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Molecular basis of UG-rich RNA recognition by the human splicing factor TDP-43

Nature Structural & Molecular Biology volume 20, pages 1443–1449 (2013)Cite this article

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Abstract

TDP-43 encodes an alternative-splicing regulator with tandem RNA-recognition motifs (RRMs). The protein regulates cystic fibrosis transmembrane regulator (CFTR) exon 9 splicing through binding to long UG-rich RNA sequences and is found in cytoplasmic inclusions of several neurodegenerative diseases. We solved the solution structure of the TDP-43 RRMs in complex with UG-rich RNA. Ten nucleotides are bound by both RRMs, and six are recognized sequence specifically. Among these, a central G interacts with both RRMs and stabilizes a new tandem RRM arrangement. Mutations that eliminate recognition of this key nucleotide or crucial inter-RRM interactions disrupt RNA binding and TDP-43–dependent splicing regulation. In contrast, point mutations that affect base-specific recognition in either RRM have weaker effects. Our findings reveal not only how TDP-43 recognizes UG repeats but also how RNA binding–dependent inter-RRM interactions are crucial for TDP-43 function.

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Figure 1: Interaction of TDP-43 RBD with UG-rich RNA.
Figure 2: Structural overview of the TDP-43 RBD–AUG12 RNA complex.
Figure 3: Recognition of UG-rich RNA by TDP-43 RBD.
Figure 4: Impairment of CFTR exon 9 splicing by alanine mutagenesis of residues at the RRM-RRM or TDP-43 RBD–AUG12 RNA interfaces.

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Acknowledgements

The authors thank C. Maris for help with setup of filtered NMR experiments and denaturing RNA purification; M. Blatter and D. Theler for help with structure calculation and validation; and A. Clery and J. Boudet for help with ITC measurement and data analysis. We also thank S. Chesnov from the Functional Genomics Center Zürich for MS analyses. This work was supported by the project 'Central European Institute of Technology' (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund, by grants from the Human Frontier Science Program (RGP0024/2008) and by the Internationalization of the Structural Biology Research Program (CZ.1.07/2.3.00/20.0042) to P.J.L. and from Agenzia di Ricerca per la Sclerosi Laterale Amiotrofica (TARMA project) and Thierry Latran Foundation (REHNPALS) to C.S., E.B. and F.E.B. and by a Sinergia grant from the Swiss National Science Foundation (CRSII3 136222) to F.H.-T.A.

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Author notes

  1. Dalia Daujotyte, James R Tollervey & Jernej Ule

    Present address: Present addresses: Lexogen GmbH, Vienna, Austria (D.D.), Buck Institute, Novato, California, USA (J.R.T.), and Institute of Neurology, University College London, London, UK (J.U.).,

Authors and Affiliations

  1. Central European Institute of Technology, Masaryk University, Brno, Czech Republic

    Peter J Lukavsky

  2. Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland

    Peter J Lukavsky, Fred F Damberger & Frédéric H-T Allain

  3. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Dalia Daujotyte, James R Tollervey & Jernej Ule

  4. International Centre for Genetic Engineering and Biotechnology, Trieste, Italy

    Cristiana Stuani, Emanuele Buratti & Francisco E Baralle

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Contributions

P.J.L., D.D., E.B. and F.H.-T.A. designed the project. J.R.T. and J.U. performed and analyzed the individual-nucleotide-resolution CLIP data. D.D. and J.R.T. expressed the initial TDP-43 protein construct and performed band-shift assays to optimize the UG-rich RNA sequence. P.J.L. optimized the TDP-43 protein construct, prepared all samples for structural studies and ITC measurements and performed all NMR experiments, NMR data analysis, structure calculations and ITC measurements and analysis. F.F.D. assisted with setup of specialized NMR experiments. F.F.D. and F.H.-T.A. helped with NMR data analysis and F.F.D. with structure calculation and validation. C.S. and E.B. designed and performed the minigene add-back experiments; E.B. and F.E.B. participated in their interpretation. P.J.L., F.F.D., E.B. and F.H.-T.A. wrote the manuscript; all authors discussed the results and approved the manuscript.

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Correspondence to Peter J Lukavsky or Frédéric H-T Allain.

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Integrated supplementary information

Supplementary Figure 1 Sequence alignment of TDP-43 RBD from different species, interaction studies with different UG-rich RNAs by EMSA and hammerhead-ribozyme design and transcription for efficient preparation of isotope-labeled single-stranded AUG12 RNA.

