EMBO/soda-nel huggingface dataset:
Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB (2014)
Figure 1

PTB RRM2 interacts with multiple RNA‐binding proteins

Schematic representation of PTB (top) and the GST‐PTB RRM2 (below), with the limits of the PTB minimal repressor domain indicated. PTB is composed of four RNA recognition motifs (RRM) with three linker regions in between them. It also contains a N‐terminus bipartite nuclear localization signal (NLS) as well as a nuclear export signal (NES). The GST‐PTB RRM2 is composed of the second RRM fused to a GST tag in the N‐terminus.
Silverstain of the GST‐PTB RRM2 pull‐down of the wild‐type RRM2 and the Y247Q mutant. Five microlitre of the pull‐down was run on a 15% SDS–PAGE and silver‐stained. Three strong bands can be seen which are due to the recombinant protein and the beads used for the pull‐down. The region encompassing 50 kDa to the top of the gel was sliced and subjected to in‐gel digestion and mass spectrometry. The two strongest bands visible in this region are labelled as Matrin3 and Raver1.
Proteins identified in the GST‐PTB RRM2 pull‐down ranked by their unique peptide number. The table shows the different proteins we found binding to RRM2, as well as when present, the sequence of the PRI motif. The indicated function is only a guideline as many have more functions than shown.
Western blot of the GST pull‐down using antibodies against Matrin3 and Raver1. Lanes 1 and 2 show 5 and 10% of input, respectively, lanes 3 and 4 show GST‐PTB full‐length pull‐down of wild‐type and Y247Q mutant, respectively, and lanes 5 and 6 show GST‐PTB RRM2 pull‐down of wild‐type and Y247Q mutant, respectively, and lane 7 with pull‐down using GST alone.

Source data are available online for this figure
Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB (2014)
Figure 2

Matrin3 interacts with PTB via a PRI motif

Schematic representation of Matrin3. Matrin3 is composed by two zinc finger (ZF) domains, two tandem RNA recognition motifs (RRM), as well as a N‐terminal nuclear export signal (NES) and a C‐terminal nuclear localization signal (NLS). A PRI motif is localized between the first ZF and the first RRM, and the sequence is aligned with the sequence from the two functional PRIs from Raver1. Conserved PRI residues are in bold.
FLAG immunoprecipitation of Matrin3 and Raver1, both with wild‐type and with PRI mutated, and FLAG‐MS2 as a negative control. The immunoprecipitated complex was separated in a SDS–PAGE and subjected to Western blot using antibody against PTB which showed interaction to wild‐type Matrin3 (lane 2) and Raver1 (lane 4). The input was also analysed by Western blot with antibodies against PTB as a loading control and against the FLAG tag to ensure equal expression of proteins.
The 20‐residue Raver1 491–511 (lane 1) and the Matrin3 346–365 (lane 3) peptide fused to MS2 were transcribed and translated in vitro (Input) and then pulled down with GST‐PTB or with GST‐SXL as a control. Effects of single mutation of the Matrin3 PRI GILPPP to GIAPPP were also tested (lane 4).

Source data are available online for this figure
Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB (2014)
Figure 3

Global splicing effects upon Matrin3 knockdown

Western blot probed for Matrin3 (top panel), PTB (middle panel) and actin (lower panel). Lanes 1–4 contain a twofold dilution of the control C2 sample (lane 1—12.5%, lane 2—25%, lane 3—50%, lane 4—100%). Lanes 4–7 contain equal amount of proteins, as can be confirmed by the anti‐actin (lower panel), of control sample (lane 4), double‐PTB and nPTB siRNA‐treated sample (lane 5), Matrin3 siRNA‐treated sample (lane 6) and triple knockdown of Matrin3, PTB and nPTB (lane 7). Lane 7 is from the same gel and exposure, but some lanes present in the original gel were cropped for clarity, and a black line to indicate cropping was placed.
Pie chart of the different categories of Matrin3‐regulated alternative splicing events (ASEs), 330 cassette exons (50%), 116 alternative promoter usage (17%), 78 terminal exons (11%), 32 alternative 5′ (5%) and 34 3′ (5%) splice site and 18 intron retention (IR; 3%) events.
Gene ontology (GO) analysis of the Matrin3‐regulated cassette exons. The x‐axis represents the P‐value in a logarithmic scale as shown.
Pie chart of the activated and repressed cassette exons by Matrin3.
Venn diagram of the overlap between the PTB‐ (blue) and Matrin3‐ (yellow) regulated cassette exons, showing the 813 events regulated only by PTB, 270 by Matrin3 only and the 61 that overlap.
RT–PCR validation of Matrin3‐regulated alternative spicing events in the ST7, ACSL3, PLEKHA3, TCF12, VWA5A, PTBP2, PTBP3, C3orf17, ZMYND8, VEZT, PIGX and DMD genes. In each case, triplicates for each condition (C—control, M—Matrin3, PTB/nPTB and Matrin3/PTB/nPTB siRNA transfection samples) were analysed and exon inclusion (EI) percentage is shown beneath the corresponding lane, along with the standard deviation (s.d.).

