 13-16, but it does always at least partially overlap such stretch of nucleotides;
    3- Two distinct 3′-binding modes seem to exist. Yet, arriving to that conclusion (that is at the core of the title) is not easy for the reader (see below);
    4- Increasing miRNA length can sometimes improve 3′ binding affinity, but it cannot substitute for other features required for high affinity to the miRNA 3′ region.
    5- Central to the paper and underlying several analyses, the authors show that parameters derived from interactions of purified RNAs in solution are not directly relevant to miRNAs associated with AGO2;
    6- GG/GC/CG dinucleotides in positions 13-16 most likely participate in productive 3' pairing, and extra Gs beyond this stretch also favor.
    7- Importantly, there is a functional difference between 3′-supplementary and 3′- compensatory pairing in regard to the presence of mismatches in the seed.
    8- By using chimeric miRNAs, the authors separate effects of seed-mismatches, to those effects derived from the length, position, offset, and nucleotide-identity preferences of the 3′ region;
    9- Finally, the two different 3' binding modes presented in this manuscript help rationalizing some aspects of target-dependent miRNA degradation (TDMD).

    The title:
    Should the term "seed mismatch" be included to highlight one of the most important aspects of the paper?

    The Introduction:
    Well-written and informative, but perhaps too long.
    The authors should explain why they have chosen Ago2 for all their experiments, when they continuously refer to "AGOs" in the Introduction.

    The results:
    Specific comments:
    The authors jump from Fig. 1A to Fig. 1C. Fig. 1B is mentioned at the Introduction. Should Fig. 1B be moved to the supplement?

    The authors mainly focus on let-7a and two well-known miRNAs: miR-1 and miR-155. The RNA bind-n-seq analysis reveals different binding behaviors. Are those miRNAs representatives? In how much the analysis provided by the authors get close to a (nearly) full picture of 3' miRNA binding modes?

    The (many) figures displaying color-gradient squares to calculate Kds are elegant but I would argue that replacing some of them by tables and numbers would be more informative and less demanding for the eye of the reader.

    I would also suggest to bring back TargetScan at the Discussion (as in the previous paper by Mc Geary et al. 2019), to highlight the benefits of the biochemical approach on top of the powerful and universally used TargetScan.

    A general comment goes towards the presentation of the data. In contrast to other manuscripts, the authors rely on a unique type of data, that emerges from binding assays on nitrocellulose membranes, and their quantification. For a better visualization, I would encourage the authors to include examples of such bindings and quantifications.

    Was this evaluation helpful?