(a) Sequence alignment of TDP-43 RBD from human, mouse, D. melanogaster and C. elegans. Phylogenetic analysis reveals a high level of conservation between species. Residues important for the interaction or discussed in the text are numbered according to the human protein sequence (NP_031401). (b) EMSA of TDP-43 RBD with a variety of UG-rich RNA substrates. Increasing concentrations of TDP-43 RBD protein were incubated with 32P labelled RNA oligonucleotides as indicated above the gel images. The corresponding RNA sequences are also shown above each gel image. Complex formation was analyzed using native polyacrylamide gel electrophoresis. The lower bands represent free, unbound RNA, whereas the upper bands represent TDP-43-bound RNA. A smear between the bound and unbound states suggests that the binding is unstable. (c) Sequence of the DNA template used for in vitro transcription of a stably folding 5'-cis hammerhead ribozyme AUG12 RNA. The different sequence elements are underlined and described below the sequence. (d) Secondary structure of the 5'-cis hammerhead ribozyme AUG12 RNA. The cleavage site is indicated by a black arrow and the desired AUG12 RNA cleavage product is boxed. (e) Optimization of Mg2+ concentration for in vitro transcription of 5'-cis hammerhead ribozyme AUG12 RNA. The optimal hammerhead ribozyme activity is reached at 16–18 mM Mg2+ concentration. The Mg2+ concentration for each trial transcription reaction is indicated above each gel lane and the individual products are described on the right. (f) Maximum hammerhead cleavage is reached upon incubation of the transcription reaction overnight at room temperature (see Online Methods). (g) Denaturing PAGE analysis of the purified, isotope-labelled AUG12 RNA indicates high purity of the final RNA product. (h) MALDI-TOF mass spectrometric analysis of the purified 15N-labelled and 13C,15N-labelled AUG12 RNA confirms homogeneity of the RNA products and shows the expected mass for 15N- and 13C,15N-labelled RNA.

Supplementary Figure 2 NMR data of the TDP-43 RBD–AUG12 RNA complex.

(a) A 2D TOCSY spectrum displaying the region of the uracil H5-H6 cross-peaks of TDP-43-bound AUG12 RNA. Four distinct cross-peaks for the four uracil residues of AUG12 RNA indicate binding of this RNA on the protein surface in a single register. The sequence of the AUG12 RNA is displayed at the top of the TOCSY spectrum. (b) Superposition of 1H-15N HSQC spectra representing the NMR titration of the 15N-labelled TDP-43 RBD with increasing amounts of unlabelled AUG12 RNA. The titration was performed at 25 °C in the gel filtration buffer (see Online Methods), because the unbound TDP-43 RBD was unstable in the low salt NMR buffer. The spectrum corresponding to the free protein is shown in purple and the spectra obtained at the different RNA:protein stoichiometries are shown in yellow (0.1 RNA), orange (0.3 RNA), red (0.6 RNA), brown (0.8 RNA) and blue (1.0 RNA), respectively. Note that complexes prepared for NMR measurements performed for assignment and structure determination were purified by size-exclusion chromatography to ensure precise 1:1 complex formation (see Online Methods). (c) Representative 13C plane of the 3D 13C NOESY HSQC recorded using the 15N-labelled TDP-43 RBD in complex with 13C,15N-labelled AUG12 RNA. Isotope labelling of the RNA was essential for the unambiguous assignment of NOEs between ribose protons and aromatic and aliphatic side chain protons of the protein as illustrated for the G5 H5',H5'' protons. (d) Two 13C planes of the 3D 13C NOESY HSQC recorded using the 15N-labelled TDP-43 RBD in complex with 13C,15N-labelled AUG12 RNA. Unusual, non-sequential i, i+2 NOEs between G1(i) H1',H4' and G3(i+2) H1',H8 protons are observed since the sequential i+1 nucleotide U2 is looped out. The corresponding cross-peaks are labelled red.

Supplementary Figure 3 Comparison of TDP-43–RNA complex with other tandem RRM–RNA complexes.

(a) Comparison of tandem RRMs from TDP-43, HuD, PABP, and Nucleolin. The protein is displayed in ribbon (protein backbone) representation with RRM1 in light blue, RRM2 in light green, and the linker in red. RNA residues at the RRM – RRM interface are labelled in light blue (5' residue), red (linker residues), and light green (3' residue). Only the TDP-43 tandem RRMs display RNA-binding in the 5' to 3' direction from RRM1 to RRM2 while all other tandem RRMs bind RNA in the opposite direction. (b) Comparison of the indirect sequence-specific recognition of a guanine base by TDP-43, hnRNP F and Fox-1. The protein is displayed in grey ribbon (protein backbone) representation. The heavy atoms of aromatic protein side chain atoms stacking with the RNA bases are displayed in green (carbon atoms), red (oxygen atoms), blue (nitrogen atoms). Hydrogen bonds are represented by orange dashed lines. The top figures shows the location of the indirect sequence-specific recognition of a guanine base on the entire RRM and the bottom figures zoom into the site of interaction on each RRM.

