CLIP-seq library preparation and sequencing
Brains from 8-week-old female C57Bl/6 mice or frozen human autopsy cortical specimens were dissociated using a cell strainer of pore size 100 μm (BD Falcon) before ultraviolet cross-linking. CLIP-seq libraries were constructed and sequenced on the Hiseq 2000 platform for 36 cycles15
. Three different antibodies to FUS/TLS were used: Ab1, a rabbit antibody (Bethyl Laboratories, A300–302A) specific for amino acids 1–50 of human FUS/TLS (per the company's description), which are almost identical between the human and the mouse FUS/TLS proteins, Ab2, a mouse monoclonal antibody specific for the C terminus of FUS/TLS (Santa Cruz Biotechnology, clone 4H11, sc-47711), and Ab3, a rabbit antibody to a peptide spanning amino acids 128–140. For each of the three CLIP-seq libraries, the brain of one mouse was used.
Injections of ASO in mice CNS and RNA-seq library preparation
All procedures were accomplished using a protocol approved by the Institutional Animal Care and Use Committee of the University of California at San Diego (Department of Health and Human Services, NIH Publication 86–23). Brains from Fus/Tls−/−
mice, in which the Fus/Tls
gene is interrupted28
, were collected at embryonic day 18.5 and RNAs and proteins were extracted using Trizol (Invitrogen). To deplete FUS/TLS in vivo
, we performed stereotactic injections of either 2 or 10 μl of ASO solution, corresponding to a total of 75 or 300 μg of ASOs, respectively, into the striatum or the right ventricle of 8–10-week-old female C57Bl/6 mice15
. Mice were monitored for any adverse effects for 2 weeks until they were killed, and striatum and adjacent cortex area or the spinal cord, respectively, were dissected and frozen in 1 ml of Trizol (Invitrogen). For the double knockdown experiment, 75 μg of TDP-43 ASO was mixed with or 50 μg of FUS/TLS ASO before injection into the striatum. A control group injected with the same dose (125 μg) of control ASO without any known target in the mouse genome was used for comparison. The ASOs are gapmers with capitalized nucleotides containing 2′-O-(2-methoxy)ethyl modifications with sequences as follows: CCTGGttatttcccaTGAGC (FUS/TLS); AAGGCttcatattgtACTTT (TDP-43). We sequenced 8 pmol of each constructed RNA-seq library51
on the HiSeq 2000 platform for 100 cycles.
RT-PCR and qRT-PCR of RNA from mice samples
To validate alternative splicing, we carried out RT-PCR amplification (24–27 cycles) using poly-A–selected and reverse transcribed (Superscript III, Invitrogen) cDNA from three mice treated with either a control or FUS/TLS ASO. Isoform products were separated on 10% polyacrylamide gels and stained with SYBR gold (Invitrogen) and quantified with ImageJ software (US National Institutes of Health). Intensity ratios between products with included and excluded exons were averaged from three biological replicates per group. qRT-PCR for mouse Tardbp
were performed using the Express One-Step SuperScript kits (Invitrogen) and thermocycler ABI Prism 7700 (Applied Biosystems), with the cyclophilin A (Ppia
) gene as a normalization control. cDNA synthesis and amplification were performed using specific primers and 5′ FAM, 3′ TAMRA-labeled probes. qRT-PCR for all other genes tested was performed with 3–5 mice for each group (treated with saline, control ASO, or ASO to either FUS/TLS or TDP-43) and two technical replicates using the iQ SYBR green supermix (Bio-Rad) on the iQ5 multicolor real-time PCR detection system (Bio-Rad). Analysis was performed using the iQ5 optical system software (Bio-Rad; version 2.1). Expression values were normalized to at least two of the following control genes: Actb
. Expression values were expressed as a percentage of the average expression of saline-treated samples. Inter-group differences were assessed by two-tailed Student's t
test. Primer sequences designed using Primer3 software (http://frodo.wi.mit.edu/primer3/
) are available in Supplementary Table 7
Antibodies for immunoblots
Immunoblots for FUS/TLS or TDP-43 were performed in a least three mice for each treatment group (). For primary antibodies, we used rabbit antibody to FUS/TLS (Bethyl Laboratories, A300-302A, 1:5,000), mouse antibody to FUS/TLS (Santa Cruz Biotechnology, clone 4H11, sc-47711, 1:500), rabbit antibody to amino acids 128–140 of FUS/TLS (serum diluted to 1:2,000), rabbit antibody to TDP-43 (Proteintech, 10782, 1:2,000), mouse DM1α antibody to tubulin (1:10,000) and mouse antibody to GAPDH (Abcam, AB8245, 1:10,000).
