EMBO/soda-nel huggingface dataset:
Siwi levels reversibly regulate secondary piRISC biogenesis by affecting Ago3 body morphology in Bombyx mori (2020)
Figure 1

Figure 1. Vret interconnects Ago3-piRISC and unloaded Siwi for secondary Siwi-piRISC production.

Silver staining of protein components in the Ago3 complex immunoisolated from BmN4 cells. P150 and P130 co-immunoprecipitated with Ago3. n.i.: non-immune IgG.

Western blots showing the presence of Vret-L (P150) and Vret-S (P130) in the Ago3 complex in A. n.i.: non-immune IgG.

Top: western blot showing Vret-L and Vret-S immunoprecipitated from BmN4 cells upon UV irradiation. Bottom: CLIP signals showing that Vret-L, but not Vret-S, binds RNAs in vivo.

Five upper panels: Northern blots showing the levels of RT3-piRNA, Bmmar6-piRNA, R2Bm-piRNA, and R1Bm-piRNA in normal (Control), Vret-L/S-depleted (Vret KD), Vret-L-depleted (Vret-L KD), and Vret-S-depleted (Vret-S KD) cells. Bantam (miRNA) was detected as a loading control. Two lower panels: western blots showing the efficiency of the Vret knockdown. β-Tub was detected as a loading control.

Two upper panels: western blots showing the abundances of Flag-Vret-L and Ago3 in both samples. Note that Flag-Vret-L is present only in the Vret-L complex but not with Ago3 isolated from total cell lysates (Fig EV1F). Bottom panel: Northern blot showing the level of PiggyBac-piRNA associated with Ago3 in total lysate (Ago3-IP) and in the Vret-L complex (Flag-Vret-IP). The ratio of the piRNA signal intensity in the two samples is indicated at the bottom.

Two upper panels: western blots showing the abundances of Flag-Vret-L and Siwi in both samples. Bottom panel: Northern blot showing the level of RT3-piRNA associated with Siwi in the total lysate (Siwi-IP) and in the Vret-L complex (Flag-Vret-IP).

Boxplot showing log2 fold-change of Vret-KD (RPM)/Control (RPM) values of piRNAs in increased, unchanged and decreased groups defined by Ago3-KD (RPM)/Control (RPM) values shown in Fig EV1I. Boxplot represents median, first and third quartile, and maximum and minimum values of the log2 fold-change of piRNAs (N = 2). Outliers are represented as open circles.

Flag-EGFP and -Vret-L WT and mutants were immunoprecipitated using the anti-Flag antibody and then western blotting was performed using anti-Flag, anti-Siwi, anti-Ago3 and anti-Vret antibodies. The domain structures of Tud1mut (Y753A/Y757A/N759A) and Tud2mut (Y973A/ Y976A/N978A) are shown.
Siwi levels reversibly regulate secondary piRISC biogenesis by affecting Ago3 body morphology in Bombyx mori (2020)
Figure 2

Figure 2. Ago3 bodies serve as the center of secondary Siwi-piRISC production.

Immunofluorescence shows that the Vret signals (green) co-localize with the Ago3 signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the image on the left-hand side. This panel arrangement applies also for panels B−H. The bar chart shows the percentage of Vret-positive nuage in Ago3-positive nuage.

Immunofluorescence shows that the Vret signals (green) partially co-localize with the Siwi signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The bar chart shows the percentage of Vret-positive nuage in Siwi-positive nuage.

Immunofluorescence showing the Ago3 signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm.

Immunofluorescence showing the Siwi signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm.

Immunofluorescence showing the Vret signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm.

Immunofluorescence showing the Siwi signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm.

Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) expressed in naïve BmN4 cells (Control). DAPI shows the nuclei (blue). Scale bar: 10 μm.

Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) in Ago3-depleted cells (Ago3 KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.
Siwi levels reversibly regulate secondary piRISC biogenesis by affecting Ago3 body morphology in Bombyx mori (2020)
Figure 3

Figure 3. Lack of Siwi causes Ago3 phosphorylation and insolubilization, causing Ago3-body enlargement.

Immunofluorescence showing the Ago3 signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the images on the left-hand side. This panel arrangement applies also for panels B, H and I. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 106; Small: IntDen value < 106).

Immunofluorescence showing the Vret signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105).

