Abstract
Unkempt is an evolutionarily conserved RNA-binding protein that regulates translation of its target genes and is required for the establishment of the early bipolar neuronal morphology. Here we determined the X-ray crystal structure of mouse Unkempt and show that its six CCCH zinc fingers (ZnFs) form two compact clusters, ZnF1–3 and ZnF4–6, that recognize distinct trinucleotide RNA substrates. Both ZnF clusters adopt a similar overall topology and use distinct recognition principles to target specific RNA sequences. Structure-guided point mutations reduce the RNA binding affinity of Unkempt both in vitro and in vivo, ablate Unkempt's translational control and impair the ability of Unkempt to induce a bipolar cellular morphology. Our study unravels a new mode of RNA sequence recognition by clusters of CCCH ZnFs that is critical for post-transcriptional control of neuronal morphology.
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Acknowledgements
We are grateful to the staff of the Northeastern Collaborative Access Team for their assistance with collection of the X-ray data; to K. Kathrein (Boston Children's Hospital) for zebrafish cDNA, E. Greer (Boston Children's Hospital) for worm cDNA and K. Roper (University of Queensland) for sponge cDNA; and to members of the laboratories of D.J.P. and Y.S. for discussion and comments on the manuscript. This study was supported by the Nancy Lurie Marks Post-doctoral Fellowship (J.M.), the LOEWE program Ubiquitin Networks (Ub-Net) of the State of Hesse (Germany) (K.Z.), US National Institutes of Health grants MH096066 (Y.S.) and GM104962 (D.J.P.), and Memorial Sloan Kettering Cancer Center support Grant/Core Grant (P3O CA008748) (D.J.P.). Y.S. is supported as an American Cancer Society Research Professor.
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Contributions
J.M. and M.T. performed the experiments; K.Z. carried out the computational analysis of sequencing data; J.M., M.T., K.Z., Y.S. and D.J.P. analyzed and interpreted the results and wrote the manuscript.
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Y.S. is a cofounder of Constellation Pharmaceuticals, Inc., and a member of its scientific advisory board. Y.S. is also a consultant for Active Motif.
Integrated supplementary information
Supplementary Figure 1 The unique topologies of the CCCH ZnF clusters of Unkempt.
(a) Disorder probability profile of the full-length mouse Unkempt protein determined by the DISOPRED prediction method within the PSIPRED protein sequence analysis workbench (http://bioinf.cs.ucl.ac.uk/psipred/). (b) Structure-based sequence alignment of ZnF1–3 and ZnF4–6 tandem ZnF cassettes. Zn-coordinating cysteine and histidine residues are highlighted in blue and the secondary structure elements are shown above the sequences. Residues involved in base stacking and hydrophobic interactions with RNA nucleotides are indicated by rectangles, while asterisks and triangles indicate residues that form hydrogen bonds (H bonds) with RNA bases via their backbone functional groups and side chains, respectively. The α, β and η symbols refer to α-helix, β-strands and 310-helix, respectively. Helices are displayed as squiggles, β-strands are rendered as arrows, strict β-turns as TT letters. (c) Superimposition of the ZnF4–6 cluster of Unkempt (light blue) and the CCCH ZnFs 5–7 of yeast Nab2 protein (red) revealing substantial differences in their topologies. (d) Comparison of ZnF1–3 of Unkempt (yellow) with the CCCH ZnFs 5–7 of yeast Nab2 protein (red) indicates structural diversity of both folds. Both comparisons were done by superimposing the most C-terminal ZnFs in each pair of clusters, i.e. ZnF3 and ZnF6 of Unkempt and ZnF7 of Nab2 protein.
Supplementary Figure 2 Intramolecular protein-protein interactions within the ZnF1–3 and ZnF4–6 clusters of Unkempt in their RNA-bound conformation.
(a) Structure of the ZnF4–6 cluster shown in a ribbon representation. ZnFs 4, 5, and 6 are colored in light blue, the N-terminal loop with α-helix 1 and the linker separating ZnFs 5 and 6 with α-helix 2 are colored in light orange, and the linker separating ZnFs 4 and 5 is colored in green. (b) Hydrophobic and hydrogen-bonding interactions of the N-terminal α-helix 1 and the loop residues (Thr209, Val212, Leu213, Tyr216 and Lys217) with the linker separating ZnFs 5 and 6 (Thr285, Phe289 and Tyr294), and with the linker separating ZnFs 4 and 5 (Asp242, Arg244 and Ser246). (c) Hydrogen-bonding interactions and van der Waals contacts of the linker separating ZnFs 4 and 5 (Arg244, Arg245, Ser246, Tyr252, Ser254 and Pro256) with the N-terminal loop and α-helix 1 (Leu213, Gly214, Tyr216 and Lys217), and with the linker between ZnFs 5 and 6 (Thr283, Glu286, Gln287, His290 and Ile293). (d) Structure-based sequence alignment of the ZnF1–3 and ZnF4–6 clusters. Cysteine and histidine residues coordinated to Zn are highlighted in blue. Secondary structure elements of ZnF1–3 and ZnF4–6 are shown above the sequences and are colored as in a and b. Residues involved in intramolecular hydrophobic interactions (rectangles) and hydrogen-bonding via their backbone functional groups (asterisks) or side chains (triangles) in both domains are color-coded for the ZnF regions, the N- and C-termini, and for the intervening linkers. (e) Structure of the ZnF1–3 cluster shown in ribbon representations. ZnFs 1, 2, and 3 are colored in light blue, the N- and C-termini and the linker separating ZnFs 2 and 3 with α-helix 3 are colored in light orange, and the linker separating ZnFs 1 and 2 is colored in green. (f) Hydrophobic and hydrogen-bonding interactions of the N-terminal α-helix 1 and loop residues (His33, Tyr36, Leu37, Phe40 and Arg41) with the linker between ZnFs 2 and 3 (Thr116, Tyr120 Tyr125), and with the linker separating ZnFs 1 and 2 (Arg71). (g) Hydrogen-bonding interactions and van der Waals contacts of the linker separating ZnFs 1 and 2 (Arg71, Arg72, Arg73, Ser74, Arg76, Tyr84, Pro86, Asp87 and Tyr89) with the N-terminal loop and α-helix 1 (Leu37, Lys38, Phe40 and Arg41), and with the linker between ZnFs 2 and 3 (Thr112, Glu117, Arg118, His121 and Tyr124). Hydrogen bonds that are equivalent between ZnF1–3 and ZnF4–6 are colored red. Cysteine and histidine side chains coordinated to Zn atoms (light blue balls) are shown in stick representation.
