Mayr F , Schütz A , Döge N , Heinemann U .

	Figure 1. Schematic representation of the domain structure of Lin28b and the processing of pre-let-7 by Dicer. (A) Domain structure of XtrLin28a and b and human (h) Lin28b with the molecular weight and amino acid positions indicated. The truncated Lin28 variants used in this study are shown below the corresponding domain representation. (B) Lin28 inhibits the Dicer-mediated pre-let-7 processing by binding to the pre-element (preE).
	Figure 2. Comparison between apo Lin28 CSDs and oligonucleotide-bound Lin28 CSD. (A) Superimposition of apo XtrLin28b CSD (gray) and hLin28b CSD (orange). Highly conserved residues that are involved in nucleic acid binding are indicated in stick representation and marked according to their position within the XtrLin28b CSD. Gln69, which corresponds to a highly conserved Phe in bacterial CSPs, is indicated in red. (B) Cartoon representation of dT7 bound to XtrLin28b CSD. The aromatic side chains that are involved in direct stacking interactions with the dT7 bases are shown in stick representation.
	Figure 3. Molecular interactions that promote binding of dT7 to XtrLin28b CSD. The electrostatic surface potential of XtrLin28b CSD upon binding to dT7 was calculated with APBS for pH 7 with a range from −10 (red) to +10 kT (blue). The bound oligonucleotide is shown in cartoon representation. Water molecules are shown as gray spheres. (A) Detailed view of T-2 bound to the hydrophobic pocket at binding subsite 2. Besides hydrophobic contacts including a three-membered stack with Phe77 and T-1, T/U-specific contacts are mediated via the backbone atoms of Arg78 and Phe77. (B) Detailed view of T-5 bound in the hydrophobic cleft created by Lys95 and Phe66. Ser93 and Phe97 form hydrogen bonds with O4 and HN3 of T-5. Binding of T-4 is only mediated via stacking interactions with His68 and a water molecule-mediated hydrogen bond. (C) Detailed view of binding subsites 6 and 7. T-6 and T-7 are mainly bound via stacking interactions with Phe48 and Trp39, respectively.
	Figure 4. Determinants of Lin28:pre-let-7f interactions. (A) EMSAs with Xtr-pre-let-7f as a probe, mixed with increasing concentrations of protein (for XtrLin28b and XtrLin28b CSD+ZKD: 0, 0.4, 0.8, 1.6, 3.2, 6.4, 13, 26 µM; for XtrLin28b CSD and ZKD: 0, 0.8, 1.6, 3.2, 6.4, 13, 26, 51 µM). All EMSAs were performed using 1 nM α-32P-ATP-labeled RNA. (B) The terminal loop sequence and secondary structure of the Xtr-pre-let-7f mutants as predicted by Mfold (49). Regions that were mutated are shaded. (C) Results from EMSA experiments shown in (A). The binding affinity was scored according to the following dissociation constant ranges: +++++, 0.8–1.6 µM; ++++, 1.6–3.2 µM; +++, 3.2–6.4 µM; ++, 6.4–12.8 µM; +, 12.8–25.6 µM; −, >25.6 µM. (D and E) In vitro pre-miRNA processing reaction on 32P 5′-end-labeled Xtr-pre-let-7f. The indicated concentrations (in micromolar) of XtrLin28b, XtrLin28b RBDs, XtrLin28b CSD and XtrLin28b ZKD were added to 1 nM Xtr-pre-let-7f in the presence or absence of human Dicer. The samples were resolved on a 10% (w/v) denaturing PAGE and visualized by autoradiography.
	Figure 5. The Lin28 CSD remodels the terminal loop of pre-let-7 and facilitates a subsequent binding of the ZKD to the conserved GGAG motif. (A–C) RNA remodeling assay. A truncated Xtr-pre-let-7g that contains only the preE sequence and 5 bp of the upper stem region (Xtr-pre-let-7g*) was incubated with increasing concentrations of the corresponding Lin28 protein. The 5′-end of the RNA was modified with the quencher dabcyl (Dab), and the adjacent 3′-end harbored a fluorescein (FAM) label. The sequence and secondary structure of the RNA are indicated and the known ZKD- and CSD-binding sites are marked in red and green, respectively. The increase of fluorescence was plotted as a function of titrated Lin28 protein. All experiments were performed using a Cary-Eclipse fluorescence spectrometer at 293 K in 20 mM Tris (pH 7.5), 60 mM KCl, 10 mM MgCl2 and 1 mM DTT. (D) EMSAs with Xtr-pre-let-7g as a probe, mixed with increasing concentrations of XtrLin28a and the indicated XtrLin28b variants (0, 0.4, 0.8, 1.6, 3.2, 6.4, 13, 26 µM). All EMSAs were performed using 1 nM α-32P-ATP-labeled RNA. (E) Apparent dissociation constants from EMSAs shown in (D). Band intensities were quantitated from three independent experiments and used to generate the binding data. (F) In vitro pre-miRNA processing reaction on Xtr-pre-let-7g. 10 µM of the indicated XtrLin28b variant was added to 1 nM 32P 5′-end-labeled Xtr-pre-let-7g in the presence or absence of human Dicer. The samples were resolved on a 10% (w/v) denaturing PAGE and visualized by autoradiography. (G) Pre-steady-state kinetics of Lin28-mediated binding and remodeling of Xtr-pre-let-7g*. After rapid mixing of Xtr-pre-let-7g* with 15 µM (final concentration) of the indicated XtrLin28b variant, the change of FAM fluorescence was monitored for 1 s using a Chirascan stopped-flow instrument. Traces of at least eight replicates were fitted to a mono- (Y133A) or biexponential curve (Xtrlin28b), respectively (solid lines). For W39A, the time courses of the first 80 ms were fitted to a single exponential association curve, while traces from 90 ms to 1 s were fitted to a one-phase decay curve (see Supplementary Figure S11).
	Figure 6. Schematic model of Lin28-mediated binding and inhibition of pre-let-7 maturation. In the absence of Lin28, the PAZ domain of Dicer recognizes the 2 nt 3′-overhang of pre-let-7 and cleaves the substrate about 22 nt from the end (Dicer cleavage site, indicated by dark red arrows). In the presence of Lin28, the CSD first binds with its preformed nucleic acid-binding platform to pyrimidine-containing single-stranded parts in the terminal loop (preE) of pre-let-7. In a fast reaction, the CSD then remodels the preE region and melts a part of the upper stem region including the Dicer cleavage site. As the binding of Lin28 to pre-let-7 is highly cooperative, the remodeling of pre-let-7 may be facilitated in trans by another Lin28 molecule. Once the conserved GGAG is freely accessible, the Lin28 ZKD mediates a specific binding to this motif and anchors Lin28 in this position. This second reaction is rather slow, as both the ZKD and the flexible basic linker between the domains have to perform larger conformational changes as judged from apo and nucleotide-bound structures. As a consequence, the Dicer cleavage site remains constantly open, and Lin28 inhibits the cleavage by Dicer. The sequence-specific interaction via the ZKD thus ensures a directional positioning of Lin28 to pre-let-7, which may allow promoting the terminal uridylation of pre-let-7 by TUT4/Zcchc11 or PUP-2.

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