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Characterizing the RNA targets and position-dependent splicing regulation by TDP-43

Abstract

TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.

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Figure 1: Comparison of TDP-43 RNA binding in brain tissue from subjects with and without FTLD-TDP.
Figure 2: TDP-43 binding motif analysis.
Figure 3: The RNA splicing map of TDP-43.
Figure 4: TDP-43 regulates splicing of non-coding and protein-coding RNAs.

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Acknowledgements

We thank K. Zarnack for advice and comments on the manuscript, F. Baralle, Y. Ayalla and A.D. Ambrogio for HeLa knockdown RNA, and J. Hadfield and the genomic team at CRI for Illumina sequencing. This work was supported by the European Research Council (206726-CLIP), the MRC, Slovenian Research Agency (P2-0209, J2-2197, L2-1112, Z7-3665), Wellcome Trust and MRC Strategic Grant Award (089701/Z/09/Z), Motor Neuron Disease Association, Heaton-Ellis Trust and Psychiatry Research Trust.

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Contributions

J.R.T. carried out TDP-43 iCLIP, microarray and PCR experiments. M.B. carried out CELF2 iCLIP. T.C. and G.R. mapped the iCLIP sequence reads to genome, evaluated random barcodes, determined cross-link clusters and annotated the data. T.C. analyzed the reproducibility, sequence and positioning of TDP-43 cross-link sites and performed gene ontology analysis. B.R., A.L.N. and V.Ž. prepared RNA from knockdown cells and brain tissue. T.H. selected, sampled and analyzed the brain samples. M.C. and M.K. analyzed splice-junction microarray data and generated the RNA splicing map. R.P. prepared the embryonic stem cells. S.C., C.E.S., B.Z., J.K. and J.U. supervised the project. J.R.T., T.C., B.R., J.K., C.E.S. and J.U. prepared the manuscript.

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Correspondence to Jernej Ule.

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The authors declare no competing financial interests.

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Supplementary Figures 1–6 and Tables 1–4 (PDF 5584 kb)

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Tollervey, J., Curk, T., Rogelj, B. et al. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat Neurosci 14, 452–458 (2011). https://doi.org/10.1038/nn.2778

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