Journal: eLife

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.7554/elife.70948

Figure Lengend Snippet: Figure 1. T6B fusion protein prevents miRNA-induced silencing complex (miRISC) assembly and impairs microRNA (miRNA) activity in vitro. (A) Schematics of T6B action: T6B competes with TNRC6 for binding to AGO proteins preventing miRISC assembly. (B) Schematics of the size-exclusion chromatography (SEC) assay for the fractionation of AGO-containing complexes according to their molecular weight. (C) SEC profiling of miRISC components upon T6B expression: total lysates from HCT116 cells expressing no fusion protein (upper panel), T6B (middle panel), or T6BMut (lower panel) were fractionated as described in (B) and immunoblotted to detect AGO2, TNRC6A, T6B, and PABP1. For each blot, the relative signal intensity was assessed by densitometric analysis. (D) RNAseq analysis of total and small RNAs isolated from mouse embryo fibroblasts (MEFs) cell lines expressing either no fusion protein, T6B, or T6BMut (n = 3 for each cell line). Upper panel: bubble plot of target de-repression against miRNA abundance. The mean log2-fold change (T6B or T6BMut vs. control) of predicted targets for each conserved miRNA family was calculated, converted to a z-score and is plotted on the x-axis against the miRNA family abundance (log of the sum of read counts for each member of the family). The size of each circle is proportional to the number of predicted targets. A positive z-score indicates that the targets for that family are preferentially upregulated upon T6B expression, while a negative score would indicate preferential downregulation. Expression of T6B, but not of T6BMut, causes preferential upregulation of miRNA targets of the most miRNA families and the effect is roughly proportional to each miRNA family abundance. Lower panel: cumulative distribution plot of predicted let-7 targets compared to background in T6B-expressing MEFs. (E) Scatter plots of miRNA abundance as determined by small-RNAseq of total RNA extracted from MEFs expressing either T6B or T6BMut (n = 3 for each cell line). Each dot represents a miRNA in miRbase. (F) Effect of T6B expression on AGO2 slicing activity. MEFs expressing either T6B or T6BMut were transfected with siRNAs targeting GAPDH mRNA (siGAPDH) or with scramble siRNA (siCTL). Levels of GAPDH, T6B, and tubulin were assessed by immunoblot 72 hr post-transfection. T6B and T6BMut have slightly different migration on PAGE, as previously observed by Hauptmann et al., 2015.

Article Snippet: DOI: https://doi.org/10.7554/eLife.70948 17 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence- based reagent Col1a1 common _F This paper PCR primers AATCATCCCAGGTG CACAGCATTGCGG Sequence- based reagent Col1a1 wildtype _R This paper PCR primers CTTTGAGGGCTCAT GAACCTCCCAGG Sequence- based reagent Col1a1 mutant _R This paper PCR primers ATCAAGGAAACCC TGGACTACTGCG Sequence- based reagent R26_F This paper PCR primers AAAGTCGCTCT GAGTTGTTAT Sequence- based reagent R26a_R This paper PCR primers GCGAAGAGTTTG TCCTCAACC Sequence- based reagent R26b_R This paper PCR primers CCTC CAAT TTTA CACC TGTTC Sequence- based reagent T6B- YFP_F This paper PCR primers GACTACAAGGACG ACGATGACAAG Sequence- based reagent T6B- YFP_R This paper PCR primers GTTACTTGTACAG CTCGTCCATG Commercial assay or kit RNAscope 2.5 HD Detection Reagent, BROWN ACD #320771 Commercial assay or kit RNAScope Igfbp5 Probe ACD #425738 Commercial assay or kit Superose 6 10/300 GL Cytiva #GE17- 5172- 01 Now available as Increase 10/300 GL, Cytiva #GE29- 0915- 96 Commercial assay or kit Novex NuPAGE SDS/ PAGE gel system Thermo Fisher #NP0321 Commercial assay or kit EnVision + HRP DAKO, Glostrup, Denmark #K401111- 2, RRID:AB_2827819 Commercial assay or kit GFP- trap Chromotek #gtma- 10 RRID:AB_2827592 Commercial assay or kit TruSeq Stranded mRNA LT Kit, Illumina #RS- 122- 2102 Software, algorithm OMERO PMID:22373911 RRID:SCR_002629 Software, algorithm STAR v2.5.3a PMID:23104886 Software, algorithm DESeq2 PMID:25516281 RRID:SCR_015687 Software, algorithm miRbase version 21 https://www.mirbase.org/ Software, algorithm TargetScan PMID:26267216 RRID:SCR_010845 Chemical compound, drug Doxycyline- containing Rodent diet Envigo #TD01306 625 mg/kg doxycycline Chemical compound, drug Dextran sulfate sodium (DSS) Cayman Chemical #23250 Chemical compound, drug Surgipath Decalcifier I Leica Biosystems #3800400 Formic acid solution Other EDTA- free complete protease inhibitors Sigma- Aldrich #11836170001 Other KnockOut DMEM GIBCO #10829018 Continued Continued on next page La Rocca, King, et al. eLife 2021;10:e70948.