Supplementary Figure 4 Structural details of the TDP-43–AUG12 RNA complex and the TDP-43 RRM-RRM interface.

(a) The aromatic residues Trp172 and His143 of TDP-43 RRM1 are located in the vicinity of the 5' end of the AUG12 RNA, but do not contribute to RNA binding (see Text). The protein is displayed in grey ribbon (protein backbone) representation. The heavy atoms of important protein side chain and backbone atoms at the RRM – RRM interface are displayed in green (carbon atoms), red (oxygen atoms), blue (nitrogen atoms) and grey (amide protons). Hydrogen bonds are represented by orange dashed lines. Secondary structure elements of the protein are labelled and interacting amino acids are indicated by amino acid type and residue number. (b) The positioning of base and ribose moiety of G9 explains unusual chemical shifts observed for the G9 H8 and H5' protons. The same representation and colour scheme is used as in (a) for the protein. The RNA heavy atoms are shown in gold (carbon atoms), red (oxygen atoms), orange (phosphorus atoms) and blue (nitrogen atoms) and protons are shown in grey. Below, the 1H-13C HSQC spectra of the ribose and aromatic region of the 13C,15N-labelled AUG12 RNA in complex with TDP-43 RBD are shown with the unusually shifted proton resonances indicated. The strong downfield shift of the G9 H5' proton arises from the deshielding effect of the aromatic ring of Phe231, while the upfield shift of the G9 H8 proton is caused by a shielding effect from the aromatic ring of Phe221. (c) Interaction between the RRMs of TDP-43 RBD. The same representation and colour scheme is used as in (a) for the protein; sulphur atoms are shown in light grey. The main interdomain contacts are the salt bridge between Arg151 and Asp247, the hydrophobic side chain packing of Leu131, Met132, Gln134 from RRM1 and Ile249, Ile253, Thr199 from RRM2 and the hydrogen bond between Lys136 and the backbone carbonyl oxygen of Arg197. In addition, a salt bridge is observed between Lys181 (linker) and Asp247 (RRM2) and hydrogen bonds occur between the side chain of Gln184 (linker) and the backbone carbonyl oxygen of Gly245 (RRM2) and from the backbone carbonyl oxygen of Gln184 (linker) to the side chain of Lys102 from the N-terminus. (d) Molecular details of the interaction between the TDP-43 linker, RRM2 and the C-terminus. The same representation and colour scheme is used as in (c).

Supplementary Figure 5 CFTR exon 9 splicing assay and western blot analysis of TDP-43 single- and double-alanine mutants in the RNA-binding sites and the RRM-RRM interface.

(a) Agarose gel analysis of CFTR exon 9 splicing efficiency of the single alanine TDP-43 mutants. HeLa cells were siRNA treated to obtain the knockdown of the endogenous TDP-43 protein. After 36 hours, the cells were again transfected with 0.5 μg of a TG11-T5 CFTR exon 9 reporter minigene and 1 μg of plasmid carrying an si-resistant wild-type TDP-43 (WT) as positive control and an RNA-binding impaired mutant (F4L) as negative control. Additional mutants bearing alanine substitutions in the residues involved in the RRM and RNA interactions as indicated below the gels were transfected in order to determine their ability to inhibit CFTR exon 9 recognition in the absence of endogenous TDP-43. The levels of CFTR exon 9 inclusion (Ex 9+) in each sample following RT-PCR amplification using minigene-specific primers were measured by densitometric scanning and analyzed using the ImageJ program. The Western blots against the endogenous TDP-43, added back flag-TDP-43, and tubulin are reported in the lower boxes to show silencing efficiency, proper transgene expression, and equal loading. For the Western blots, 15 μg of total protein extract were analyzed in each lane. (b) Agarose gel analysis of CFTR exon 9 splicing efficiency of TDP-43 variants with single alanine mutations of amino acids at the RRM interface. The add-back experiments and Western blots were performed and analyzed as described in (a). (c) Agarose gel analysis of CFTR exon 9 splicing efficiency of TDP-43 variants with double alanine mutations. The add-back experiments and Western blots were performed and analyzed as described in (a). (d) Original Fig. 4c with uncropped Western blot gels. The add-back experiments and Western blots were performed and analyzed as described in (a).

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Lukavsky, P., Daujotyte, D., Tollervey, J. et al. Molecular basis of UG-rich RNA recognition by the human splicing factor TDP-43. Nat Struct Mol Biol 20, 1443–1449 (2013). https://doi.org/10.1038/nsmb.2698

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