1. Volume 18, Issue 11, Pages 2664-2675 (March 2017)
LIN28 Zinc Knuckle Domain Is Required and Sufficient to Induce let-7 Oligouridylation 
Longfei Wang, Yunsun Nam, Anna K. Lee, Chunxiao Yu, Kira Roth, Casandra Chen, Elizabeth M. Ransey, Piotr Sliz 
Cell Reports 
Volume 18, Issue 11, Pages (March 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 2664-2675DOI: (10.1016/j.celrep.2017.02.044)
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3. Figure 1
The ZKD of LIN28 Is Critical to Reduce the Dissociation Rate of the LIN28:pre-let-7 Complex
(A) Schematic representation of mouse LIN28A and truncations used for EMSA.
(B) EMSAs with mouse pre-let-7g as probe, mixed with increasing concentrations (16, 33, 65, 130, 260, 520, 1,004, 2,100, and 4,200 nM) of LIN28, CSD, and ZKD. ∗Free probe; ∗∗complex. A second CSD molecule can bind to pre-let-7g at high CSD concentrations, which results in a 2:1 CSD:pre-let-7g complex.
(C) FP assays with FAM-labeled mouse preE-let-7f-1 as probe, mixed with increasing concentrations of mouse LIN28A, CSD, and ZKD.
(D) Comparison of the dissociation constants (KD) of mouse LIN28A, CSD, and ZKD for let-7 RNA from EMSA and FP assay (Figure S1).
(E) Comparison of the dissociation rates between mouse LIN28A mutants using SPR assay, with preE-let-7f-1 immobilized to the sensor surface, followed by the injection of LIN28 mutants.
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4. Figure 2
Recombinant LIN28 and TUT4 Are Sufficient for Oligouridylation of pre-let-7 by TUT4
(A) Schematic representation of mouse TUT4 and N-terminal TUT4 (NTUT4) constructs purified (Figures S3 and S4).
(B) SDS-PAGE and western blot of purified mouse TUT4 and its catalytic dead construct DADA. Each lane of the SDS-PAGE and western blot shows the same sample but in different gels.
(C) Oligouridylation assays with pre-let-7g as probe, carried out using recombinant TUT4 and UTP (Figure S5). ∗Oligouridylated pre-let-7.
(D) Oligouridylation assay with pre-let-7g as probe, carried out using recombinant TUT4 and ribonucleotide triphosphate (ATP, GTP, CTP, and UTP).
(E) Super elongation complex (SEC) chromatogram and SDS-PAGE of recombinant N-terminal TUT4.
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5. Figure 3
The LIN28 ZKD Mediates the Formation of the LIN28:pre-let-7:TUT4 Ternary Complex
(A) EMSAs with pre-let-7g as probe, mixed with mouse LIN28A truncation constructs and TUT4.
(B) View of ZKD interacts with GGAG RNA. GGAG RNA is in orange. The ZKD is represented by gray. The tested RNA binding and surface residues are in red. The model used is mouse LIN28A (PDB: 3TS2) (Nam et al., 2011).
(C) EMSAs with pre-let-7g as probe, mixed with mouse LIN28A mutants and TUT4.
(D) EMSAs with pre-let-7g mutants as probe, mixed with mouse LIN28A and TUT4.
(E) Surface view of the LIN28A ZKD. The surface residues E151 is in magenta. The model used is mouse LIN28A (PDB: 3TS2) (Nam et al., 2011).
(F) EMSAs with pre-let-7g as probe, mixed with increasing concentrations (16, 33, 65, 130, 260, 520, and 1,004 nM) of mouse LIN28A variants.
∗Free probe; ∗∗LIN28A:pre-let-7g complex; ∗∗∗LIN28A:pre-let-7g:TUT4 complex.
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6. Figure 4 The N-terminal Region of TUT4 Binds LIN28:pre-let-7 Complexes
(A) Schematic representation of N-terminal TUT4 and C-terminal TUT4 constructs used in EMSAs.
(B) EMSAs with pre-let-7g as probe, mixed with mouse LIN28A and NTUT4.
(C) EMSAs with pre-let-7g as probe, mixed with ZKD and NTUT759 truncations.
(D) EMSAs with pre-let-7g as probe, mixed with mouse LIN28A, NTUT4 and CTUT4 truncations.
(E) Size exclusion chromatograms of NTUT759 (gray) and LIN28A:NTUT759:pre-let-7g ternary complex (black).
(F) Conservation analysis of the 36 residues of the C-termini of NTUT759. The analysis was performed using ConSurf. All residues are color coded based on conservation score.
(G) EMSAs with pre-let-7g as probe, mixed with LIN28A and NTUT759 truncations.
(H) EMSAs with pre-let-7g as probe, mixed with LIN28A surface mutants and NTUT759 mutants. Concentrations for each NTUT759 mutants are 3, 1, 0.33, and 0.11 μM.
∗Free probe; ∗∗LIN28A:pre-let-7g complex (B, D, G, and H) and ZKD:pre-let-7g complex (C); ∗∗∗LIN28A:NTUT4:pre-let-7g ternary complex (B, D, G, and H) and ZKD:NTUT759:pre-let-7g ternary complex (C).
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7. Figure 5
Assembly of the LIN28:pre-let-7:TUT4 Ternary Complex Requires the Stem Region of RNA
(A) EMSAs using pre-let-7g and preE-let-7g, mixed with mouse LIN28A and TUT4.
(B) Schematic view of all pre-let-7 truncation constructs used in the EMSAs. GGAG sequences are boxed (Figure S6).
(C) EMSAs with pre-let-7g truncations as probe, mixed with LIN28A and NTUT759.
(D) EMSAs with pre-let-7g that has stem region of miR-21, mixed with LIN28A and NTUT759.
∗Free probe; ∗∗LIN28A:pre-let-7g and LIN28A:preE-let-7g complexes (A) and LIN28:pre-let-7g complex (C and D); ∗∗∗LIN28A:pre-let-7g:TUT4 ternary complex (A) and LIN28:pre-let-7g:NTUT759 ternary complex (C and D).
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8. Figure 6 The ZKD Plays Distinct Roles in Dicer and TUT4 Regulation
(A) Oligouridylation assays with pre-let-7g as probe, carried out using full-length mouse TUT4 and LIN28A truncation constructs.
(B) Oligouridylation assays with pre-let-7g as probe, carried out using full-length mouse TUT4 and LIN28A mutants.
(C) Oligouridylation assays with pre-let-7g mutants as probe, carried out using mouse TUT4 and LIN28A.
(D) Dicer processing assays with pre-let-7g and LIN28A full-length and truncation constructs.
∗Oligouridylated pre-let-7g.
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9. Figure 7 ZKD Is a Potential Therapeutic Target
(A) Overview of human LIN28ΔΔ:preE-let-7f-1. The CSD is in blue, the ZKD is in green, zinc is represented as gray spheres, and preE-let-7f-1 is in orange.
(B) Surface potential map of the ZKD RNA binding site. Blue represents a positively charged surface, and red indicates a negatively charged surface. The GGAG RNA is in orange.
(C) Identification of the druggable pocket within the ZKD. Pocket identification was performed using Sitemap (Schrödinger). Red crosses represent the pocket identified as suitable for targeting. The surface of ZKD is in white, and all residues are in green.
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