EMBO/soda-nel huggingface dataset:
The FUS gene is dual-coding with both proteins contributing to FUS-mediated toxicity (2020)
Figure 6

Figure 6: FUS mutations, synonymous for FUS but missense for altFUS, potentiate TDP-43 cytoplasmic aggregation

A, Graphical representation of FUS synonymous mutations (yellow) found in ALS patients. The canonical FUS mRNA is represented in dark blue (ENST00000254108 or NM_004960). The FUS protein coding sequence is indicated in light blue, and the altFUS protein coding sequence is indicated in green.

B, Images by confocal microscopy of TDP-43 (white), FUS (GFP tagged - green) and altFUS (Flag tagged - red) in HeLa cells over-expressing GFP-FUS(Flag), GFP-FUS(P31L-Flag)-S44=, GFP-FUS(A38V-Flag)-G51=, GFP-FUS(A46V-Flag)-G59=, GFP-FUS(R64P-Flag)-S77= (representative images from n=3). White arrows indicate some TDP-43 aggregates. The white scale bar corresponds to 10 μm.

C, Quantification of cells with TDP-43 aggregates in HeLa cells (see panel B). The data are represented as the fold-change compared to the GFP-FUS(Flag) expressing cells. Statistical significance is indicated above the bars (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, two-way ANOVA with Tukey's multiple comparison correction).

Expanded View Figure Legends
tRIP-seq reveals repression of premature polyadenylation by co-transcriptional FUS-U1 snRNP assembly (2020)
Figure 2

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.

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.
tRIP-seq reveals repression of premature polyadenylation by co-transcriptional FUS-U1 snRNP assembly (2020)
Figure 4

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 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.
tRIP-seq reveals repression of premature polyadenylation by co-transcriptional FUS-U1 snRNP assembly (2020)
Figure 6

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.

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.

Expanded View Figure Legends
ALYREF links 3'-end processing to nuclear export of nonpolyadenylated mRNAs (2019)
Figure 1

Figure 1. ALYREF binds to a region next to the SL on histone mRNAs.

(A) The Venn diagram depicts the overlapping of histone mRNAs detected in a previously performed rRNA-depleted nuclear RNA-seq (RPM>1) and in ALYREF-iCLIP-seq (with clustered binding sites in at least two out of three biological replicates).

(B) The ratio of unique histone iCLIP-seq read population to unique histone RNA-seq read population was calculated and shown, with the unique histone RNA-seq read population set as "1". Error bars represent standard deviations from three replicates of ALYREF iCLIP data. Statistical analysis was performed using Student's t-test. ***P< 0.001.

(C) ALYREF RIP-qPCRs to examine ALYREF binding on histone mRNAs. Relative RIP efficiencies are shown. A tRNA was used as a negative control for ALYREF binding. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01, ***P< 0.001, n.s., not significant.

(D) The distribution of histone reads in 5' UTR, CDS and 3' UTR in rRNA-depleted nuclear RNA-seq and ALYREF-iCLIP-seq.

(E) Metagene analysis of normalized ALYREF signals on histone mRNA based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot.

(F) Screenshots of two histone mRNAs showing clustered ALYREF binding sites. The 3' UTR regions are highlighted.

(G) Distribution profile of clustered ALYREF binding sites on histone mRNA 3' UTRs relative to the cleavage site (CS). The CS is set as "0" in the x axis, and the relative position to the CS is marked. The y axis shows the total occurrence of binding sites in three repeats at each nucleotide.

(H) Graphic displays ALYREF binds next to SLBP on histone mRNAs.
ALYREF links 3'-end processing to nuclear export of nonpolyadenylated mRNAs (2019)
Figure 2

Figure 2. SLBP interacts with ALYREF and facilitates its binding on histone mRNAs.

(A, B) IPs from RNase A-treated S-phase HeLa cell lysate using the SLBP antibody or IgG (A), or the ALYREF antibody or IgG (B). Western blotting was performed with indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blots (Source data Fig 2 A, B).

(C) Pull-downs of in vitro translated 35S labelled SLBP and luciferase (Cntl) using MBP or MBP-ALYREF in the presence of RNase A. The proteins pulled down were visualized by coomassie staining (left) or phosphoimager (right). 3% of input was loaded.

(D) GST-SLBP, GST-UAP56 and GST were used for pull-down of purified MBP-ALYREF or MBP in the presence of RNase A. Proteins pulled down were separated by SDS-PAGE, followed by coomassie staining. 37.5% of input proteins were loaded.

(E) (Top) Domain schematic representation of SLBP. (Bottom) Flag IPs from RNase A-treated HeLa cell lysate individually expressing indicated Flag tagged proteins, followed by western blotting using Flag and ALYREF antibodies. 3% of input was loaded. The * indicates a band probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot (Source data Fig 2E).

(F) Same as (E), except that Flag IPs were carried out from HA-SLBP stable expression cells transfected with plasmids expressing ALYREF fragments.