Source data are available online for this figure
Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB (2014)
Figure 5

Matrin3 and PTB splicing maps

Matrin3 crosslinking in Matrin3‐regulated pre‐mRNAs where position of crosslinked nucleotides was mapped onto the regulated exon and the 500 nucleotides upstream and downstream of its 3′ss and 5′ss, respectively, the upstream flanking exon and 500 nucleotides downstream of its 5′ss and the downstream flanking exon with 500 nucleotides upstream of its 5′ss. The iCLIP tags were mapped onto silenced ASE (blue, n = 421), enhanced ASE (red, n = 187) and to control ASE (grey, n = 16,242), and percentage of occupancy is plotted.
Same as in (A) but using iCLIP tags obtained from PTB iCLIP and mapped onto PTB/nPTB‐regulated ASE, silenced ASE (blue, n = 729), enhanced (red, n = 820) and control ASE (grey, n = 14,599).
Matrin3 crosslinked nucleotides mapped onto PTB‐regulated ASE.
PTB crosslinked nucleotides mapped onto Matrin3‐regulated ASE.
Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB (2014)
Figure 6

Matrin3 splicing function requires PRI and RRMs

A Schematic representation of Matrin3 with its domains in the wild‐type construct (WT) and the mutants used in overexpression, mPRI which has corresponding domain deleted in each one of the mutants, mPRI where the PRI motif is mutated from GILGPPP to GAAAPPA, ΔZ1 where the first zinc finger is deleted, ΔRRM where both the RRM domains are deleted and ΔZ2 where the second zinc finger is deleted.
B Western blot of the overexpressed Matrin3 and its mutants probed with anti‐FLAG and anti‐actin antibodies.
C, D RT–PCR analysis using primers specific to the ST7 exon 11 (C) and ABI2 exon 8 (D) splicing reporters, respectively, of samples where FLAG‐tagged Matrin3 and its mutants have been overexpressed. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 compared with control sample (−).
E Western blot of overexpressed FLAG‐tagged Matrin3 and PTB, in control siRNA and PTB/nPTB siRNA‐transfected cells, probed with anti‐FLAG to detect overexpressed proteins, anti‐PTB to detect the knockdown levels and anti‐actin to confirm protein loading. A titration of control sample (lane 1—12.5%, lane 2—25%, lane 3—50% and lane 4—100%) is also included to assess the levels of knockdown. The band present above the PTB doublet in lane 6 is FLAG‐tagged PTB.
F, G RT–PCR analysis using primers specific to the ST7 exon 11 (F) and ABI2 exon 8 (G) splicing reporters, respectively, of samples where Matrin3 or PTB expression vectors were co‐transfected, in control siRNA and PTB/nPTB siRNA‐transfected cells. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 when compared with control sample in each condition (‐).

Source data are available online for this figure
CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping (2020)
Figure 1

Figure 1 - CRY-independent circadian timekeeping occurs cell-autonomously

(A) Representative double-plotted actograms showing wheel-running activity of wild type (WT) and CRY-deficient (CRY Knockout; CKO) mice during constant light (yellow shading) and thereafter in constant darkness. Note the 48 hour x-axis for WT versus 32 hour for CKO. Full figure showing CKO data in modulo 24h is presented in Figure EV1A.

(B) Mean period and amplitude (±SEM) of mouse behavioural data (n=4). P-values were calculated by two-way ANOVA.

(C) Longitudinal bioluminescence recordings of organotypic SCN slices from WT (black) and CKO (red) PER2::LUC mice (RLU; relative light units).

(D) Mean period and amplitude (±SEM) of rhythmic SCN bioluminescence traces. P-values were calculated by two-way ANOVA.

(E) Circadian PER2::LUC expression in immortalised WT and CKO adult lung fibroblasts. Left panel shows two raw traces of a representative longitudinal bioluminescence recording, right panel shows same data detrended with a 24-hour moving average to remove differences in baseline expression.