CLIP-seq and RNA-seq read mapping
Reads from CLIP-seq and RNA-seq libraries had adaptor sequences trimmed and homopolymeric runs removed before mapping to the repeat-masked mouse genome (mm9) using bowtie (version 0.12.2), with parameters –q –p 4 –e 100 –a –m 10 –best–strata. Reads with identical sequences were collapsed into a single read. Reads were allowed to map to multiple locations, but only the best scoring alignment was kept for downstream analysis. The entire trimmed library was mapped against reference snRNA sequences rather than the genome, and reads mapped to the U1 snRNA reference sequence were plotted as cumulative read coverage per nucleotide. Mismatch sites provided evidence of incorrect base incorporation during the reverse transcriptase step, as a result of protein adducts left by ultraviolet cross-linking52
CLIP-seq cluster identification, motif and pattern analysis
CLIP cluster identification was performed as previously described15,17,53,54
. Significant clusters of reads were calculated using a Poisson distribution using two different frequencies to determine a P
value. First, a transcriptome-wide frequency was calculated by dividing the total length of all pre-mRNAs by the total CLIP reads mapping in them. Second, a gene-specific frequency was calculated by dividing the size of a particular pre-mRNA by the total CLIP reads in it. A significant cluster had sufficient reads to satisfy a bonferroni-corrected P
for both frequencies. To determine CLIP clusters outside of annotated pre-mRNAs, we calculated a genomic frequency using genome size divided by total genomic CLIP reads, as well as a local frequency using a ±1-kb region surrounding a potential cluster divided by the total CLIP reads overlapping this region. We used 150-nt regions surrounding the center of CLIP clusters to identify strand-specific motifs against a nucleotide frequency–matched background of all pre-mRNAs, using the software HOMER55
. To visualize sawtooth-like patterns, we determined intron distributions of CLIP clusters by assigning a cluster to a 1%-resolution intron bin dependent on the length of the intron. For each cluster, a score was added to this bin equal to the fraction this cluster represents versus all clusters on the entire gene to normalize for differing bin sizes. Motifs (defined from HOMER) and RNA-seq reads were analyzed the same way.
RNA-seq analysis and annotation of candidate intron retention events
Differentially expressed genes computed from RPKM56
values were defined by a z
score > 1.96 (P
< 0.05). A subset of 17 genes was excluded from further analysis, as they also changed in control oligo–treated samples compared with saline-treated samples. To identify putative intron-retention/alternative 3′ UTR events, RPKMs of individual regions, not entire genes, were calculated for known 3′ UTRs and their distribution was plotted. The most frequent RPKM value was chosen as the cutoff (natural log RPKM > 2) that we used to distinguish expressed intronic regions (1,866 regions) from non-expressed ones.
Splicing-sensitive microarray analysis
Microarray analysis was performed as previously described, filtering events with a q
value >0.05 and an absolute separation score > 0.5 (ref. 57
). An RNA splicing map was produced as previously reported17
, overlapping FUS/TLS CLIP-seq clusters around splicing array–detected cassette exons promoted by FUS/TLS (skipped following knockdown, n
= 63), repressed by FUS/TLS (included following knockdown, n
= 114) or unchanged by FUS/TLS (n
Generation of NPCs and neurons from pluripotent stem cells
Human pluripo-tent embryonic stem cells (HUES6 line) and iPS cells were induced into neural progenitors and differentiated into mature neurons using a pan-neuronal protocol as previously described58,59
. Stem cells grown on matrigel-coated plates (BD) with mTeSR1 growth media (Stem Cell Technologies) at 37 °C and 5% CO2
were removed by treatment with collagenase IV (Sigma). Whole colonies were grown on ultra low–attachment plates in DMEM/F12 + GlutaMAX supplemented with N2 for 1 week as embryoid bodies, before being transferred onto polyornathine/laminin-coated plates and DMEM/F12 + GlutaMAX supplemented with N2 and FGF-2 (20 ng ml−1
). After 1 week, neural rosettes were manually picked, enzymatically dissociated and re-plated in DMEM/F12 + GlutaMAX with N2, B27-RA and FGF-2. To differentiate the rosette derived NPCs into mature neurons, 10 μM ROCK inhibitor (Y27632) was added to the media for 2 d and cultured for an additional 4 weeks without FGF-2. To generate iPS cells, human fibroblasts from a non-ALS individual were subjected to infection by retroviruses harboring the reprogramming factors c-Myc, Klf4, Oct4 and Sox2 and replated onto mouse embryonic fibroblasts after 5 d. Colonies with ES cell–like morphology, including well-defined borders, high nuclear to cytoplasmic ratio, and phase-bright characteristics were isolated after 4 weeks of reprogramming and propagated separately on Matrigel-coated plates and mTeSR1 growth media.
