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Multi-domain utilization by TUT4 and TUT7 in control of let-7 biogenesis

Nature Structural & Molecular Biology volume 24, pages 658–665 (2017)Cite this article

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Abstract

The uridyl transferases TUT4 and TUT7 (collectively called TUT4(7)) switch between two modes of activity, either promoting expression of let-7 microRNA (monoU) or marking it for degradation (oligoU). Lin28 modulates the switch via recruitment of TUT4(7) to the precursor pre-let-7 in stem cells and human cancers. We found that TUT4(7) utilize two multidomain functional modules during the switch from monoU to oligoU. The catalytic module (CM) is essential for both activities, while the Lin28-interacting module (LIM) is indispensable for oligoU. A TUT7 CM structure trapped in the monoU activity staterevealed a duplex-RNA-binding pocket that orients group II pre-let-7 hairpins to favor monoU addition. Conversely, the switch to oligoU requires the ZK domain of Lin28 to drive the formation of a stable ternary complex between pre-let-7 and the inactive LIM. Finally, ZK2 of TUT4(7) aids oligoU addition by engaging the growing oligoU tail through uracil-specific interactions.

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Figure 1: Assembly of the Lin28–pre-let-7/TUT4 ternary complex.
Figure 2: Structure of human TUT7 CM in the monoU-addition state.
Figure 3: Structure of human TUT7 CM in the oligoU-addition state.
Figure 4: Role of ZK2 in Lin28-dependent oligoU addition compared with monoU addition.
Figure 5: Active site interactions in CM–U2.
Figure 6: Active site comparison of TUT7 CM with CID1 and T. brucei TUT4.
Figure 7: Recognition of group II pre-let-7 substrate.
Figure 8: Model of the TUT4(7) activity switch.

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References

  1. Büssing, I., Slack, F.J. & Grosshans, H. let-7 microRNAs in development, stem cells and cancer. Trends Mol. Med. 14, 400–409 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Shell, S. et al. Let-7 expression defines two differentiation stages of cancer. Proc. Natl. Acad. Sci. USA 104, 11400–11405 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Thomson, J.M. et al. Extensive post-transcriptional regulation of microRNAs and its implications for cancer. Genes Dev. 20, 2202–2207 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Johnson, S.M. et al. RAS is regulated by the let-7 microRNA family. Cell 120, 635–647 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Viswanathan, S.R. et al. Lin28 promotes transformation and is associated with advanced human malignancies. Nat. Genet. 41, 843–848 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Piskounova, E. et al. Lin28A and Lin28B inhibit let-7 microRNA biogenesis by distinct mechanisms. Cell 147, 1066–1079 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Heo, I. et al. Lin28 mediates the terminal uridylation of let-7 precursor MicroRNA. Mol. Cell 32, 276–284 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Newman, M.A., Thomson, J.M. & Hammond, S.M. Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing. RNA 14, 1539–1549 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rybak, A. et al. A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment. Nat. Cell Biol. 10, 987–993 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Viswanathan, S.R., Daley, G.Q. & Gregory, R.I. Selective blockade of microRNA processing by Lin28. Science 320, 97–100 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Piskounova, E. et al. Determinants of microRNA processing inhibition by the developmentally regulated RNA-binding protein Lin28. J. Biol. Chem. 283, 21310–21314 (2008).

    Article  CAS  PubMed  Google Scholar 

  13. Heo, I. et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 138, 696–708 (2009).

    Article  CAS  PubMed  Google Scholar 

  14. Hagan, J.P., Piskounova, E. & Gregory, R.I. Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nat. Struct. Mol. Biol. 16, 1021–1025 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chang, H.-M., Triboulet, R., Thornton, J.E. & Gregory, R.I. A role for the Perlman syndrome exonuclease Dis3l2 in the Lin28-let-7 pathway. Nature 497, 244–248 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Faehnle, C.R., Walleshauser, J. & Joshua-Tor, L. Mechanism of Dis3l2 substrate recognition in the Lin28-let-7 pathway. Nature 514, 252–256 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ustianenko, D. et al. Mammalian DIS3L2 exoribonuclease targets the uridylated precursors of let-7 miRNAs. RNA 19, 1632–1638 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Heo, I. et al. Mono-uridylation of pre-microRNA as a key step in the biogenesis of group II let-7 microRNAs. Cell 151, 521–532 (2012).

