1. Evaluation Summary:

    MicroRNAs (miRNAs) control gene expression during development and play crucial roles in disease. Loss-of-function mutations in key pathway components are embryonically lethal. Here, La Rocca et al. establish an elegant mouse model that enables acute and reversible inhibition of miRNA-guided silencing. Analysis of this model has convincingly demonstrated that miRNA activity is dispensable for homeostasis in most adult tissues, with the notable exception of heart and skeletal muscle. This work provides an extremely useful tool for the study of miRNAs in vivo and provides new insights into the roles of miRNAs in adult mammalian tissues. The findings presented will impact many fields given the well-established roles of miRNAs in normal development and diseases.

    (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 #1 and Reviewer #2 agreed to share their names with the authors.)

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  2. Reviewer #1 (Public Review):

    In this manuscript the authors developed an inducible and reversible system for inhibition of miRNA-mediated gene repression in vivo. The authors achieved their aims by demonstrating the use of this system in inhibition of global miRNA activity in adult animals. Interestingly, they found that global loss of miRNA function has different effects on different tissues and organs and in some tissues the effect is context dependent. Overall, the findings presented are interesting and data presented is high quality. The results are presented support their conclusions. It is expected that this strategy is a significant advance and will impact many fields including but not limited to RNA biology, organ homeostasis and cancer biology.

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  3. Reviewer #2 (Public Review):

    The authors have engineered a mouse line harboring an inducible transgene encoding a peptide that blocks AGO-TNRC6 interaction. Activation of this transgene allows the rapid and reversible inhibition of miRNA function in vivo. This approach offers several advantages over inducible genetic loss of function of miRNA biogenesis factors, the more standard approach to impair miRNA function in vivo. Most notably, the extreme stability of most miRNAs generally results in long-term residual miRNA expression even after deletion of miRNA processing factors. This new model circumvents this limitation, allowing much more rapid inhibition of miRNA silencing. Using this system, the authors confirm that miRNA-mediated regulation is needed for embryogenesis, but is surprisingly dispensable for homeostasis in most adult tissues, with the notable exception of heart and skeletal muscle.

    This work is of extremely high quality, the analyses are thorough and convincing, and the findings are significant.

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  4. Reviewer #3 (Public Review):

    Here, La Rocca and colleagues establish a versatile mouse model to probe the function of miRNA-guided gene silencing in vivo. Guided by results from protein structures, the Meister group previously designed a peptide that directly inhibits the function of miRNA-guided silencing complexes (miRISC) by competing for the interaction with the effector factor TNRC6/GW182. The Ventura lab now adapted this clever approach to generate an elegant in vivo model. Their transgenic mouse expresses the inhibitory peptide (T6B) in a doxycycline inducible manner, and allows for acute and reversible inhibition of miRNA-silencing.

    The authors perform a careful characterization of their model system, and then investigate the consequences of miRISC inhibition in homeostasis and during regeneration after injury in different tissues. Interestingly, the acute inhibition of miRISC activity is well tolerated by most tissues under homeostatic conditions. However, upon injury - at the example of dextran sulfate sodium (DSS)-induced colitis- lack of miRNA-guided regulation results in increased tissue damage and severity of clinical symptoms. Similarly, hematopoiesis fails to regenerate after 5-fluoro-uracil treatment.

    Finally, the authors generate a second adjusted mouse model to interrogate miRISC function in heart and skeletal muscle. Inhibition of miRISC induces severe cardiomyopathy and degenerative lesions in skeletal muscles. Taken together, this well-designed and carefully executed study elucidated a differential requirement for miRNA-guided gene regulation in adult tissues under homeostatic and stressed conditions.

    Results from this study generate important hypotheses for the pathophysiology of heart and skeletal muscle diseases, and tissue regeneration. The established mouse presents a valuable resource for a broad scientific community and can be used to study any cell type, tissue or disease in vivo.

    It is a pleasure to learn from this important, careful and well-presented study.

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