(G) Western blotting to examine the KD efficiency of SLBP. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot (Source data Fig 2G).

(H, I) Cntl- or SLBP-siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to western analysis (H) and RT-qPCRs (I). Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001, n.s., not significant.

(J) Metagene analysis of normalized ALYREF signals on histone mRNAs in Cntl and SLBP KD cells based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot.

(K) Graphic displays that SLBP functions in recruiting ALYREF to histone mRNAs.
ALYREF links 3'-end processing to nuclear export of nonpolyadenylated mRNAs (2019)
Figure 3

Figure 3. ALYREF is required for proper 3'-end processing of histone pre-mRNAs. (A) A diagram of the RNA-seq experimental approach.

(B) List of histone genes with defective 3'-end processing detected by polyA+ and polyA- RNA-seq. PolyA+ ALYREF/Cntl shows polyA+ RNA-seq RPM ratio of each histone gene in ALYREF KD versus Cntl.

(C) Screenshots of two exemplified histone mRNAs with 3'-end processing defects are shown. Transcription direction is indicated. Black arrowheads and the corresponding lines mark the position of CS.

(D) RPM of each histone mRNA in Cntl cells and RPM ratio of each histone mRNA in KDs to Cntl detected in 3'READS+.

(E, F) RT-qPCRs to detect histone mRNA levels in the polyA+ (E) or the polyA- fraction (F) in cells treated with Cntl, ALYREF or ARS2 siRNA. The arrowhead and arrows indicate the cleavage site and primer location, respectively. The bars show the relative abundance of polyA+ forms to the actin mRNA (E) and that of polyA- forms relative to the 18S rRNA (F).

(G, H) Western blotting to examine ALYREF protein level (G) and RT-qPCRs to detect polyA+ histone mRNA levels (H) in Flag-Cntl (eIF4A3) or siRNA-resistant Flag-ALYREF stable expression cells treated with Cntl or ALYREF siRNA. The bars show the relative abundance of histone mRNAs to the GAPDH mRNA. The white line delineates the boundary where irrelevant lanes have been removed from the same blot (Source data Fig 3G).

Data information: (E-F, H) Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.
ALYREF links 3'-end processing to nuclear export of nonpolyadenylated mRNAs (2019)
Figure 4

Figure 4. ALYREF directly interacts with Lsm11 and facilitates U7-snRNP recruitment.

(A, B) IPs were carried out from RNase A-treated S-phase HeLa cell lysate using the Lsm11 antibody and IgG (A), or the ALYREF antibody and IgG (B), followed by western blotting with indicated antibodies. 2 % of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blot (Source data Fig 4A, B).

(C) Pull-down of purified His-Cntl (PGK1) or His-Lsm11 using equal amount of GST and GST-ALYREF proteins in the presence of RNase A. Proteins pulled down were separated by SDS-PAGE followed by coomassie staining. * indicates a nonspecific band.

(D, E) Cntl or ALYREF siRNA treated Flag-Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody. The immunoprecipitates were subjected to RT-qPCRs (E) and western analysis (D). * indicates the antibody heavy chain. The bars show the relative IP efficiencies of histone pre-mRNAs to U7 snRNA. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.

(F) Graphic displays that ALYREF interacts with Lsm11 and facilitates U7-snRNP recruitment.
ALYREF links 3'-end processing to nuclear export of nonpolyadenylated mRNAs (2019)
Figure 5

Figure 5. The TREX complex promotes histone mRNA export.

(A) (Top) Illustration of the HIST2H2AA3 reporter construct. (Bottom) FISH to detect the distribution of the HIST2H2AA3 mRNA in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. DAPI staining served as a nuclear marker. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were determined for 20 cells in each experiment.

(B) Distribution of the endogenous HIST2H2AA3 mRNA was detected with the transcript-specific probe in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were calculated the same as (A).

(C, D) Western analysis to examine the purities of nuclear and cytoplasmic fractions (C), and RT-qPCRs to examine N/C distribution of each histone mRNA (D) in Cntl and UAP56 KD cells. MTR4 and Tubulin were used as the nuclear and cytoplasmic marker, respectively.

(E) Graphic displays that TREX promotes histone mRNA export.

(F) Relative NXF1 iCLIP read populations on histone mRNAs in Cntl, ALYREF and SLBP KD cells.

(G) NXF1 iCLIP signals along the histone mRNA. The histone mRNA is divided into 10 fractions based on their mRNA size. Each fraction represents 10% of each histone mRNA. The tenth fractions that contains the SL is boxed by green dashed lines.

Data information: Error bars represent standard deviations from biological repeats (n=3 in A, B and D; n=2 in F). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.
ALYREF links 3'-end processing to nuclear export of nonpolyadenylated mRNAs (2019)
Figure 6

Figure 6. 3'-end processing promotes TREX recruitment and histone mRNA export.

