Journal: Molecular cell

Article Title: Structural and functional basis of mammalian microRNA biogenesis by Dicer.

doi: 10.1016/j.molcel.2022.10.010

Figure Lengend Snippet: Figure 1. DExD/HEL1 domain of Dicer but not its ATPase activity is essential for miRNA homeostasis and normal mouse development (A) Studied mouse Dicer protein variants and mutants. Dicer (full-length) and DicerO lacking HEL1 are endogenous isoforms. DicerGNT and DicerFH-DQCH carry point mutations in HEL1 abolishing its function as ATPase. DicerSOM and DicerDHEL1 are HA-tagged mutants with modified sequence encoding the N terminus. The engineered allele is designated DicerDHEL1 to distinguish it from the endogenous DicerO isoform, which is transcribed from an oocyte-specific promoter. (B) Western blot analysis of Dicer expression in different tissues of a heterozygote DicerDHEL1/SOM mouse. (C) Production of endo-siRNAs from MosIR, a dsRNA-expressing plasmid (Flemr et al., 2013) in DicerDHEL1/DHEL1 ESCs. The y axis depicts reads per million (RPMs) per small RNA sequencing library. Data points, mean ± SD. (D) Breeding performance of different heterozygous mutants. (E) MA plot of small RNA-seq analysis of whole DicerGNT/GNT E15.5 embryos compared with wild-type embryos (n = 3 for each genotype). Depicted are changes in levels of annotated murine miRNAs (miRBase 22.1; Kozomara et al., 2019). Significantly dysregulated 5p and 3p miRNAs (DESeq p value 0.05) are shown as oriented blue and red triangles, respectively. Mirtron non-canonical miRNAs are depicted by green triangles. (F) Weight of embryos normalized to wild-type littermates shows relative retardation of DicerDHEL1/DHEL1 embryos—the growth retardation does not appear to be a simple proliferation defect (Figure S2B). Although a lower weight in DicerDHEL1/DHEL1 embryos is apparent at E10.5, the main reduction of growth appears between stages E12.5 and E14.5. Data points, mean ± SD.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Hairpin-expressing plasmid CAG-EGFPMosMos Addgene Cat# 120515 Hairpin-expressing plasmid CAG-EGFPRlucIR this paper N/A MosIR plasmid Addgene Cat# 120516 pCIneo 5’-DICER1(dHEL1)-2xFLAG this paper N/A pCIneo 5’-DICER1(dHEL2)-2xFLAG this paper N/A pCIneo 5’-DICER1(dDExD)-2xFLAG this paper N/A pEF1-MH.Bl-mDcrOO Addgene Cat# 120541 pEF1-MH.Bl-mDcrSOM Addgene Cat# 120540 pFastBACT1 5’-TwinStrep-HA-TEVDicerO-TEV-2xFLAG-8xHis this paper N/A pFastBACT1 5’-TwinStrep-HA-TEV-DicerTEV-2xFLAG-8xHis this paper N/A pFastBACT1 TwinStrep-HA-TEVDicer(E1560A, E1807A)-TEV2xFLAG-8xHis this paper N/A pFastBACT1 TwinStrep-HA-TEVDicerO(E1560A, E1807A)-TEV2xFLAG-8xHis this paper N/A pFastBACT1 plasmid Invitrogen N/A puromycin selection plasmids Taborska et al., 2019 N/A Renilla luciferase reporter - RL-Lin28 plasmid Addgene Cat# 120520 Software and algorithms Alphafold Jumper et al., 2021; Varadi et al., 2022 https://alphafold.ebi.ac.uk/ Coot 0.9.6.2 Emsley et al., 2010 https://www2.mrc-lmb.cam.ac.uk/ personal/pemsley/coot/ crYOLO 1.7.6 Wagner et al., 2019 https://cryolo.readthedocs.io/en/stable/ cryoSPARC Punjani et al., 2017 https://cryosparc.com/ cutadapt version 1.8.3 Martin, 2011 N/A DESeq2 Love et al., 2014 N/A EMAN2 Tang et al., 2007 https://blake.bcm.edu/emanwiki/EMAN2 fastx-toolkit version 0.0.14 http://hannonlab.cshl.edu/fastx_toolkit N/A featureCounts v.2.0.0 Liao et al., 2014 N/A GCTF Zhang, 2016 https://www2.mrc-lmb.cam.ac.uk/ research/locally-developed-software/ zhang-software/ GraphPad Prism 9.1.0 GraphPad Software https://www.graphpad.com/scientific- software/prism/ ISOLDE 1.1.0 Croll, 2018 https://isolde.cimr.cam.ac.uk/static/isolde/ doc/isolde.html Molprobity Davis et al., 2004, 2007;Williams et al., 2018 http://molprobity.biochem.duke.edu/ ?fbclid=IwAR23TiIo_fFJl0iW0JjnMBt So2JRdRKoxNt2tsD4m7hPt3FzRzvJG 08IDpU MotionCor2 Zheng et al., 2017 https://emcore.ucsf.edu/ucsf-software Multi Gauge v3.2 Fujifilm, Tokyo, Japan N/A pcaExplorer Marini and Binder, 2019 N/A PHENIX 1.19.2-4158-000 Liebschner et al., 2019 https://phenix-online.org/ (Continued on next page) Molecular Cell 82, 4064–4079.e1–e13, November 3, 2022 e6