(F) Period of rhythmic fibroblast bioluminescence traces from at least 31 experiments (n≥3 per experiment, individual values ±SEM shown). P-values were calculated by an unpaired t test with Welch correction. Standard deviations differ significantly between WT and CKO (F test: p <0.0001).
CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping (2020)
Figure 2

Figure 2 - CRY-less oscillations are temperature compensated and entrained

(A) Detrended traces of bioluminescence recordings of WT and CKO fibroblast at different constant temperature conditions within the physiological range (n=4, solid lines: mean, dashed lines: ±SEM). Temperature was changed from 37°C to 32°C halfway through the experiment, as depicted by red/blue shading. Arrows represent medium changes. Note the lack of rhythmicity in the first three days in CKO and the appearance of rhythmicity after the first medium change.

(B) Quantification of period from recordings presented in (A). Both WT and CKO oscillations are temperature compensated with respective Q10s of 1.05 and 0.95 (n=3, mean ±SEM). P-values were determined by two-tailed t-test.

(C) Bioluminescence of WT and CKO PER2::LUC cells during temperature entrainment (12h 32°C (blue) - 12h 37°C (red)) (n=3, solid lines: mean, dashed lines: ±SEM).
CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping (2020)
Figure 3

Figure 3 - CRY-independent rhythms are regulated post-transcriptionally

(A) Mean number of PER2::LUC molecules per cell at the estimated peak of PER2 expression for each cell line (mean of three experiments, n=3 each). P-values were calculated by paired t test.

(B) PER2::LUC binding to BMAL1 in WT and CKO cells. Cells were harvested at the peak of PER2 expression, BMAL1 was immunoprecipitated, and PER2::LUC binding was measured by bioluminescence measurements (n=3, mean ±SD). P-values were calculated by unpaired t test.

(C) Per2 mRNA levels in WT (left) and CKO (right) cells were determined by qPCR over one circadian cycle (bottom), while PER2::LUC bioluminescence (top, min-max normalised) was recorded from parallel cultures. Per2 mRNA reported relative to Rns18s (bottom), n=3, ±SEM; PER2::LUC (top) presented as mean (solid) ±SEM (dashed), n=3. The WT mRNA trace was preferentially fit by a circadian damped sine wave compared with straight line (p=0.0412, extra sum-of-square F-test) whereas CKO data was not (ns).

(D) Detrended Per2 and Nr1d1 promoter activity in WT, CKO and quadruple Cry1/2-Per1/2 knockout (CPKO) mouse embryonic fibroblasts (MEFs) recorded at 37°C, n=3, mean (solid) ±SEM (dashed). Nr1d1 data were preferentially fit with a circadian damped sine wave over straight line (p<0.0001, extra sum-of-squares F-test) (right hand graphs, solid lines; error bars, SEM). Similar recordings performed at 32°­­C and an expanded view of Per2 data are presented in figures EV3E and F.
CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping (2020)
Figure 4

Figure 4 - PER2::LUC stability oscillates in CRY-deficient cells

(A) Actual PER2::LUC levels (dark symbols (3-hour moving average, n=3 ±SEM, 4 outliers removed)) as assayed in acute luciferase assays on cell lysates from cells harvested every hour over 48 hours, compared with parallel longitudinal co-recordings from cells in the presence of 0.1 mM luciferin (light lines (n=6, mean ±SEM)).

(B) PER2::LUC recording of asynchronous WT and CKO cells pulsed with proteasome inhibitor MG132 (10 µM, applied at the arrow) (n=3, mean ±SEM).

(C) Quantification of relative PER2::LUC induction upon proteasome inhibition, n=3, mean (solid) ±SEM (dashed). P-value was calculated by unpaired t test.

(D) Phase-dependent PER2::LUC half-life was determined by inhibiting translation at different circadian phases and fitting the resulting data with a one-phase exponential decay curve (n=3, mean ±SEM). Left image depicts the timing of cycloheximide (CHX, 10 µM) pulses (labelled I (PER2 levels going up) and II (PER2 levels going down)), plotted on PER2::LUC bioluminescence traces of control cells (dark colours). A representative trace of CHX-treated cells at time point I is shown in light colours. See FIG EV4D and F for more raw data and time points. Right image shows quantifications, p-values were calculated by unpaired t test.

(E) A simple model incorporating mRNA, protein translation and PER2::LUC stability that were measured experimentally (inputs) shows that the observed oscillating stability of PER2 is sufficient to generate rhythmic PER2::LUC expression (output).
CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping (2020)
Figure 5

Figure 5 - A role for CK1 and GSK3 in the cytoplasmic oscillator

(A) Period (n=3, mean ±SEM) analyses of WT and CKO PER2::LUC cells in the presence or absence of CK1δ/ε inhibitor PF670462 (0.3 µM; PF). P-values were calculated by unpaired t test.