    Article  CAS  PubMed  Google Scholar 

  19. Stagno, J., Aphasizheva, I., Aphasizhev, R. & Luecke, H. Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases. Proc. Natl. Acad. Sci. USA 104, 14634–14639 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Stagno, J., Aphasizheva, I., Bruystens, J., Luecke, H. & Aphasizhev, R. Structure of the mitochondrial editosome-like complex associated TUTase 1 reveals divergent mechanisms of UTP selection and domain organization. J. Mol. Biol. 399, 464–475 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Stagno, J., Aphasizheva, I., Rosengarth, A., Luecke, H. & Aphasizhev, R. UTP-bound and Apo structures of a minimal RNA uridylyltransferase. J. Mol. Biol. 366, 882–899 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Rajappa-Titu, L. et al. RNA editing TUTase 1: structural foundation of substrate recognition, complex interactions and drug targeting. Nucleic Acids Res. 44, 10862–10878 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Deng, J., Ernst, N.L., Turley, S., Stuart, K.D. & Hol, W.G.J. Structural basis for UTP specificity of RNA editing TUTases from Trypanosoma brucei. EMBO J. 24, 4007–4017 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lunde, B.M., Magler, I. & Meinhart, A. Crystal structures of the Cid1 poly (U) polymerase reveal the mechanism for UTP selectivity. Nucleic Acids Res. 40, 9815–9824 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Munoz-Tello, P., Gabus, C. & Thore, S. Functional implications from the Cid1 poly(U) polymerase crystal structure. Structure 20, 977–986 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Yates, L.A. et al. Structural basis for the activity of a cytoplasmic RNA terminal uridylyl transferase. Nat. Struct. Mol. Biol. 19, 782–787 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Munoz-Tello, P., Gabus, C. & Thore, S. A critical switch in the enzymatic properties of the Cid1 protein deciphered from its product-bound crystal structure. Nucleic Acids Res. 42, 3372–3380 (2014).

    Article  CAS  PubMed  Google Scholar 

  28. Thornton, J.E. et al. Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4). Nucleic Acids Res. 42, 11777–11791 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jones, M.R. et al. Zcchc11-dependent uridylation of microRNA directs cytokine expression. Nat. Cell Biol. 11, 1157–1163 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Liu, X. et al. A MicroRNA precursor surveillance system in quality control of MicroRNA synthesis. Mol. Cell 55, 868–879 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lim, J. et al. Uridylation by TUT4 and TUT7 marks mRNA for degradation. Cell 159, 1365–1376 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Pirouz, M., Du, P., Munafò, M. & Gregory, R.I. Dis3l2-mediated decay is a quality control pathway for noncoding RNAs. Cell Rep. 16, 1861–1873 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Łabno, A. et al. Perlman syndrome nuclease DIS3L2 controls cytoplasmic non-coding RNAs and provides surveillance pathway for maturing snRNAs. Nucleic Acids Res. 44, 10437–10453 (2016).

    PubMed  PubMed Central  Google Scholar 

  34. Ustianenko, D. et al. TUT-DIS3L2 is a mammalian surveillance pathway for aberrant structured non-coding RNAs. EMBO J. 35, 2179–2191 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ameres, S.L. et al. Target RNA-directed trimming and tailing of small silencing RNAs. Science 328, 1534–1539 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. van Wolfswinkel, J.C. et al. CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs. Cell 139, 135–148 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. Ren, G., Chen, X. & Yu, B. Uridylation of miRNAs by hen1 suppressor1 in Arabidopsis. Curr. Biol. 22, 695–700 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhao, Y. et al. The Arabidopsis nucleotidyl transferase HESO1 uridylates unmethylated small RNAs to trigger their degradation. Curr. Biol. 22, 689–694 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Iliopoulos, D., Hirsch, H.A. & Struhl, K. An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation. Cell 139, 693–706 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Urbach, A. et al. Lin28 sustains early renal progenitors and induces Wilms tumor. Genes Dev. 28, 971–982 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Guo, Y. et al. Identification and characterization of lin-28 homolog B (LIN28B) in human hepatocellular carcinoma. Gene 384, 51–61 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Wang, Y.-C. et al. Lin-28B expression promotes transformation and invasion in human hepatocellular carcinoma. Carcinogenesis 31, 1516–1522 (2010).