Techniques: Activity Assay, Sequencing, Western Blot, Expressing, Plasmid Preparation, RNA Sequencing

Journal: Molecular cell

Article Title: Structural and functional basis of mammalian microRNA biogenesis by Dicer.

doi: 10.1016/j.molcel.2022.10.010

Figure Lengend Snippet: Figure 2. HEL1 restricts processing of mirtrons and 3p passenger strand loading (A) Strongly upregulated mirtrons have extended stems and larger loops. Secondary structures were adopted from miRBase (Kozomara et al., 2019). (B) miR-3102 is a unique mirtron cleaved by Dicer twice at points indicated by black arrowheads. (C) Changes of miR-3102 levels in DicerDHEL1/DHEL1 ESCs. Relative expressions are shown in counts per million (CPMs) estimated by DESeq; 21–23 nt small RNA sequencing data were mapped on the genomic sequence (represented as the genomic locus in 5p–3p orientation), collapsed, and normalized per million of 21–23 nt reads. Gray columns represent sequences aligned with the annotated mature miRNA sequence; black represent sequences outside the annotated miRNA sequence. (D) In vitro cleavage of miR-7068 precursor. Radiolabeled in vitro synthesized precursor (final conc. 5 nM) was incubated for 30 min with full-length Dicer (WT) or with DicerDHEL1 (DHEL1). Reaction was resolved by PAGE and visualized by phosphorimaging. Black arrowheads depict two cleavage products corresponding to cleavage positions at the 30 end of 5p miRNAs, a gray arrowhead depicts a product of an asymmetric cleavage. The experiment was repeated 3 times; a representative gel is shown. Apparent higher size of mir-7068 and cleavage products is caused by altered migration due to high G content. (E) MA plots depicting relative changes of dominant miRNAs (left) and passenger strands (miRNA*, right) in DicerDHEL1/DHEL1 E15.5 embryos. 5p and 3p origins of significantly changed miRNAs or miRNA*s are distinguished by color and triangle orientation as depicted. (F) Relative changes of dominant miRNAs and their passenger strands in DicerDHEL1/DHEL1 E15.5 embryos. Each triangle represents one miRNA:miRNA* pair, its color corresponds to the dominant strand (blue = 5p and red = 3p main strand). Triangle positions indicate relative changes of the dominant miRNA (x axis) and its miRNA* (y axis). Deep colors indicate significantly dysregulated miRNAs.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Hairpin-expressing plasmid CAG-EGFPMosMos Addgene Cat# 120515 Hairpin-expressing plasmid CAG-EGFPRlucIR this paper N/A MosIR plasmid Addgene Cat# 120516 pCIneo 5’-DICER1(dHEL1)-2xFLAG this paper N/A pCIneo 5’-DICER1(dHEL2)-2xFLAG this paper N/A pCIneo 5’-DICER1(dDExD)-2xFLAG this paper N/A pEF1-MH.Bl-mDcrOO Addgene Cat# 120541 pEF1-MH.Bl-mDcrSOM Addgene Cat# 120540 pFastBACT1 5’-TwinStrep-HA-TEVDicerO-TEV-2xFLAG-8xHis this paper N/A pFastBACT1 5’-TwinStrep-HA-TEV-DicerTEV-2xFLAG-8xHis this paper N/A pFastBACT1 TwinStrep-HA-TEVDicer(E1560A, E1807A)-TEV2xFLAG-8xHis