(B) As in (A, n=4), GSK3 inhibitor CHIR99021 (5 µM; CHIR).

(C) As in (A, n=4), in presence of CRY inhibitor KL001 (1 µM).

(D) Schematic model integrating CRY-independent timekeeping into the existing canonical model of the circadian clock. The CRY-dependent gene expression feedback loop (TTFL) is required for most circadian regulation of transcriptional clock controlled genes (CCGs) and therefore for robustness and behavioural and physiological rhythmicity. However, it is dispensable for circadian timekeeping per se, as reported by residual oscillations in PER2 protein levels, suggestive of the existence of a coupled underlying (cytosolic) timekeeping mechanism involving CK1 and GSK3 (cytoscillator).

Data information: See FIG EV5 for raw data.

EXPANDED VIEW FIGURE LEGENDS
Structure of a bacterial ribonucleoprotein complex central to the control 
of cell envelope biogenesis (2022)
Figure 3

Figure 3. RapZ-GlmZ interactions support regulatory activity of RapZ in vivo. (a) Interaction of the RapZ-CTD dimers with SLI (top) and SLII (bottom) of GlmZ. The indicated residues were substituted in RapZ and resulting variants were tested for regulatory activity in vivo. The rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in b.-d. (b) Northern blotting experiment detecting GlmZ (top panel) and 5S rRNA (bottom panel; loading control) to assess the degree of GlmZ cleavage triggered by the RapZ variants. Processed GlmZ is indicated with an asterisk. (c) GlmS protein (MW = 66.9 kDa) levels in the same cultures as revealed by SDS-PAGE of total protein extracts followed by Coomassie blue staining. (d) β-Galactosidase activities produced by the same cultures and replicates (mean values and standard deviations are shown). Cells carry a glmS'-lacZ fusion on the chromosome, which requires base-pairing with sRNA GlmZ for high expression. Strains Z8 (wild-type, lanes/columns 1, 2) and Z28 (∆rapZ, all other lanes/columns) were used. Strain Z28 carried plasmid pBAD33 (lanes/columns 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variants (cf. Table S2 for corresponding plasmids).
HUWE1 is a critical colonic tumour suppressor gene that prevents MYC signalling, DNA damage accumulation and tumour initiation. (2016)
Figure 1

Figure 1. Huwe1 is an intestinal tumour suppressor gene.

(a) Kaplan-Meier survival plot of cohorts of induced Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant reduction in survival of these animals (Vil Apc vs Vil Apc Huwe1het / Vil Apc Huwe1hom, Log Rank, p < 0.001, n ≥ 10). (b) Wholemount isolation of small intestines from Vil Apc and Vil Apc Huwe1hom mice culled at clinical endpoint. Note the huge numbers of tiny macroscopic adenomas visible in the Vil Apc Huwe1hom intestine (red arrows). (c) H&E staining of intestinal cross sections from Vil Apc and Vil Apc Huwe1hom mice demonstrating the increased adenoma burden following Huwe1 deletion. Black arrows indicate individual adenomas. Scale bars = 200 µm. (d) Quantification of total tumour numbers per gut in sacrificed Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant increase in the number of tumours per gut (Mann Whitney, n ≥ 5).
HUWE1 is a critical colonic tumour suppressor gene that prevents MYC signalling, DNA damage accumulation and tumour initiation. (2016)
Figure 2

Figure 2. Huwe1 deletion leads to perturbed intestinal homeostasis and promotes stem cell transformation.

(a) H&E staining of control and Huwe1 deleted intestinal epithelium. Shortened villi in Huwe1 deficient tissue are indicated. Scale bars = 100 µm. (b) BrdU IHC of control and Huwe1 deleted intestinal epithelium. Scale bars = 100 µm. (c) PAS staining identifying Goblet cells. No gross changes were observed. Scale bars = 100 µm. (d) Lysozyme staining (Paneth cell marker) of control and Huwe1 deleted small intestine. Note the occurrence of lysozyme positive cells away from the crypt base (black arrows). Scale bars = 100 µm. (e) Dual Periodic acid–Schiff / Alcian blue staining to identify Paneth cell secretory vesicles (light blue / pink – marked with black arrows) and goblet cells (dark blue / purple – marked with red arrows). Note Paneth cell secretory vesicles are restricted to crypt base in both control and Huwe1 deficient small intestines (inset – black arrows). Scale bars = 100µm. (f) MMP7 staining of control and Huwe1 deleted small intestine. Note MMP7 staining is restricted to crypt base in Huwe1 deficient intestines. Scale bars = 100µm.
HUWE1 is a critical colonic tumour suppressor gene that prevents MYC signalling, DNA damage accumulation and tumour initiation. (2016)
Figure 3

Figure 3. Increased MYC protein expression following Huwe1 deletion.

