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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 regulates splicing of non-coding and protein-coding RNAs(a) TDP-43 crosslinking in MIAT ncRNA. UG repeat lengths are shown in pink bars and crosslink clusters are shown as black bars. Analysis of MIAT exon 10 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (b) TDP-43 crosslinking in MEF2D protein-coding transcript. UG repeat lengths are shown in pink bars and crosslink clusters are shown as black bars. Analysis of MEF2D exon 11 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (c) Analysis of BIM exon 3 splicing (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells, and in healthy (C23, C25, C30) and FTLD-TDP (F19, F20, F21) brain samples.
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Figure 4: TDP-43 regulates splicing of non-coding and protein-coding RNAs(a) TDP-43 crosslinking in MIAT ncRNA. UG repeat lengths are shown in pink bars and crosslink clusters are shown as black bars. Analysis of MIAT exon 10 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (b) TDP-43 crosslinking in MEF2D protein-coding transcript. UG repeat lengths are shown in pink bars and crosslink clusters are shown as black bars. Analysis of MEF2D exon 11 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (c) Analysis of BIM exon 3 splicing (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells, and in healthy (C23, C25, C30) and FTLD-TDP (F19, F20, F21) brain samples.

Mentions: TDP-43 crosslink clusters were also present in introns of 7,499 protein-coding genes and 3′ UTRs of 1,172 genes. 67% of the genes with 3′ UTR clusters (785/1,172) also had intronic clusters. For instance, two crosslink clusters were present in the intron downstream of the alternative exon 11 and three clusters in the 3′ UTR of myocyte enhancer factor 2D (MEF2D) mRNA (Fig. 4a). We identified a 29% decrease in inclusion of the MEF2D exon 11 in TDP-43 knockdown SH-SY5Y cells (Fig. 4a). The additional binding sites in the 3′ UTR indicate that TDP-43 can remain associated with the MEF2D mRNA after splicing is finished and thereby regulate additional aspects of RNA processing.


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 regulates splicing of non-coding and protein-coding RNAs(a) TDP-43 crosslinking in MIAT ncRNA. UG repeat lengths are shown in pink bars and crosslink clusters are shown as black bars. Analysis of MIAT exon 10 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (b) TDP-43 crosslinking in MEF2D protein-coding transcript. UG repeat lengths are shown in pink bars and crosslink clusters are shown as black bars. Analysis of MEF2D exon 11 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (c) Analysis of BIM exon 3 splicing (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells, and in healthy (C23, C25, C30) and FTLD-TDP (F19, F20, F21) brain samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108889&req=5

Figure 4: TDP-43 regulates splicing of non-coding and protein-coding RNAs(a) TDP-43 crosslinking in MIAT ncRNA. UG repeat lengths are shown in pink bars and crosslink clusters are shown as black bars. Analysis of MIAT exon 10 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (b) TDP-43 crosslinking in MEF2D protein-coding transcript. UG repeat lengths are shown in pink bars and crosslink clusters are shown as black bars. Analysis of MEF2D exon 11 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (c) Analysis of BIM exon 3 splicing (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells, and in healthy (C23, C25, C30) and FTLD-TDP (F19, F20, F21) brain samples.
Mentions: TDP-43 crosslink clusters were also present in introns of 7,499 protein-coding genes and 3′ UTRs of 1,172 genes. 67% of the genes with 3′ UTR clusters (785/1,172) also had intronic clusters. For instance, two crosslink clusters were present in the intron downstream of the alternative exon 11 and three clusters in the 3′ UTR of myocyte enhancer factor 2D (MEF2D) mRNA (Fig. 4a). We identified a 29% decrease in inclusion of the MEF2D exon 11 in TDP-43 knockdown SH-SY5Y cells (Fig. 4a). The additional binding sites in the 3′ UTR indicate that TDP-43 can remain associated with the MEF2D mRNA after splicing is finished and thereby regulate additional aspects of RNA processing.

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