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, 15 (11), 1488-97

Divergent Roles of ALS-linked Proteins FUS/TLS and TDP-43 Intersect in Processing Long pre-mRNAs

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Divergent Roles of ALS-linked Proteins FUS/TLS and TDP-43 Intersect in Processing Long pre-mRNAs

Clotilde Lagier-Tourenne et al. Nat Neurosci.

Abstract

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of >950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in TDP-43 aggregate-containing motor neurons in sporadic ALS, supporting a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS.

Figures

Figure 1
Figure 1
FUS/TLS RNA targets in mouse and human brain. (a) FUS/TLS protein domains used as antigens to generate antibodies Ab1, Ab2 and Ab3. Q/G/S/Y, glutamine, glycine, serine, tyrosine; G, glycine; E, nuclear export signal; RRM, RNA recognition motif; R/G, arginine/glycine; ZF, zinc finger; L, nuclear localization signal. (b) Autoradiograph of FUS/TLS protein–RNA complexes from mouse brain immunoprecipitated with Ab1 and trimmed with increasing concentrations of micrococcal nuclease (MNase) (first panel). Complexes highlighted by the red box were used for sequencing. Beads coated with IgG antibodies did not detect protein-RNA complexes (second panel). Immunoprecipitated FUS/TLS-RNA complexes migrated at the expected FUS/TLS mobility (third panel), and no FUS/TLS remained after immunoprecipitation (fourth panel). (c) FUS/TLS (green) and TDP-43 (purple) binding to low molecular-weight neurofilament subunit (Nefl) RNA. Vertical red lines show the positions of GUGGU motifs. The scale bar represents the read coverage per base. (d) Flow chart illustrating reads analyzed from three CLIP-seq experiments to define FUS/TLS clusters. (e) Positional distribution of the GUGGU motif in FUS/TLS CLIP clusters in human and mouse brain. (f) Percentages of TDP-43 and FUS/TLS CLIP clusters in pre-mRNAs regions as defined in the top panel. (g) FUS/TLS binding in human and mouse brain in orthologous exon 5 of the Fmr1 RNA. (h) Venn diagrams showing genes with TDP-43 and FUS/TLS CLIP clusters overlapping by at least one nucleotide (left) or genes with both TDP-43– and FUS/TLS-binding sites (right). (i) Overlapping TDP-43 and FUS/TLS clusters in neighboring, but distinct, intronic positions in the Gria3 pre-mRNA.
Figure 2
Figure 2
FUS/TLS binding patterns in mouse and human brain. (a) Mouse (light green) and human (dark green) FUS/TLS or TDP-43 (purple) binding to FUS/TLS RNA in a highly conserved region that represents either an alternative 3′ UTR, or a retained intron. (b) FUS/TLS bound to long transcripts such as neuroligin 1 (Nlgn1) in a characteristic sawtooth-like pattern. (c) Model for the deposition of FUS/TLS on long introns during transcriptional elongation and co-transcriptional splicing. A collection of nascent transcripts at different stages of elongation accumulated to produce a sawtooth-like pattern consistent with FUS/TLS deposition co-transcriptionally. (d) Graph showing enrichment of FUS/TLS, but not TDP-43 or RBFOX2, binding at the 5′ end of long introns. (e) Graph displaying a uniform density of FUS/TLS motif frequency across long and short introns measured by the signal-to-noise ratio (SNR) of the per gene fraction of clusters per 1% bin.
Figure 3
Figure 3
Changes in the expression of FUS/TLS and TDP-43 targets after depletion (knockdown, KD) of either FUS/TLS or TDP-43 in brain and spinal cord. (a,b) Strategy for depletion of FUS/TLS in mouse striatum (a) and spinal cord (b) by injection of ASOs into the striatum or the lateral ventricle, respectively. (c) Immunoblot of FUS/TLS after FUS/TLS ASO, control ASO or saline treatment. (d) RNA-seq analysis identified 355 and 275 genes that were significantly upregulated (red) or downregulated (green) following FUS/TLS depletion in the striatum. (e) qRT-PCR for Kcnip4, Park2, Mal and Smyd3 in striatum and spinal cord with FUS knockdown compared with control ASO. Error bars represent s.d. in three biological replicas. (f) Correlation between differentially expressed genes and FUS/TLS-binding sites. Genes were ranked on their degree of regulation after FUS/TLS depletion (x axis) and the mean number of intronic CLIP clusters (green line) or the mean total intron length (blue line) for the next 100 genes were plotted. Inset, cluster count for each upregulated and downregulated gene. (g) Scatter plot comparing RNA level alterations following TDP-43 or FUS/TLS depletion. Venn diagrams showing the number of overlapping genes misregulated following depletion of either TDP-43 or FUS/TLS, with only 45 and 41 genes that were similarly downregulated or upregulated, respectively, and few genes regulated in opposite directions (yellow diamonds). Bar graphs showing the high density of TDP-43 and FUS/TLS clusters and the increased intronic length in genes commonly downregulated, compared to all other quadrants. (h) Immunoblot in mouse brains treated with ASOs targeting FUS/TLS, TDP-43 or both. (i) qRT-PCR for Kcnip4, Park2, Smyd3, Nrxn3, Nlgn1, Nkain2 and Csmd1 in FUS/TLS-, TDP-43–, or both FUS/TLS- and TDP-43–depleted tissues. Error bars represent s.d. in three biological replicas. *P < 0.05, **P < 0.01, t test.
