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

Tollervey JR, Curk T, Rogelj B, Briese M, Cereda M, Kayikci M, König J, Hortobágyi T, Nishimura AL, Zupunski V, Patani R, Chandran S, Rot G, Zupan B, Shaw CE, Ule J - Nat. Neurosci. (2011)

Bottom Line: 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.We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge, UK.

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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TDP-43 binding motif analysis(a) z-scores of penamer occurrence within the 61 nt sequence surrounding all crosslink sites (−30 nt to +30 nt) are shown for healthy and FTLD-TDP brain iCLIP. The sequences of the two most enriched pentamers and the Pearson correlation coefficient (r) between the two samples are given. (b) Enrichment of crosslinking compared to randomised data in UG repeats of different lengths in TDP-43 iCLIP experiments from healthy brains (blue) and FTLD-TDP brains (red), and CELF2 experiments in healthy brain (green). (d) Enrichment of crosslinking compared to randomised data on UG repeats of different lengths in TDP-43 iCLIP experiments from SH-SY5Y and hES cells, where we either included all crosslink sites (dark grey), or only those mapping to crosslink clusters (light grey). (e) Analysis of positions of UGUGU enrichment compared to randomised data around TDP-43 crosslink sites in healthy brains (blue) and FTLD-TDP brains (red), and CELF2 experiments in healthy brain (green). (e) Analysis of positions of UGUGU enrichment compared to randomised data in TDP-43 iCLIP experiments from SH-SY5Y and hES cells, where we either included all crosslink sites (dark grey), or only those mapping to crosslink clusters (light grey). (f) TDP-43 crosslinking in its own transcript (TARDBP). The replicate experiments are summed and shown in a single track. The RNA sequence underlying the peak crosslinking site is shown, with UG and GU dinucleotides in pink.
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Figure 2: TDP-43 binding motif analysis(a) z-scores of penamer occurrence within the 61 nt sequence surrounding all crosslink sites (−30 nt to +30 nt) are shown for healthy and FTLD-TDP brain iCLIP. The sequences of the two most enriched pentamers and the Pearson correlation coefficient (r) between the two samples are given. (b) Enrichment of crosslinking compared to randomised data in UG repeats of different lengths in TDP-43 iCLIP experiments from healthy brains (blue) and FTLD-TDP brains (red), and CELF2 experiments in healthy brain (green). (d) Enrichment of crosslinking compared to randomised data on UG repeats of different lengths in TDP-43 iCLIP experiments from SH-SY5Y and hES cells, where we either included all crosslink sites (dark grey), or only those mapping to crosslink clusters (light grey). (e) Analysis of positions of UGUGU enrichment compared to randomised data around TDP-43 crosslink sites in healthy brains (blue) and FTLD-TDP brains (red), and CELF2 experiments in healthy brain (green). (e) Analysis of positions of UGUGU enrichment compared to randomised data in TDP-43 iCLIP experiments from SH-SY5Y and hES cells, where we either included all crosslink sites (dark grey), or only those mapping to crosslink clusters (light grey). (f) TDP-43 crosslinking in its own transcript (TARDBP). The replicate experiments are summed and shown in a single track. The RNA sequence underlying the peak crosslinking site is shown, with UG and GU dinucleotides in pink.

Mentions: In order to compare the RNA sequence specificity of TDP-43 in the different iCLIP experiments, we assessed the enrichment of all possible pentamers within 30 nt on either side of all crosslink sites. In all iCLIP data sets, the most significantly enriched pentamers were GUGUG and UGUGU (Fig. 2a, Supplementary Fig. 3a–c). The pentamer enrichment scores were correlated between healthy and FTLD-TDP brain (r=0.91, Fig. 2a).


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

Tollervey JR, Curk T, Rogelj B, Briese M, Cereda M, Kayikci M, König J, Hortobágyi T, Nishimura AL, Zupunski V, Patani R, Chandran S, Rot G, Zupan B, Shaw CE, Ule J - Nat. Neurosci. (2011)

TDP-43 binding motif analysis(a) z-scores of penamer occurrence within the 61 nt sequence surrounding all crosslink sites (−30 nt to +30 nt) are shown for healthy and FTLD-TDP brain iCLIP. The sequences of the two most enriched pentamers and the Pearson correlation coefficient (r) between the two samples are given. (b) Enrichment of crosslinking compared to randomised data in UG repeats of different lengths in TDP-43 iCLIP experiments from healthy brains (blue) and FTLD-TDP brains (red), and CELF2 experiments in healthy brain (green). (d) Enrichment of crosslinking compared to randomised data on UG repeats of different lengths in TDP-43 iCLIP experiments from SH-SY5Y and hES cells, where we either included all crosslink sites (dark grey), or only those mapping to crosslink clusters (light grey). (e) Analysis of positions of UGUGU enrichment compared to randomised data around TDP-43 crosslink sites in healthy brains (blue) and FTLD-TDP brains (red), and CELF2 experiments in healthy brain (green). (e) Analysis of positions of UGUGU enrichment compared to randomised data in TDP-43 iCLIP experiments from SH-SY5Y and hES cells, where we either included all crosslink sites (dark grey), or only those mapping to crosslink clusters (light grey). (f) TDP-43 crosslinking in its own transcript (TARDBP). The replicate experiments are summed and shown in a single track. The RNA sequence underlying the peak crosslinking site is shown, with UG and GU dinucleotides in pink.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108889&req=5

Figure 2: TDP-43 binding motif analysis(a) z-scores of penamer occurrence within the 61 nt sequence surrounding all crosslink sites (−30 nt to +30 nt) are shown for healthy and FTLD-TDP brain iCLIP. The sequences of the two most enriched pentamers and the Pearson correlation coefficient (r) between the two samples are given. (b) Enrichment of crosslinking compared to randomised data in UG repeats of different lengths in TDP-43 iCLIP experiments from healthy brains (blue) and FTLD-TDP brains (red), and CELF2 experiments in healthy brain (green). (d) Enrichment of crosslinking compared to randomised data on UG repeats of different lengths in TDP-43 iCLIP experiments from SH-SY5Y and hES cells, where we either included all crosslink sites (dark grey), or only those mapping to crosslink clusters (light grey). (e) Analysis of positions of UGUGU enrichment compared to randomised data around TDP-43 crosslink sites in healthy brains (blue) and FTLD-TDP brains (red), and CELF2 experiments in healthy brain (green). (e) Analysis of positions of UGUGU enrichment compared to randomised data in TDP-43 iCLIP experiments from SH-SY5Y and hES cells, where we either included all crosslink sites (dark grey), or only those mapping to crosslink clusters (light grey). (f) TDP-43 crosslinking in its own transcript (TARDBP). The replicate experiments are summed and shown in a single track. The RNA sequence underlying the peak crosslinking site is shown, with UG and GU dinucleotides in pink.
Mentions: In order to compare the RNA sequence specificity of TDP-43 in the different iCLIP experiments, we assessed the enrichment of all possible pentamers within 30 nt on either side of all crosslink sites. In all iCLIP data sets, the most significantly enriched pentamers were GUGUG and UGUGU (Fig. 2a, Supplementary Fig. 3a–c). The pentamer enrichment scores were correlated between healthy and FTLD-TDP brain (r=0.91, Fig. 2a).

Bottom Line: 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.We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge, UK.

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.

Show MeSH
Related in: MedlinePlus