Techniques: Activity Assay, In Vitro, Binding Assay, Size-exclusion Chromatography, Fractionation, Molecular Weight, Expressing, Isolation, Control, Transfection, Western Blot, Migration

Journal: eLife

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.7554/elife.70948

Figure Lengend Snippet: Figure 4. T6B-induced block of miRNA-induced silencing complex (miRISC) assembly leads to impaired intestinal regeneration. (A) R26T6B and R26CTL mice (n = 6 for each genotype) kept on doxycycline diet were treated with dextran sulfate sodium (DSS) for 5 days to induce inflammatory colitis and their weight was monitored daily. Data are presented as mean ± SD. p-Values (from left to right): *p=0.034, *p=0.005, *p=0.029, *p=0.024, *p=0.011, from unpaired t-test. (B) Kaplan–Meier curves of animals treated with DSS as described in panel (A). p-Value from log-rank test (C) Representative hematoxylin-eosin-stained sections of intestine of R26T6B and R26CTL mice (n = 3 for each genotype) at different time points pre- and post-DSS treatment. (D) Ki67 immunostaining of section of intestine at the indicated time points. (E) Sections from the large intestine of control and T6B mice euthanized at day 13 were subjected to RNA in situ hybridization with a probe against the IGFBP5 transcript. The results show increased levels of IGFBP5 mRNA in ulcerated areas of R26T6B as compared to controls (n = 4 for each genotype).

Article Snippet: DOI: https://doi.org/10.7554/eLife.70948 17 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence- based reagent Col1a1 common _F This paper PCR primers AATCATCCCAGGTG CACAGCATTGCGG Sequence- based reagent Col1a1 wildtype _R This paper PCR primers CTTTGAGGGCTCAT GAACCTCCCAGG Sequence- based reagent Col1a1 mutant _R This paper PCR primers ATCAAGGAAACCC TGGACTACTGCG Sequence- based reagent R26_F This paper PCR primers AAAGTCGCTCT GAGTTGTTAT Sequence- based reagent R26a_R This paper PCR primers GCGAAGAGTTTG TCCTCAACC Sequence- based reagent R26b_R This paper PCR primers CCTC CAAT TTTA CACC TGTTC Sequence- based reagent T6B- YFP_F This paper PCR primers GACTACAAGGACG ACGATGACAAG Sequence- based reagent T6B- YFP_R This paper PCR primers GTTACTTGTACAG CTCGTCCATG Commercial assay or kit RNAscope 2.5 HD Detection Reagent, BROWN ACD #320771 Commercial assay or kit RNAScope Igfbp5 Probe ACD #425738 Commercial assay or kit Superose 6 10/300 GL Cytiva #GE17- 5172- 01 Now available as Increase 10/300 GL, Cytiva #GE29- 0915- 96 Commercial assay or kit Novex NuPAGE SDS/ PAGE gel system Thermo Fisher #NP0321 Commercial assay or kit EnVision + HRP DAKO, Glostrup, Denmark #K401111- 2, RRID:AB_2827819 Commercial assay or kit GFP- trap Chromotek #gtma- 10 RRID:AB_2827592 Commercial assay or kit TruSeq Stranded mRNA LT Kit, Illumina #RS- 122- 2102 Software, algorithm OMERO PMID:22373911 RRID:SCR_002629 Software, algorithm STAR v2.5.3a PMID:23104886 Software, algorithm DESeq2 PMID:25516281 RRID:SCR_015687 Software, algorithm miRbase version 21 https://www.mirbase.org/ Software, algorithm TargetScan PMID:26267216 RRID:SCR_010845 Chemical compound, drug Doxycyline- containing Rodent diet Envigo #TD01306 625 mg/kg doxycycline Chemical compound, drug Dextran sulfate sodium (DSS) Cayman Chemical #23250 Chemical compound, drug Surgipath Decalcifier I Leica Biosystems #3800400 Formic acid solution Other EDTA- free complete protease inhibitors Sigma- Aldrich #11836170001 Other KnockOut DMEM GIBCO #10829018 Continued Continued on next page La Rocca, King, et al. eLife 2021;10:e70948.

Techniques: Blocking Assay, Staining, Immunostaining, Control, RNA In Situ Hybridization