Figure 4
Figure 4
FUS/TLS-dependent alternative splicing in mouse brain. (a) Schematics (left) and bar plots (right) displaying the percentage of alternatively excluded (cassette) or constitutively spliced, or all of the exons that contain FUS/TLS clusters within 2 kb (top). Bottom, percentage of exons that were excluded, unchanged or included following FUS/TLS depletion, as detected by splicing-sensitive microarray that contain FUS/TLS clusters in 2 kb. (b) Venn diagram showing the overlap of significantly changing alternative splicing events following FUS/TLS depletion in adult mouse brain and in embryonic brain from Fus/Tls−/− mice. The number of events changing in the same direction (co-regulated) and opposite direction (anti-regulated) are indicated. (c) Semi-quantitative RT-PCR analyses of alternative splicing changes following ASO-mediated FUS/TLS depletion in adult mouse brain and embryonic brain from Fus/Tls−/− mice compared with the respective controls. Graphs show the quantification (ratio of inclusion to exclusion) of three biological replicas per group; error bars represent s.d. (d) Venn diagram comparing the overlap of significantly changing alternative splicing events as a result of FUS/TLS depletion and previously published TDP-43 depletion in adult mouse brain. (e–h) Semi-quantitative RT-PCR validations of splicing events that changed only after FUS/TLS depletion (e), changed in similar direction when either FUS/TLS or TDP43 was depleted (f), changed in opposite directions when FUS/TLS or TDP-43 was depleted (g), or changed only after TDP-43 depletion (h).
Figure 5
Figure 5
Alterations of TDP-43 and FUS/TLS human targets in neurons derived from ES cells. (a) Schematic for generation of embryoid bodies (EBs), NPCs and neurons from human ES cells, followed by transduction with lentiviruses encoding shRNAs targeting TDP-43 (shTDP-43) or FUS/TLS (shFUS), or a control shRNA (shControl). (b) qRT-PCR validation of FUS/TLS or TDP-43 depletion in NPCs. (c) qRT-PCR of PARK2, SMYD3, NRXN3, NLGN1, NKAIN2, ATXN1, KCND2 and IPW after TDP-43 or FUS/TLS depletion in NPCs. (d) qRT-PCR validation of FUS/TLS or TDP-43 depletion in differentiated neurons. (e) qRT-PCR of PARK2, SMYD3, NRXN3, NLGN1, NKAIN2, ATXN1, KCND2 and KCNIP4 after either TDP-43 or FUS/TLS depletion in differentiated neurons. Error bars represent s.d. in at least two biological replicas in each group. *P < 0.01 by t test.
Figure 6
Figure 6
Reduction of TDP-43 and FUS/TLS RNA targets in motor neurons from sporadic ALS patients. (a–o) Immunofluorescence localization in spinal cord autopsy samples from control, non-ALS individuals (a,f,k) or sporadic ALS (sALS) patients (b–e,g–j,l–o) for TDP-43 (red, a–o) and either KCNIP4 (green, a–e), Parkin (green, f–j) or SMYD3 (green, k–o). In both control and sALS motor neurons without TDP-43 aggregation, KCNIP4, Parkin and SMYD3 showed the expected, mainly cytoplasmic localization. Neurons bearing TDP-43 inclusions had markedly decreased levels of KCNIP4, Parkin or SMYD3. d, e, i, j, n and o are higher magnifications of the areas highlighted in b, c, g, h, l and m, respectively. (p–s) Triple immunofluorescence for images from a sALS spinal cord autopsy sample for Parkin (green, p), tubulin (blue, q) and TDP-43 (red, r). The merged images are shown in s. Notice that the cell on the left, which contained TDP-43 aggregates and had reduced Parkin levels, contained normal levels of tubulin. (t) Quantification of KCNIP4, Parkin, SMYD3 or tubulin levels in individual motor neurons from a total of 11 sALS patients and 3 control individuals revealed that the majority (~60–70%) of neurons with TDP-43 inclusions had reduced levels of KCNIP4, Parkin or SMYD3 staining. In contrast, the majority of TDP-43 inclusion–bearing cells (>80%) showed normal levels of tubulin staining, which is not a TDP-43 target and whose levels were not expected to be altered by TDP-43 loss.

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