    Article  CAS  PubMed  Google Scholar 

  43. West, J.A. et al. A role for Lin28 in primordial germ-cell development and germ-cell malignancy. Nature 460, 909–913 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Fu, X. et al. miR-26a enhances miRNA biogenesis by targeting Lin28B and Zcchc11 to suppress tumor growth and metastasis. Oncogene 33, 4296–4306 (2014).

    Article  CAS  PubMed  Google Scholar 

  45. Astuti, D. et al. Germline mutations in DIS3L2 cause the Perlman syndrome of overgrowth and Wilms tumor susceptibility. Nat. Genet. 44, 277–284 (2012).

    Article  CAS  PubMed  Google Scholar 

  46. Thornton, J.E., Chang, H.-M., Piskounova, E. & Gregory, R.I. Lin28-mediated control of let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) and Zcchc6 (TUT7). RNA 18, 1875–1885 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Nam, Y., Chen, C., Gregory, R.I., Chou, J.J. & Sliz, P. Molecular basis for interaction of let-7 microRNAs with Lin28. Cell 147, 1080–1091 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kim, B. et al. TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms. EMBO J. 34, 1801–1815 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Yeom, K.-H. et al. Single-molecule approach to immunoprecipitated protein complexes: insights into miRNA uridylation. EMBO Rep. 12, 690–696 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Nakel, K., Bonneau, F., Eckmann, C.R. & Conti, E. Structural basis for the activation of the C. elegans noncanonical cytoplasmic poly(A)-polymerase GLD-2 by GLD-3. Proc. Natl. Acad. Sci. USA 112, 8614–8619 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Hamill, S., Wolin, S.L. & Reinisch, K.M. Structure and function of the polymerase core of TRAMP, a RNA surveillance complex. Proc. Natl. Acad. Sci. USA 107, 15045–15050 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Bai, Y., Srivastava, S.K., Chang, J.H., Manley, J.L. & Tong, L. Structural basis for dimerization and activity of human PAPD1, a noncanonical poly(A) polymerase. Mol. Cell 41, 311–320 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Lapkouski, M. & Hällberg, B.M. Structure of mitochondrial poly(A) RNA polymerase reveals the structural basis for dimerization, ATP selectivity and the SPAX4 disease phenotype. Nucleic Acids Res. 43, 9065–9075 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Yates, L.A. et al. Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity. Nucleic Acids Res. 43, 2968–2979 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Wang, L. et al. LIN28 zinc knuckle domain is required and sufficient to induce let-7 oligouridylation. Cell Rep. 18, 2664–2675 (2017).

    Article  CAS  PubMed  Google Scholar 

  56. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Terwilliger, T.C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69 (2008).

    Article  CAS  PubMed  Google Scholar 

  58. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr. 67, 293–302 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  CAS  PubMed  Google Scholar 

  62. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  PubMed  Google Scholar 

  63. Davis, I.W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank members of the Joshua-Tor laboratory and the CSHL proteomics facility. We thank A. Héroux for help at the National Synchrotron Light Source, which is supported by the Department of Energy, Office of Basic Energy Sciences. We thank the Berkeley Center for Structural Biology, which is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and by the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Office of Science, Office of Basic Energy Sciences of the Department of Energy under contract no. DE-AC02-05CH11231. We thank the staff of the Structural Biology Center (beamline 19ID) at the Advanced Photon Source at Argonne National Laboratory. Argonne is operated by UChicago Argonne, LLC, for the Department of Energy, Office of Biological and Environmental Research, under contract no. DE-AC02-06CH11357. Plasmids containing the open reading frames of mouse TUT4 and human TUT7 were kind gifts from R.I. Gregory (Harvard Medical School) and V.N. Kim (Seoul National University), respectively. This work was supported by NIH grant R01-GM114147 (to L.J.), the Cold Spring Harbor Laboratory Women in Science Award (to L.J.) and the Watson School of Biological Sciences (to J.W. and L.J.). L.J. is an investigator of the Howard Hughes Medical Institute.