(A) (Top) Illustration of the reporter constructs. The black and red lines indicate H2AA3-SL and H2AA3-SLD PCR products, respectively. The dashed line indicates the FISH probe. (Bottom) FISH to detect the distribution of the corresponding mRNA at 2 hr after injection of H2AA3-SL and H2AA3-SLD PCR products. C and N indicate cytoplasmic and nuclear FISH signals, respectively. C/N ratios were determined for 20 cells in each experiment.

(B) (Top) Illustration of the reporter constructs. The dashed line indicates the FISH probe. FISH to detect mRNA distribution at 24 hr after transfection of the H1C-SL-Rb (cH1C) and H1C-SLD-Rb (pH1C) constructs. C/N ratios were quantified same as (A).

(C) Same as (A), except that the PCR products of cG-SL and cG-SLD were used instead. cG, β-globin cDNA.

(D, E) H1C-SL-Rb or H1C-SLD-Rb construct, together with VSV M were co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. The immunoprecipitates were subjected to western analysis to detect ALYREF (D) and RT-qPCRs to detect RNAs (E). The bars in the graph shows the relative ALYREF IP efficiency, with that for the H1C-SL-Rb mRNA set as "1".

(F) H1C-SLD-Rb construct with wild-type (H1C-SLDWT-Rb) or mutant (H1C-SLDmut-Rb) U7-binding sequence, together with VSV M, was co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. The immunoprecipitates were subjected to western analysis (left panel) and RT-qPCRs (right panel). The bars in the right graph show the relative ALYREF IP efficiency, with that for the H1C-SLDWT-Rb mRNA set as "1".

Data information: (A-C, E-F) Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.
ALYREF links 3'-end processing to nuclear export of nonpolyadenylated mRNAs (2019)
Figure 7

Figure 7. ALYREF ensures histone protein production.

(A) Western blotting to examine the expression levels of H2B and H4 in Cntl, ALYREF and ARS2 KD cells. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot (Source data Fig 7A).

(B) Growth curve of Cntl and ALYREF KD cells. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01.

(C) Cell cycle analysis of Cntl, ALYREF and ARS2 KD cells. Quantifications of cells in G0/G1 (1N), G2 (2N) and S-phase (intermediate) of the cell cycle are shown in the upper right corner of each histogram.

(D) Model for ALYREF promoting histone mRNA 3'-end processing and nuclear export. See text for details.

Expanded View Figure legends
Auto-inhibition of Mif2/CENP-C ensures centromere-dependent kinetochore assembly in budding yeast (2020)
Figure 2

Figure 2 - Ame1 and Mif2 share overlapping Mtw1c binding motifs.

(A) GST-Mif2_1-41 was immobilized on GSH Sepharose beads and incubated with Mtw1-Nnf1 complex (MN) in the presence of increasing concentrations of Ame1_1-20 peptide (lanes 4-9). GST-Ame1_1-30 served as control (lane 2 and 3). After washing, bound complexes were eluted by adding SDS sample buffer. Protein bands in Coomassie stained gel are highlighted in the indicated color. Integrated density of Mtw1 bands was quantified using ImageJ and correlated to GST-Mif2 amounts in the same lane.

(B) Binding of FITC-Mif21-41 to MN was directly determined in a fluorescence polarization (FP) assay. This interaction at EC50 was than competed with increasing concentration of Ame11-20 unlabeled peptide. Both experiments were performed in triplicates of which the mean and standard deviation is shown.

(C) Multiple sequence alignment of the N-termini of Mif2 and Ame1 from distantly related yeast species. Conserved residues are highlighted. A dashed box indicates a conserved consensus sequence of RxxG.

(D) Equivalent point mutations replacing Arg11 in Mif2 or Arg10 in Ame1 with Alanine prevent binding to the MN complex. Mif2 and Ame1 wild-type N-termini serve as a control, protein bands are highlighted in the indicated color.
Auto-inhibition of Mif2/CENP-C ensures centromere-dependent kinetochore assembly in budding yeast (2020)
Figure 3

Figure 3 - Analysis of Mtw1 interface mutants in vivo

(A) Structure of the Mtw1c head I bound to Mif2 and list of key residues constituting Interface I (Mif2 binding) and Interface II (putative Ame1 binding) from Dimitrova et al.

(B) Analysis of these Mtw1 mutants in vivo using the anchor-away technique. Mtw1-FRB strains containing RPL13-FKBP12 for cytoplasmic anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 °C.

(C) Tetrad analysis of Mtw1-wt or mutants. Surviving spores in which the Mtw1-wt or mutant rescue construct was the only source of Mtw1 are indicated in pink circles.

(D) Western blotting confirming the expression of Mtw1-6x-Flag wt or mutant proteins in the Mtw1-FRB strain background.

(E) Analytical SEC runs and accompanying SDS-PAGE of Mtw1c-wt or -5A mutant (blue), AO (grey) or a stoichiometric combination of both complexes (green) at 10 µM each. Boxes indicate corresponding fractions in left and right panel. Note that the same Ame1-Okp1 elution profile and SDS-PAGE (middle panel) is displayed in both sets to improve clarity.