this paper N/A pFastBACT1 TwinStrep-HA-TEVDicerO(E1560A, E1807A)-TEV2xFLAG-8xHis this paper N/A pFastBACT1 plasmid Invitrogen N/A puromycin selection plasmids Taborska et al., 2019 N/A Renilla luciferase reporter - RL-Lin28 plasmid Addgene Cat# 120520 Software and algorithms Alphafold Jumper et al., 2021; Varadi et al., 2022 https://alphafold.ebi.ac.uk/ Coot 0.9.6.2 Emsley et al., 2010 https://www2.mrc-lmb.cam.ac.uk/ personal/pemsley/coot/ crYOLO 1.7.6 Wagner et al., 2019 https://cryolo.readthedocs.io/en/stable/ cryoSPARC Punjani et al., 2017 https://cryosparc.com/ cutadapt version 1.8.3 Martin, 2011 N/A DESeq2 Love et al., 2014 N/A EMAN2 Tang et al., 2007 https://blake.bcm.edu/emanwiki/EMAN2 fastx-toolkit version 0.0.14 http://hannonlab.cshl.edu/fastx_toolkit N/A featureCounts v.2.0.0 Liao et al., 2014 N/A GCTF Zhang, 2016 https://www2.mrc-lmb.cam.ac.uk/ research/locally-developed-software/ zhang-software/ GraphPad Prism 9.1.0 GraphPad Software https://www.graphpad.com/scientific- software/prism/ ISOLDE 1.1.0 Croll, 2018 https://isolde.cimr.cam.ac.uk/static/isolde/ doc/isolde.html Molprobity Davis et al., 2004, 2007;Williams et al., 2018 http://molprobity.biochem.duke.edu/ ?fbclid=IwAR23TiIo_fFJl0iW0JjnMBt So2JRdRKoxNt2tsD4m7hPt3FzRzvJG 08IDpU MotionCor2 Zheng et al., 2017 https://emcore.ucsf.edu/ucsf-software Multi Gauge v3.2 Fujifilm, Tokyo, Japan N/A pcaExplorer Marini and Binder, 2019 N/A PHENIX 1.19.2-4158-000 Liebschner et al., 2019 https://phenix-online.org/ (Continued on next page) Molecular Cell 82, 4064–4079.e1–e13, November 3, 2022 e6

Techniques: RNA Sequencing, Sequencing, In Vitro, Synthesized, Incubation, Migration

Journal: Nature Communications

Article Title: The Lin28/let-7 axis is critical for myelination in the peripheral nervous system

doi: 10.1038/ncomms9584

Figure Lengend Snippet: ( a ) Forty most abundant miRNAs in the SN of wild-type mice at PN4 corrected for values in SN of PN4 mice with SC-specific deletion of Dicer (Dicer KO; see the Methods section). ( b ) Mean levels of abundant let-7 isoforms and the Lin28-dependent miR-98-5p at embryonic day (ED) 17.5, PN1, PN4, PN10, PN30 and PN60 in SN of wild-type mice in reads per million (r.p.m.). ( c ) Levels of Lin28B mRNA and of mature let-7f-5p and let-7i-5p during SN development in wild-type mice, normalized to GAPDH mRNA for Lin28B and to snoRNA-202 for let-7 miRNAs ( n =3 mice per time point). ( d ) Cumulative distribution of differential expression of all expressed mRNAs and let-7 targets, predicted with TargetScan (TS) or miRWalk (MW), in Dicer KO versus control SN at PN1. The rightward shifts in the curves for targets indicate enrichment compared with all expressed mRNAs. ( e ) Hmga2 mRNA levels during wild-type mouse SN development ( n =3 mice per time point). Error bars: s.e.m. ( c , e ). One-way ANOVA with Dunnett's multiple comparison test ** P <0.01 ( e ).