(a) HUWE1 and MYC Western blot analysis of epithelial cell extractions from control and Huwe1 deficient intestines. Levels of MYC protein are significantly increased in normal intestines lacking HUWE1 (Mann Whitney, p = 0.04, n = 3). (b) QRT-PCR analysis of Myc expression in control and Huwe1 deficient intestines. (c) MYC IHC in tumours from Vil Apc and Vil Apc Huwe1hom mice. Scale bars = 50 µm. (d) MYC Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours. Levels of MYC protein are significantly increased in tumours lacking HUWE1 (Mann Whitney, p = 0.04, n = 3). (e) QRT-PCR analysis of Myc expression in tumours from Vil Apc and Vil Apc Huwe1hom mice.
HUWE1 is a critical colonic tumour suppressor gene that prevents MYC signalling, DNA damage accumulation and tumour initiation. (2016)
Figure 4

Figure 4. MYC upregulation following Huwe1 deletion drives accelerated tumourigenesis.

(a) Kaplan-Meier survival plot of cohorts of induced Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc led to a specific and significant increase in survival of Huwe1 deleted animals (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc, Log Rank, p < 0.001, n ≥ 16). (b) Quantification of total tumour numbers per gut in sacrificed Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc did not reduce the number of tumours in Huwe1 deleted mice (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc. Mann Whitney, n ≥ 10). (c) Kaplan-Meier survival plot of cohorts of induced AhCre-ERT Apc Pten and AhCre-ERT Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC led to a significant reduction in survival (Log Rank, p < 0.001, n = 14 vs 21). (d) Quantification of total tumour numbers per gut in sacrificed Apc Pten and Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC did not increase the number of tumours in Apc Pten mice (Mann Whitney, n = 9 vs 19). (e) cBioportal OncoPrint showing mutual exclusivity of HUWE1 mutations and increased MYC copy number and / or RNA expression in CRC samples (Log Odds Ratio: -1.144 [some tendency toward mutual exclusivity] , p = 0.042).
HUWE1 is a critical colonic tumour suppressor gene that prevents MYC signalling, DNA damage accumulation and tumour initiation. (2016)
Figure 5

Figure 5. Huwe1 deletion leads to an increase in DNA damage.

(a) H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells. Band intensity relative to βactin displayed under each lane (Mann-Whitney, p = 0.04, n = 3 vs 3). (b) γ-H2AX IHC showing increased positivity in Huwe1 deleted intestinal cells. Scale bars = 50 µm. (c) γ-H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells (Mann-Whitney, p = 0.04, n = 3 vs 3). (d) γ-H2AX IHC of Vil Apc and Vil Apc Huwe1hom tumours, note increased positivity in Huwe1 deficient tumours. Scale bars = 50 µm.
HUWE1 is a critical colonic tumour suppressor gene that prevents MYC signalling, DNA damage accumulation and tumour initiation. (2016)
Figure 6

Figure 6. Huwe1 deficient tumours are sensitive to cisplatin treatment.

(a) Caspase 3 IHC of tumours from Lgr5 Apc and Lgr5 Apc Huwe1 mice either untreated or 6h post treatment with 7.5mg/kg cisplatin. Arrows identify caspase 3 positive cells. Scale bars = 50 µm. (b) Quantification of cisplatin treatment showing a significant increase in apoptosis in Huwe1 deficient tumour cells (Mann-Whitney, n = 3 vs 5). (c) Comparison of somatic mutation rate (mutations / Mb) between human tumours carrying HUWE1 mutations or not. Note the significant increase in mutational burden in HUWE1 mutated tumours (Mann-Whitney, p = 0.0002, n ≥ 15). (d) Comparison of somatic mutation rate (mutations / Mb) between human tumours carrying HUWE1 mutations or not grouped according to MLH1 status. Note that the increased mutation rate is found primarily in tumours where MLH1 is not silenced indicating increased mutation rate (and HUWE1 mutation itself) is not due to silencing of MLH1 (Mann-Whitney, p = 0.0007, n ≥ 11). (e) Survival analysis of colorectal cancer patients treated with adjuvant chemotherapy divided by HUWE1 expression levels. Note the lowest HUWE1 expressing quartile of patients respond significantly better to chemotherapy. (f) Survival analysis of colorectal cancer patients not treated with adjuvant chemotherapy divided by HUWE1 expression levels. Note the lowest HUWE1 expressing quartile of patients do not survive significantly longer than those expressing higher levels of HUWE1 if not treated with chemotherapy.