Supplementary Figure 3 Stereo views comparing the structures of ZnF4–6 and ZnF1–3 bound to their RNA substrates.
(a) Superimposition of structures of Unkempt’s ZnF1–3 bound to a U1-U2-A3 RNA element (yellow) and ZnF4–6 bound to a U1-U2-A3-G4 RNA element (light blue) showing similarities in spatial arrangement of both clusters and in the positions of the bound RNA substrates. (b) Conformations of the bound RNA substrates in the ZnF1–3 and ZnF4–6 complexes as shown in panel (a). (c) Superimposition of the RNA-contacting amino acid residues in their orientations as shown in the complexes in panel (a). The UAG motif (dark blue) bound on the surface of the ZnF4–6 cluster (light blue) highlighting the key residues contacting the RNA. Positions of corresponding residues in the ZnF1–3 cluster are shown in yellow.
Supplementary Figure 4 Base composition of the U-rich motif in HeLa and SH-SY5Y cells.
Heatmaps illustrate the positional frequency of each of the 64 possible trimers within Unkempt binding sites between 15 nts upstream and downstream of the binding site maxima in HeLa (a) and SH-SY5Y cells (b). Traces of individual trimers are displayed in the same order as in Fig. 3a. Plots above each heatmap profile the mean enrichment of different clusters of triplets color-coded as shown in the heatmap (UAG in red, the 13 enriched U-rich triplets in blue, and all other triplets in gray). The scale below the heatmap indicates fold enrichment over the median triplet frequency in a 103-nt window around the binding site maxima.
Supplementary Figure 5 Profiles of individual RNA trimers within Unkempt-binding sites in the mouse embryonic brain.
Each of the 64 profiles indicates the positional frequency of a trimer in a 101-nt window around the binding site maxima. The different clusters of triplets are color-coded: UAG in red and the 13 enriched U-rich triplets in blue.
Supplementary Figure 6 Effects of structure-guided mutations on RNA-binding affinity and protein translation.
(a) Anti-correlation between the in vivo RNA-binding affinity of Unkempt (Fig. 5d) and steady-state protein levels shown in Fig. 6a. Relative protein levels are quantified band intensities normalized to the corresponding actin control. All values are compared with no Dox control. Also shown are color-coded equations of linear regression trendlines with corresponding correlation coefficients for each target protein. (b-e) Impact of Y216A mutation on RNA-binding affinity of Unkempt. (b) Binding of the wild-type or Y216A mutant recombinant ZnF1–6 of Unkempt to HSPA8 18-mer RNA. Binding affinities were measured by ITC. (c) Quantification of the results shown in b. (d) The morphologies of HeLa cells inducibly expressing GFP alone or GFP and either wild-type or Y216A Unkempt mutant were quantified at 48 hours of Dox treatment by calculating their axial ratios (see also Fig. 6c). The results are compared with GFP control. Error bars, s.d. (n = 30 GFP-positive cells). *P < 0.0005; **P < 0.00001 by two-tailed Student’s t-test. (e) Immunoblot analysis showing similar expression levels of the induced Unkempt protein in cells used for the morphologic analysis in d.
Supplementary Figure 7 Sequence conservation of the Unkempt protein across species.
Full-length sequence of each Unkempt ortholog is shown. Sequence numbering corresponds to consecutive residue position in human (H. sapiens) and mouse (M. musculus) Unkempt orthologs. White letters on blue background highlight Zn-coordinating cysteine and histidine residues, white letters on red background indicate identical residues, red letters on white background indicate residues with similar physicochemical properties, and blue frames indicate global similarity. Multiple sequence alignment was performed using the Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) and re-drawn using the ESPript 3.0 software (http://espript.ibcp.fr/ESPript/ESPript/index.php).
Supplementary Figure 8 Molecular models of RNA recognition by the six CCCH ZnFs of Unkempt.
Molecular models of RNA recognition by the six CCCH ZnFs of Unkempt. Two models illustrate our proposed binding mode of the ZnF1–6 domain to different RNA substrates in which the UAG and the UUA motifs are separated by spacers of different lengths, one nucleotide in the model shown in panel a, and three nucleotides in the model shown in panel b. The bound RNA nucleotides are colored yellow with the nitrogens in blue, oxygens in red, and phosphorous in orange. The modeled 1- and 3-nt spacers between the bound UAG and UUA motifs are colored gray. The protein and RNA molecules are drawn to scale.
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Supplementary Figures 1–8 and Supplementary Note (PDF 2240 kb)
Supplementary Data Set 1
Uncropped scans with size marker indications (PDF 710 kb)
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Murn, J., Teplova, M., Zarnack, K. et al. Recognition of distinct RNA motifs by the clustered CCCH zinc fingers of neuronal protein Unkempt. Nat Struct Mol Biol 23, 16–23 (2016). https://doi.org/10.1038/nsmb.3140
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DOI: https://doi.org/10.1038/nsmb.3140
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