Lentiviral shRNA–mediated depletion of human TDP-43 and FUS/TLS
Lentiviral shRNA constructs (Open Biosystems) toward TDP-43
(TRCN0000016038 and TRCN0000016041) and FUS/TLS
(TRCN0000010450) in the pLKO.1 vector system was used to produce lentivirus as previously described53
. The control virus used was pLKO.1 containing a shRNA targeting GFP
. The efficacy of the lentivirus was tested by infection of NPCs at varying viral concentrations and qRT-PCR quantitation of TDP-43
5 d after infection.
qRT-PCR in human neuronal progenitor cells and differentiated mature neurons
qRT-PCR was performed on 7900HT Fast Real-time PCR system (Applied Biosystems) with Fast SYBR-Green PCR Master Mix (Applied Biosystems) in triplicate from polyA-selected, reverse-transcribed cDNA from control, FUS/TLS-depleted and TDP-43–depleted human neural progenitor cells and differentiated mature neurons. Gene expression values were normalized using either SMYD5, BMI or GAPDH levels.
Human tissue acquisition and processing
Human CNSs were obtained and archived via an institutional review board and Health Insurance Portability and Accountability Act-compliant process. The ALS tissues were obtained from patients who met the modified El Escorial criteria for definite ALS60
. The diagnosis was confirmed post-mortem through histologic analysis of TDP-43 aggregation2,3
. Mutations in SOD1
genes were excluded. Control tissues were obtained from non-neurologically affected patients out of the hospital's critical care unit when life support was withdrawn, or patients on hospice. Autopsies were performed immediately following death, with an average post-mortem interval of 4 h. For histological studies, gross segments were fixed in 10% neutral buffered formalin (wt/vol) for 14 d, embedded in paraffin, and stored at 20–25 °C. For biochemical analysis, tissue segments were embedded in optimal cutting temperature medium (Tissue Tek, 4583) and frozen.
Immunofluorescence from human autopsy samples
We performed double immunofluorescence on formalin-fixed, paraffin-embedded lumbar spinal cord sections from SALS or control patients using an antibody to TDP-43 and one to either Parkin, KCNIP4, SMYD3 or tubulin. The slides were deparaffinized, rehydrated through a descending ethanol wash series, permeabilized by immersion in 0.2% Triton X-100 (vol/vol) for 10 min, and washed twice in phosphate-buffered saline (PBS). Antigen retrieval was performed in ScyTek Citrate Buffer pH 6.0 (CBB999) for 20 min at 125 °C in a Dako Pascal pressure chamber. After washing and blocking the slides were incubated overnight with one of the following primary antibodies: TDP-43 (CosmoBio, TIP-PTD-P01, phospho–TDP-43 rabbit polyclonal IgG, 1:50; in-house FL4 antibody, mouse monoclonal IgG, dilution of ascites 1:1,000), Parkin (R&D, AF1438, goat polyclonal IgG, 1:50), KCNIP4 (Sigma, HPA022862, rabbit polyclonal IgG, 1:200), SMYD3 (Abcam, ab16027, rabbit polyclonal IgG, 1:200) and tubulin (Sigma Aldrich, T8660, mouse monoclonal IgG, 1:1,000). Secondary antibodies (Alexa 488–conjugated goat antibody to rabbit, Cy3-conjugated donkey antibody to mouse, Cy3-conjugated donkey antibody to rabbit, Alexa 488–conjugated donkey antibody to goat or Cy5-conjugated donkey antibody to mouse) were diluted directly in PBS and applied at a 1:500 dilution ratio. Slides were stained using 10 μg ml−1 DAPI in ddH20 for 10 min, rinsed twice in ddH20, autofluorescence-masked in 0.1% Sudan Black (vol/vol, Fisher, BP109-10) dissolved in 70% ethanol for 10 s, and rinsed twice in PBS. Visualization and imaging was performed using a Nikon Eclipse Ti confocal microscope system. Motor neurons were counted and classified upon the presence or absence of TDP-43 inclusion and the qualitative difference of staining for Parkin, SMYD3, KCNIP4 or tubulin. The result presented in is the average for each category of motor neurons determined by two independent investigators.