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Author notes

  1. Jack Walleshauser

    Present address: Plexxikon Inc., Berkeley, California, USA

  2. Christopher R Faehnle and Jack Walleshauser: These authors contributed equally to this work.

Authors and Affiliations

  1. W.M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Christopher R Faehnle, Jack Walleshauser & Leemor Joshua-Tor

  2. Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Jack Walleshauser

  3. Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Leemor Joshua-Tor

  4. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Christopher R Faehnle, Jack Walleshauser & Leemor Joshua-Tor

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Contributions

C.R.F., J.W. and L.J. designed all experiments. C.R.F. and J.W. conducted all experiments. All authors contributed to data analysis and wrote the paper.

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Correspondence to Leemor Joshua-Tor.

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Integrated supplementary information

Supplementary Figure 1 Biochemical characterization of the monoU and oligoU activity switch of TUT4(7).

(a) Mouse TUT4 and human TUT7 were assayed for monoU and Lin28-dependent oligoU addition activities for group II pre-let-7g (1 nt 3’-end overhang) and group I pre-let-7g substrate with a pre-existing 2 nt overhang. (b) Sequence and secondary structure prediction (mFold) of pre-let-7g used in the activity assays. The GGAG element within the terminal loop is highlighted (red shadow). Pre-let-7g is a group II miRNA, containing a 1 nt 3’-end overhang. The green box highlights the stem region, which was the basis for design of dsRNA mimics for crystallization. (c) Shown is the stem region of pre-let-7g with a pre-existing 2 nt overhang end structure, as used in the activity assays. (d) Shown is the sequence of the palindromic RNA used in crystallization to obtain the CM-dsRNA structure. (e) Domain layout of mouse TUT4 (mTUT4). The Lin28-interating module (LIM) and catalytic module (CM) are indicated. The LIM is composed of the CCHH zinc finger motif (pink) and inactive NTD1 (light purple, white hash marks). The CM contains the zinc knuckle domains ZK1-ZK3 (purple) and the active NTD2 (green). The mTUT4 truncation constructs analyzed in this study are labeled (mT1-mT7). We found that construct mT1 (truncation of C-terminal domain up to ZK3) has the same activity as full-length mTUT4, but is easier to purify and more stable. We therefore used mT1 as the backbone for other mutant constructs used in this study. (f) MonoU addition time course (0-15 minutes) assay conducted with the indicated mTUT4 truncation construct and pre-let-7g. Reactions were resolved to single nucleotide resolution on 10% sequencing gels. 1 nt vs. 2nt products are labeled. (g) Lin28-dependent oligoU addition time course (0-15 minutes) assays conducted with the indicated mTUT4 truncation construct and pre-let-7g (pre-incubated with mouse Lin28). Reactions were resolved to single nucleotide resolution on 10% sequencing gels. OligoU and monoU products are labeled as 1) oligoU, 2) oligoUshort, and 3) monoU. Gels shown are representative of three technical replicates. Uncropped source gels and replicate gels are shown in Supplementary Data Set 1.

Supplementary Figure 2 Analysis of Lin28 domains controlling the TUT4 oligoU switch.

