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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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The RNA splicing map of TDP-43The map of crosslink clusters at positions within 500 nt of alternative exons and flanking exons. The cassette exons with ΔIrank > 1 (enhanced exons, red clusters) or ΔIrank < −1 (silenced exons, blue clusters) with at least one crosslink cluster in these regions are shown. The exons are grouped by sequential analysis of crosslink cluster positions in three regions: group S1 is identified by clusters in region 1, 150nt upstream till the exon and within the exon; group S2 by clusters in region 2, 150–500nt downstream of the exon; and group E by clusters present 0–150nt downstream of the exon.
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Figure 3: The RNA splicing map of TDP-43The map of crosslink clusters at positions within 500 nt of alternative exons and flanking exons. The cassette exons with ΔIrank > 1 (enhanced exons, red clusters) or ΔIrank < −1 (silenced exons, blue clusters) with at least one crosslink cluster in these regions are shown. The exons are grouped by sequential analysis of crosslink cluster positions in three regions: group S1 is identified by clusters in region 1, 150nt upstream till the exon and within the exon; group S2 by clusters in region 2, 150–500nt downstream of the exon; and group E by clusters present 0–150nt downstream of the exon.

Mentions: To study how TDP-43 binds pre-mRNAs to regulate splicing, we visualised the positions of crosslink clusters in the form of an RNA splicing map, where the crosslink sites are plotted within 500 nt of the silenced (blue clusters) or enhanced (red clusters) cassette exons and the flanking exons (Fig. 3). The crosslink clusters identified three regions on the RNA splicing map where TDP-43 binding was mainly restricted to silenced or enhanced exons (Fig. 3). Binding in the region 1, 0–150 nt upstream or within the alternative exons, was present at 11 silenced and 1 enhanced exon (group S1). Binding in the region 2, 150–500 nt downstream of the alternative exons, was present at 7 silenced and 1 enhanced exon (group S2). Five of the silenced exons contained multiple crosslink clusters that spanned a sequence of 100 nt or longer in this region. This indicates that ‘deep’ intronic binding involving multiple dispersed binding sites might contribute to the ability of TDP-43 to silence exon inclusion. Finally, binding in the region 3, 0–150 nt downstream of the alternative exons (but not region 1 or 2), was present at four enhanced exons (group E).


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)

The RNA splicing map of TDP-43The map of crosslink clusters at positions within 500 nt of alternative exons and flanking exons. The cassette exons with ΔIrank > 1 (enhanced exons, red clusters) or ΔIrank < −1 (silenced exons, blue clusters) with at least one crosslink cluster in these regions are shown. The exons are grouped by sequential analysis of crosslink cluster positions in three regions: group S1 is identified by clusters in region 1, 150nt upstream till the exon and within the exon; group S2 by clusters in region 2, 150–500nt downstream of the exon; and group E by clusters present 0–150nt downstream of the exon.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: The RNA splicing map of TDP-43The map of crosslink clusters at positions within 500 nt of alternative exons and flanking exons. The cassette exons with ΔIrank > 1 (enhanced exons, red clusters) or ΔIrank < −1 (silenced exons, blue clusters) with at least one crosslink cluster in these regions are shown. The exons are grouped by sequential analysis of crosslink cluster positions in three regions: group S1 is identified by clusters in region 1, 150nt upstream till the exon and within the exon; group S2 by clusters in region 2, 150–500nt downstream of the exon; and group E by clusters present 0–150nt downstream of the exon.
Mentions: To study how TDP-43 binds pre-mRNAs to regulate splicing, we visualised the positions of crosslink clusters in the form of an RNA splicing map, where the crosslink sites are plotted within 500 nt of the silenced (blue clusters) or enhanced (red clusters) cassette exons and the flanking exons (Fig. 3). The crosslink clusters identified three regions on the RNA splicing map where TDP-43 binding was mainly restricted to silenced or enhanced exons (Fig. 3). Binding in the region 1, 0–150 nt upstream or within the alternative exons, was present at 11 silenced and 1 enhanced exon (group S1). Binding in the region 2, 150–500 nt downstream of the alternative exons, was present at 7 silenced and 1 enhanced exon (group S2). Five of the silenced exons contained multiple crosslink clusters that spanned a sequence of 100 nt or longer in this region. This indicates that ‘deep’ intronic binding involving multiple dispersed binding sites might contribute to the ability of TDP-43 to silence exon inclusion. Finally, binding in the region 3, 0–150 nt downstream of the alternative exons (but not region 1 or 2), was present at four enhanced exons (group E).

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