Article Snippet: Lin28 expression vector was acquired from Addgene (Addgene plasmid 26358)ref. .

Techniques: Quantitative Proteomics, Control, Comparison

Journal: Nature Communications

Article Title: The Lin28/let-7 axis is critical for myelination in the peripheral nervous system

doi: 10.1038/ncomms9584

Figure Lengend Snippet: ( a – c ) Electron micrographs of SN of Ctr, Lin28 tg and Dicer KO at PN5. Examples of myelinated axons (black arrows) and 1:1 SC-axon relations (white arrows) are highlighted. ( d ) Levels of mature let-7f-5p, let-7i-5p and let-7d-5p in SN of Ctr and Lin28 tg at PN14, normalized to snoRNA-202 ( n =3 mice per condition). ( e ) Cumulative distribution of differential expression of all expressed mRNAs and let-7 targets, predicted with TargetScan (TS) or miRWalk (MW), in Lin28 tg versus control SN at PN1. The rightward shifts in the curves for targets indicate enrichment compared with all expressed mRNAs. ( f , g ) Quantification of myelinated axons per cross-sections ( f ) and g-ratios (ratio of the inner axonal diameter to the total outer nerve fibre diameter) ( g ), determined on SN electron micrographs at PN5 ( n =3 mice per condition). ( h – j ) Immunoblots of E-cadherin ( h ), Krox20 ( i ), MBP ( j ) and β-actin as loading control, from PN5 SN of Lin28 tg, Dicer KO and their respective controls. Numbers refer to estimated apparent molecular weights (kDa), except for MBP where the location of the 20 kDa size marker band is indicated. For quantifications, see . Scale bar, 2 μm ( a – c ). Error bars: s.e.m. Two-sided two-sample Student's t -test * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 ( d , f , g ).

Article Snippet: Lin28 expression vector was acquired from Addgene (Addgene plasmid 26358)ref. .

Techniques: Quantitative Proteomics, Control, Western Blot, Marker

Journal: Nature Communications

Article Title: The Lin28/let-7 axis is critical for myelination in the peripheral nervous system

doi: 10.1038/ncomms9584

Figure Lengend Snippet: ( a – f ) Immunostainings for neurofilament (NF; green) and MBP (red) of dissociated wild-type DRG explants treated with Anti-let-7 or control antagomir (Anti-ctr), prior to induction of myelination. ( g ) Quantification of MBP-positive area normalized to the NF-positive area ( n =4 embryos). For individual embryonal cultures, Anti-ctr values were set to 1.( h , i ) Electron micrographs of SN of Lin28 tg mice at PN10, injected with lentivirus expressing let-7S21L (7S21L inj) or control virus (Control inj) at PN3. Examples of myelinated axons (black arrows) and axons at the pro-myelinating stage (white arrows) are highlighted. ( j ) Quantification of the number of myelinated axons in let-7S21L ( n =4 mice) or control injected ( n =3 mice) SN, normalized to the number in the respective contralateral nerve. ( k ) Levels of Krox20 mRNA in primary SCs, induced for 1 h with Nrg1 48 h after transfection with Lin28, let-7 TuD or GFP-expressing vector. Primary SCs were grown and three randomly selected individual cultures per condition derived, individually processed and analysed. Two such sets of experiments were performed and the results of a representative one are shown. Scale bars equal 50 μm ( a – f ) and 3 μm ( h , i ); two-sided two-sample Student's t -test ** P <0.01, *** P <0.001 ( g , j ). One-way ANOVA with Dunnett's multiple comparison test * P <0.05 ( k ). Error bars: s.e.m. ( g , j , k ).

Article Snippet: Lin28 expression vector was acquired from Addgene (Addgene plasmid 26358)ref. .