(a) Domain layout of mTUT4. The construct used for the experiments presented in this figure is indicated (mT1). (b) Domain layout of full length mouse Lin28 (CSD, brown and CCHCx2, blue) and mutant constructs used for experiments presented in panels c-g (mL1-mL5). Point mutants of the Lin28 CCHC zinc-binding motifs (mL3, C139A, C142A) and (mL4, C161A, C164A) are indicated by a red X. (c) OligoU addition assay of mT1 and pre-let-7g pre-incubated with mL1. (d) OligoU addition assay of mT1 and pre-let-7g pre-incubated with mL2. (e) OligoU addition assay of mT1 and pre-let-7g pre-incubated with mL3. (f) OligoU addition assay of mT1 and pre-let-7g pre-incubated with mL4. (g) OligoU addition assay of mT1 and pre-let-7g pre-incubated with mL5. Shown in each panel is a schematic of mTUT4 (green and light purple oval), pre-let-7g (black cartoon), and the Lin28 construct (CSD, brown and CCHCx2, blue) used in the corresponding assay. The product generated in each reaction is represented by either oligoU tail (orange ribbon cartoon) or monoU addition (orange U). Reactions were resolved to single nucleotide resolution on 10% sequencing gels. OligoU and monoU products are labeled for each gel. Gels shown are representative of three technical replicates. Uncropped source gels and replicate gels are shown in Supplementary Data Set 1.

Supplementary Figure 3 Structure of human TUT7 CM.

(a) The structure of CM-apo (collected at the Zn2+ absorption edge, λ=1.28 Å) was determined by MR using a model output from the Autobuild routine in Phenix derived from SAD phasing of a Se-Met derivative. The N-lobe (green), C-lobe (light blue), and ZK2 (purple) are labeled. As described (see Methods), all datasets collected on crystals that were grown and cryoprotected in high lithium sulfate conditions contained a sulfate (yellow stick) and two iodide ions (gold spheres) in place of the desired ligand in the substrate-binding site. To capture substrate complexes we devised a soaking procedure in low ionic strength buffer conditions (see Methods). To verify the identity and position of Zn2+ coordinated by ZK2 (green sphere), we calculated an anomalous difference map (red mesh, 4.0 σ). (b) The asymmetric unit of CM-dsRNA, contains one palindromic RNA duplex (5’-pGCGAAGCGCUUCGCU-3’, purple and orange strands) and two CMs. Each CM is bound to opposite ends of the duplex, with the 1 nt overhang (U15) positioned in the active site. The difference electron density map (orange and purple mesh) calculated after MR and before inclusion of RNA in the model is shown at 2.0 σ. In this manuscript we describe only one CM (N-lobe, green and C-lobe, light blue) and the RNA duplex as the relevant monoU addition state structure. However, the remaining copy of the CM (light brown) is nearly equivalent to the one discussed in the text. We discuss the CM that exhibited the highest quality electron density map by visual inspection. (c) Close-up view of the difference electron density map (contoured at 2.0 σ) prior to inclusion of the RNA model in refinement. The final refined RNA duplex is displayed (orange and purple sticks).

Supplementary Figure 4 Comparison of TUTase folds and PAP folds.

(a) Structure of hTUT7 CM with the N-lobe (green) and C-lobe (light blue) highlighted. The ZK2 domain, unique to mammalian TUTases is also labeled (purple ribbon). (b) Structure of S. pombe Cid127 with lobes colored as in panel a. (c) Structure of TbTUT422 with lobes colored as in panel a. (d) Structure of TbTUT122 with lobes colored as in panel a. Also shown are the RRM domain (gray cartoon) and the unique CCHH ZnF domain (yellow cartoon). A CCHH zinc finger is also present in the TUT4(7) LIM. (e) Structure of TbRET223 with lobes colored as in panel a. Also shown is the RRM domain (white cartoon). (f) Structure of TbMEAT120 with lobes colored as in panel a. Also shown is helical insertion unique to TbMEAT1 (yellow cartoon). (g) Structure of mtPAP54 with lobes colored as in panel a. MtPAP is known to form a homodimer, but only one subunit is displayed. (c) Structure of GLD-251 with lobes colored as in panel a. GLD-2 was determined in complex with GLD-3 (red cartoon), which is required for GLD-2 activation. (c) Structure of Trf4p52 with lobes colored as in panel a. Trf4p was determined in complex with the CCHC zinc knuckle protein, Air2p (red cartoon), a modulator of Trf4p polymerase activity.

