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Figure Legends
36
Figure 1. Identification of protein-RNA interactions by tRIP-seq
A. Schematic representation of Terminator 5′-phosphate-dependent exonuclease (TEX) treatment
of immunoprecipitated RNA. TEX digestion stops at the antibody-tethered nucleotide, while TEX
eliminates the non-specific RNA and linkers remaining on the beads. Green circle represents m6A
nucleotide.
B. Distribution of RBFOX2-eCLIP reads and RBFOX2-tRIP reads on chr10: 75,544,000–
75,635,000 (GRCh37/hg19).
C. Distributions of FUS-HITS-CLIP reads, FUS-tRIP reads, and ContAb-tRIP reads on chr15:
26,165,000–28,520,000 (GRCm38/mm10). The number of N2A cells is indicated.
D. Correlation of read densities constituting MACS-defined peaks between RBFOX2-tRIP and
RBFOX2-eCLIP. Scatter plot indicates RPM of tRIP-seq and eCLIP constituting each peak.
E. Correlation in read densities constituting MACS-defined peaks between FUS-tRIP and FUSHITS-CLIP. Scatter plot indicates RPM of tRIP-seq and HITS-CLIP constituting each peak.
F. The RBFOX2 motif identified by MEME in the peaks of RBFOX2-tRIP.
G. The FUS motif identified by MEME in the peaks of FUS-tRIP of 1 × 108 cells.
H. Distributions of FUS-tRIP reads (upper panel) and ContAb-tRIP reads (lower panel) mapped to
the relative positions of all coding genes in mouse. The ngs.plot tool [58] was used to calculate the
average RPM for a gene structure. The average RPM at each position was normalized based on the
total RPM mapped to each gene. The standard error of normalized RPM is shown as a semitransparent shade around the average curve. TSS, transcriptional start site; TTS, transcriptional
termination site.
37
38
Figure 2. Identification of protein-RNA interactions in the RNAPII machinery by tRIP-seq
A. Schematic representation of immunoprecipitation of protein-RNA complexes engaged to
RNAPII shown in (B). With UV-crosslinking of protein-RNA interactions (UV+) or without (UV-),
the chromatin fraction is isolated from the cells and treated with (Pre-RNase+) or without RNase A
(Pre-RNase-). RNAPII, shown in gray, is 1st immunoprecipitated. The immunoprecipitants are
treated with RNase A to release a protein, shown in green, from the protein-RNA-RNAPII complex.
B. RNAPII-RBP-RNA complex was immunoprecipitated by anti-RNAPII antibody or control
antibody (cont), and subjected to immunoblotting. The input lanes contained 0.4% of lysates used in
the immunoprecipitation experiments.
C. Generation of tRIP-seq libraries of RNAPII-RBP-RNA complexes. After UV crosslinking,
protein-RNA complexes were immunoprecipitated with anti-RNAPII (1st IP). RNA of the isolated
RNAPII-RBP-RNA complexes was partially digested with RNase on beads, and RBP-RNA
complexes dissociated from the bead were further immunoprecipitated with anti-FUS or anti-U1C
antibody bound to the other beads (2nd IP). The immunoprecipitated FUS-RNA or U1C-RNA
complex was treated with TEX, and then subjected to tRIP-seq library generation. The bottom
panels show PCR-amplification of tRIP-seq libraries generated from RNAPII-FUS-RNA complex
(left panel) and RNAPII-U1C-RNA complex (right panel).
D. Read distributions of Chr-FUS-tRIP, U1C-FUS-tRIP, and RNAPII-FUS-tRIP mapped to the
relative positions of all coding genes in mouse. The ngs.plot tool [58] was used to calculate the
average RPM for a gene structure. The average RPM at each position was normalized for the total
RPM mapped to each gene. The standard error of normalized RPM is shown as a semi-transparent
shade around the average curve. Arrowhead indicates a peak before the transcription termination
site (TTS) in RNAPII-FUS-tRIP.
39
E. Read distributions of RNAPII-FUS-tRIP (purple lines) and Chr-FUS-tRIP (black lines) around
the APA sites repressed (bold lines) or activated (thin lines) by FUS. The p-values for the
differences between RNAPII-FUS-tRIP and Chr-FUS-tRIP around FUS-repressed APA sites are
indicated by circles. An arrowhead indicates a peak upstream to APA sites in RNAPII-FUS-tRIP.
F. Read distributions of RNAPII-CPSF160-tRIPs generated from Fus-silenced cells (siFus, pink
line) and those of control siRNA-treated cells (siCont, green line). The p-values for the differences
between siFus and siCont are indicated by circles. An arrowhead indicates a peak before APA sites
in RNAPII-CPSF160-tRIP of siFus-treated cells.