Techniques: Control, Injection, Expressing, Virus, Transfection, Plasmid Preparation, Derivative Assay, Comparison

Journal: Nature Communications

Article Title: The Lin28/let-7 axis is critical for myelination in the peripheral nervous system

doi: 10.1038/ncomms9584

Figure Lengend Snippet: ( a ) Heatmap representation of significantly ( P <0.05) regulated Notch pathway members in sequencing data from PN1 Lin28 tg and Dicer KO versus control SN ( n =3 mice per condition). Colour intensities represent the Z-score for each row, red indicating higher expression and blue indicating lower expression. ( b , c ) Levels of Notch1 mRNA in SN of Lin28 tg and respective controls ( n =4 mice per condition) ( b ) and in Dicer KO and respective controls ( n =3 mice per condition) ( c ) at PN5. ( d , e ) Immunoblots of NICD ( d ), Hes1 ( e ), and β-actin as a loading control, from PN5 SN of Lin28 tg, Dicer KO and their respective controls. Numbers refer to estimated apparent molecular weights (kDa). For quantifications see . Error bars: s.e.m. Two-sided two-sample Student's t -test * P <0.05 ( b , c ).

Article Snippet: Lin28 expression vector was acquired from Addgene (Addgene plasmid 26358)ref. .

Techniques: Sequencing, Control, Expressing, Western Blot

Journal: Nature Communications

Article Title: The Lin28/let-7 axis is critical for myelination in the peripheral nervous system

doi: 10.1038/ncomms9584

Figure Lengend Snippet: ( a , b ) Electron micrographs of PN5 Lin28 tg SN after injection of Notch signalling inhibitor LY411575 or DMSO solvent at both, PN3 and PN4. ( c ) Quantification of myelinated axons in LY411575- or DMSO-treated Lin28 tg per SN cross-section ( n =3 mice per condition). ( d ) Krox20 mRNA levels in primary SCs transduced with GFP or let-7 TuD-expressing lentivirus prior to 1 h Nrg1 stimulation, with or without prior application of 10 μM LY411575. Primary SC were grown and three randomly selected individual cultures per condition derived, individually processed and analysed. ( e , f ) Electron micrographs of PN10 Lin28 tg SN, injected with lentivirus expressing dnMAML (dnMAML inj) or control virus (control inj) at PN3. ( g ) Quantification of myelinated axons in dnMAML- or control-injected SN, normalized to the respective contralateral nerve ( n =3 mice per condition). ( h ) Krox20 mRNA levels in dnMAML-GFP-positive versus GFP − cells, sorted 72 h after dnMAML lentivirus delivery to PN3 Lin28 tg SN ( n =4 mice). ( i ) Notch1 mRNA levels 48 h after transfection of SpL201 cells with GFP or Lin28, or after application of control antagomir (Anti-control) or let-7f antagomir (Anti-let-7; n =3 cultures per condition). ( j , k ) Predicted let-7 seed interactions (allowing Wobble base pairs and one binding site per region) in Notch1 3′-UTR ( j ). Scheme of predicted seed interactions, conserved indicated in grey ( k ). RNAhybrid-predicted interactions are listed in . ( l , m ) Luciferase activity in primary SCs, transfected 48 h before with let-7 mimics or scrambled mimics, and empty pmirGLO or pmirGLO harbouring the Notch1 3′-UTR. Primary SCs were grown and three randomly selected individual cultures per condition derived, individually processed and analysed. Three such sets of experiments were performed and the results of a representative one are shown. ( n ) Model summarizing the role of the Lin28B/let-7 axis in myelination. Solid lines (active mechanisms), dotted lines (inactive mechanisms). Myelinated axons (black arrows), pro-myelinating stage axons (white arrows), scale bars, 2 μm ( a , b , e , f ). Error bars: s.e.m., two-sided two-sample Student's t -test * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 ( c , d , g , h , i , l , m ).

Article Snippet: Lin28 expression vector was acquired from Addgene (Addgene plasmid 26358)ref. .

Techniques: Injection, Solvent, Transduction, Expressing, Derivative Assay, Control, Virus, Transfection, Binding Assay, Luciferase, Activity Assay