Supplementary Figure 5 Difference electron density maps for CM-U2 and CM-U5 structures.

(a) Overall structure of CM-U2 with N-lobe (green), C-lobe (light blue), and ZK2 (purple) labeled. The difference electron density map (red mesh) accounting for UMPNPP (yellow stick) and U2 RNA (orange stick), calculated prior to their inclusion in refinement, is shown (2.0 σ contour level). (b) Close-up view of difference map and UMPNPP-U2 from panel a. (c) Overall structure of CM-U5 with N-lobe (green), C-lobe (light blue), and ZK2 (purple) labeled. The difference electron density map (red mesh) accounting for U5 RNA (orange stick), calculated prior to their inclusion in refinement, is shown (2.0 σ). (d) Close-up view of difference map and U5 from panel c. Clear density could only account for U2-U5 in the final refined model. (e) Overlay of UMPNPP-U2 (yellow stick, UMPNPP and orange stick, U2) and U5 RNA (gray stick) as a result of superposition of CM-U2 vs. CM-U5 structures. Also shown is the ZK2 (purple cartoon and stick).

Supplementary Figure 6 OligoU activities of mTUT4 zinc binding mutants.

(a) Domain layout of mTUT4. Indicated are mT1 and mutant constructs of the CCHH zinc finger (CCHHmut) and CCHC zinc knuckle (ZK1-ZK2mut, ZK1-ZK3mut, ZK2-ZK3mut) domains used for experiments shown in this figure. We made double mutants of the ZK domains, because single mutants had only modest effects on oligoU activity (data not shown). Mutations made are as follows: CCHHmut (C326A, C329A), ZK1mut (C932A, C935A), ZK2mut (C1312A, C1315A), and ZK3mut (C1360A, C1363A). (b) OligoU activity assays of mT1 and the indicated mutant. CCHHmut is severely impaired for oligoU addition as reported previously47. Mutation of ZK2, either as ZK1-ZK2mut or ZK2-ZK3mut, reproducibly leads to oligoUshort products. OligoU and monoU products are labeled as 1) oligoU, 2) oligoUshort, and 3) monoU. Gels shown are representative of three technical replicates. Uncropped source gels and replicate gels are shown in Supplementary Data Set 1.

Supplementary Figure 7 Substrate recognition by TUT7 CM.

(a) Structural superposition of CM-dsRNA vs. CM-U5. The dsRNA (orange and purple transparent cartoon) and U5 RNA (gray cartoon) substrates are displayed. (b) Superposition of CM-dsRNA vs. CM-U5. Shown are the UTP (yellow stick) and dsRNA (3’-strand orange cartoon, 5’-strand gray) from CM-dsRNA and ZK2 (purple cartoon, sticks) from CM-U5. (c) Overall structure of CM-dsRNA. The 5’-anchor (dark green) is indicated. Also highlighted are (red box) the group II miRNA binding site and the groove loop (blue box). (d) Close-up view of the active site interactions in CM-dsRNA. Incoming nucleotide (UTP, yellow stick) in the +1 position, dsRNA (orange stick), and CM substrate interacting residues (N-lobe residues; green sticks, C-lobe; gray sticks) are shown. General H-bond interactions (gray dashed lines) are displayed, as are U-specific interactions with the +1 position (red dashed lines) and the -1 position (black dashed line). Also displayed are the 5’-anchor (green cartoon and sticks) and the hydrophobic platform (green sticks). (e) Close-up view of the groove loop interactions with dsRNA (purple and orange strands). Groove loop residues (blue sticks) primarily make van der Waals’ contact with the minor groove of dsRNA.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7. (PDF 1922 kb)

Supplementary Dataset 1

Uncropped source and replicate gels. (PDF 3867 kb)

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Faehnle, C., Walleshauser, J. & Joshua-Tor, L. Multi-domain utilization by TUT4 and TUT7 in control of let-7 biogenesis. Nat Struct Mol Biol 24, 658–665 (2017). https://doi.org/10.1038/nsmb.3428

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