40
Figure 3. Identification of protein-RNA interactions in the RNAPII machinery by tRIP-seq to
investigate FUS-dependent enhancement of U1 snRNP-RNA interactions around alternative
splice sites
A. Read distributions of Chr-FUS-tRIP mapped around constitutive splice sites (CS sites, gray
dotted line) and alternative splice sites (AS sites, black solid line). Positions of CS sites and AS
sites are according to the ENSEMBL annotations on GRCm38/mm10. 3SS, 3′ splice site; 5SS, 5′
splice site.
B. Read distributions of RNAPII-FUS-tRIP (RNAPII-FUS, purple line), U1C-FUS-tRIP (U1CFUS, blue line), and Chr-FUS-tRIP (Chr-FUS, black line) around AS sites. The p-values for the
41
differences between RNAPII-FUS and Chr-FUS are indicated by circles.
C. Read distributions of Chr-U1C-tRIPs generated from Fus-silenced cells (siFus, pink line) and
those of control siRNA-treated cells (siCont, green line). Arrowhead indicates a peak at the 5′ splice
site, which disappears upon Fus-silencing.
D. Read distributions of RNAPII-U1C-tRIPs generated from Fus-silenced cells (siFus, pink line)
and control siRNA-treated cells (siCont, green line).
Data information: C and D. Left panels show read distributions of tRIP-seqs mapped to the
relative positions of all coding genes in mouse. The standard error of mean is shown as a semitransparent shade around the average curve. Right panels show read distributions of tRIP-seqs
mapped around AS sites. The p-values for the differences between siFus and siCont are indicated by
circles.
42
Figure 4. FUS-U1 snRNP complex on RNAPII suppresses APA
A and B. PolyA-seq was performed using N2A cells to examine the effect of downregulation of
FUS and U1 snRNP on polyA site usage. Cells were treated with siRNA against Fus (siFus) and/or
U1 antisense morpholino (U1AS). Control cells (Cont) were treated with both control siRNA and
control antisense morpholino. A. Correlation of changes in polyA site usage between U1-inhibition
43
and Fus-silencing. Fold change in the RPM of a polyA site between siFus and Cont (siFus/Cont)
was plotted against that between U1AS and Cont (U1AS/Cont). Pearson’s correlation coefficient (r)
and the slope of the regression line are shown. B. Correlation of changes in polyA site usage
between U1-inhibition alone and the combination of Fus-silencing and U1-inhibition. Fold change
in the RPM of a polyA site between siFus+U1AS and Cont (siFus+U1AS/Cont) was plotted against
that between U1AS and Cont (U1AS/Cont). Pearson’s correlation coefficient (r) and the slope of
the regression line are shown. A slope of 1.0 indicates that Fus-silencing has no additive effect on
U1-inhibition.
C. Read distributions of RNAPII-FUS-tRIP (left) and RNAPII-U1C-tRIP (right) around APA sites
repressed [the first quadrant in (A)] or activated [the third quadrant in (A)] by both FUS and U1
snRNP. The p-values for the differences between the repressed and the activated APA sites are
indicated by circles. Arrowheads indicate noticeable peaks of RNAPII-FUS and RNAPII-U1C
upstream to the repressed APA sites.
D. Effects of Fus-silencing (pink line) and U1-inhibition (yellow line) on read distributions of
RNAPII-FUS-tRIP (left) and RNAPII-U1C-tRIP (right) around APA sites repressed by both FUS
and U1 snRNP [the first quadrant in (A)]. The p-values for the differences between contAS and
U1AS (left panel), as well as those between siCont and siFus (right panel), are indicated by circles.
E. Read distributions of U1A-FUS-tRIP (left) and U1C-FUS-tRIP (right) around APA sites
repressed [the first quadrant in (A)] or activated [the third quadrant in (A)] by both FUS and U1
snRNP. The p-values for the differences between repressed and activated APA sites are indicated
by circles. Arrowheads indicate noticeable peaks of U1A/U1C-FUS upstream to the repressed APA
sites.
44
Figure 5. A proposed model for the repression of APA by a complex composed of FUS and U1
snRNP.
FUS interacts with U1 snRNP, and binds upstream to APA to repress transcription termination and
polyadenylation, which allows further elongation of nascent RNA.
45
Figure 6. The ALS mutation in FUS, R495X, aberrantly activates APA sites, which are
normally repressed by FUS-U1 snRNP complex
A. Localization of FUS in R495X_N2A cells (R495X), and wild-type N2A cells (WT). Cells were
fractionated to separate nucleus and cytoplasm. Equal proportion of cell extracts from nuclear and
cytoplasmic fractions were immunoblotted with indicated antibodies.
B. Interactions between FUS and U1 snRNP in the R495X_N2A cells (R495X) and wild-type N2A
cells (WT). Total cell lysates were immunoprecipitated with anti-U1-70K antibody or a control
antibody (cont), and immunoblotted with indicated antibodies.
C. Effects of the R495X mutation on four categories of polyA sites. Four categories of polyA sites
are comprised of (1) FUS-repressed (rep) /U1-repressed sites, (2) FUS-activated (act) /U1-repressed
sites, (3) FUS-repressed/U1-activated sites, and (4) FUS-activated/U1-activated sites, according to
Fig 4A. Fold change of each polyA site usage by the R495X mutation (R495X/WT) is shown in
violin plots. Bars indicate the median and 95% confidential intervals for each category. *** p <
0.001, **** p < 0.0001, based on the Steel-Dwass test for comparing all 6 pairs of categories.
46
D. Change in polyA site usage by the R495X mutation. Fold change of polyA site usage by the
R495X mutation (R495X/WT) on the relative positions of all mouse coding genes is plotted. The
average is shown in blue line. The standard error of the mean is shown in semi-transparent blue
shade.
47
Expanded View Figure Legends
Figure EV1. Schematic
representation of the tRIP workflow
RNA binding protein (RBP)-RNA complex is immunoprecipitated with an antibody against the
RBP. Then, the immunoprecipitated RNA is partially digested with RNase on beads. After stringent
washes, a 64-nt linker is ligated to the 3’ end of the RNA fragment, and immunoprecipitants are
treated with TEX to eliminate non-specifically remaining RNA and free linkers. The TEX digests
immunoprecipitated RNA up to the RBP-tethered site. The immunoprecipitants are then treated
48
with proteinase K, followed by column purification of RNA. After the first-strand synthesis with
reverse transcription, a polyA tail is added to the 3’ end of the first-strand cDNA. After the secondstrand synthesis with the tagging primer, the double-strand cDNAs are PCR-amplified and
subjected to high-throughput sequencing analysis.
49
Figure EV2. Read distributions of m6A-tRIP and PTBP1-tRIP
A. The enrichment of m6A motif (GGAC) around the 5′ ends of mapped reads obtained from m6AtRIP and m6A-MeRIP (RNA-seq of conventional RNA immunoprecipitation using At-m6A
antibody).
B. Correlation of read densities constituting MACS-defined peaks between m6A-MeRIP and m6AtRIP. Scatter plot indicates reads per million mapped reads (RPM) of tRIP and MeRIP constituting
each peak. Pearson’s correlation coefficient (r) is indicated.
C. The CU-rich PTBP1 motifs (upper panels) and the m6A motifs (lower panels) identified by
MEME in the peaks of PTBP1-tRIPs and m6A-tRIPs, respectively, performed using indicated
number of C2C12 cells.
D and E. Distributions of PTBP1-tRIP reads (D) and m6A-tRIP reads (E) mapped to the relative
positions of all mouse coding genes. The ngs.plot tool [58] was used to calculate average RPM on a
50
gene structure. Shown are tRIP-seqs of indicated number of C2C12 cells. The standard error of
average RPM is shown as a semi-transparent shade around the average curve.
51
Figure EV3. The recruitment of FUS upstream to polyA sites slows down transcription and
inhibits the recognition of polyA signal by CPSF
A. Read distributions of Chr-RNAP II-tRIPs of Fus-silenced cells (siFus, pink line) and control
siRNA-treated cells (siCont, green line) around APA sites repressed (left graph) or activated (right
graph) by FUS. The p-values for the differences between siFus and siCont are indicated by circles.
An arrowhead indicates a peak upstream to the repressed APA sites detected in Chr-RNAP II-tRIP
of siCont-treated cells.
52
B. Expanded view of read distributions of RNAP II-FUS-tRIP (Fig 2E, bold purple line), RNAP IItRIP of siCont-treated cells (Fig EV3A, green line), and RNAP II-CPSF160-tRIP of Fus-silenced
cells (Fig 2F, pink line) around FUS-repressed APA sites. The bottom graph shows frequency of the
AAUAAAA polyadenylation signal (blue line). Arrowheads indicate noticeable peaks detected in
the respective tRIP-seqs.
C. Read distributions of Chr-CPSF160-tRIPs of Fus-silenced cells (siFus, pink line) and control
siRNA-treated cells (siCont, green line) around APA sites repressed (left graph) or activated (right
graph) by FUS. The p-values for the differences between siFus and siCont are indicated by circles.
An arrowhead indicates a peak upstream to the repressed APA sites detected in Chr-CPSF160-tRIP
of Fus-silenced cells.
D. A proposed model for FUS-dependent repression of APA. The recruitment of FUS upstream to
polyA sites slows down transcription by RNAP II and inhibits the recognition of polyA signal by
CPSF, which suppresses subsequent APA and keeps elongation by RNAP II.
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