Western blotting was performed before and after Siwi KD using the anti-Ago3 antibody. Total BmN4 lysate (Total), supernatant (Sup) and pellet (Pellet) fractions upon centrifugation were used.

Western blotting was performed using the anti-Ago3 antibody on Ago3 immunoisolated from Siwi-depleted cells. Ago3 immunoprecipitated was untreated (−) or treated with phosphatase (+) prior to western botting.

Western blot showing the degree of phosphorylation of Flag-Ago3 WT and the 8SA mutant in Siwi-depleted cells (Siwi KD).

Western blots showing the degree of phosphorylation of Flag-Ago3 WT and its DDH and KA mutants in Siwi-depleted cells (Siwi KD). The second panel from the top corresponds to the top panel with a longer exposure time. Siwi and β-Tub as a loading control were also detected also by western blotting.

Western blotting was performed using anti-Ago3, anti-Vret and anti-Siwi antibodies on lysates of BmN4 cells before (Control) and after Siwi (Siwi KD) and Siwi/Vret (Siwi/Vret KD) depletion. β-Tub was detected as a loading control.

Immunofluorescence showing the Flag-Ago3 signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.

Immunofluorescence showing the Flag-Ago3 8SA and 8SE mutant signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.
Siwi levels reversibly regulate secondary piRISC biogenesis by affecting Ago3 body morphology in Bombyx mori (2020)
Figure 4

Figure 4. The absence of Siwi rapidly stalls Ago3-piRISC in a form bound with cleaved RNA fragments.

Western blot showing Ago3 immunopurified from BmN4 cells before (Control) and after Siwi knockdown (Siwi KD). 32P-labeling of RNAs isolated from Ago3 (a mixture of unphosphorylated and phosphorylated) immunopurified from Siwi-depleted cells. 15-20-nt RNAs (indicated by a black line) were isolated and sequenced.

80.4% of the 15-20-nt RNAs corresponding to transposon sequences were antisense to transposon mRNAs.

The measurement of distances between the 3′-ends of Target-S (15-20-nt RNAs in A) and the 5′-ends of Ago3-bound piRNAs.

32P-labeling of RNAs isolated from Ago3. Target-Ss are isolated from Siwi-depleted BmN4 cells but are not isolated from Siwi/Vret double knockdown cells.

Western blot showing Ago3 immunopurified from BmN4 cells before (Control) and after Siwi knockdown (Siwi KD). Northern blot showing Bmmar6-piRNA precursors in the Ago3 complex. 50-100-nt RNAs (indicated by a black line) were isolated and sequenced.

88.6% of the transposon-matching piRNA precursors were antisense to transposon mRNAs.

The measurement of distances between the 5′-ends of Target-L and the 5′-ends of Ago3-bound piRNAs.

The measurement of distances between the 5′- and 3′-ends of Target-L fragments on the same genomic strand. Heat map displays 5′-ends of Target-L in a window around the most abundant 1,000 3′-ends of Target-L.

The measurement of distances between the 5′-ends and 3′-ends of Target-S fragments on the same genomic strand. The heat map displays 5′-ends of Target-S in a window around the most abundant 1,000 3′-ends of Target-S.

The measurement of distances between the 5′-ends of Target-L and the 3′-ends of Target-S on the same genomic strand. The heat map displays 5′-ends of Target-L in a window around the most abundant 1,000 3′-ends of Target-S.

The measurement of distances between the 5′-ends of Target-S and the 3′-ends of Target-L on the same genomic strand. The heat map displays 5′-ends of Target-S in a window around the most abundant 1,000 3′-ends of Target-L.
Siwi levels reversibly regulate secondary piRISC biogenesis by affecting Ago3 body morphology in Bombyx mori (2020)
Figure 5

Figure 5. The aberrant Ago3-body morphology is rescued by Siwi restoration.

Immunofluorescence showing the Ago3 signals (green) in Siwi-depleted BmN4 cells (Siwi KD) and Siwi KD cells where Flag-Siwi (red) was expressed. DAPI shows the nuclei (blue). Scale bar: 10 μm. The panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the images on the left-hand side. This arrangement applies also for panel C. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105). A significant difference was found plus/minus Flag-Siwi expression by the Fisher's exact test (P < 0.05).

Western blots show that the degree of Ago3 phosphorylation was reduced upon Siwi KA mutant as well as Siwi WT and DDH mutant expression.

Immunofluorescence showing the Ago3 signals (green) in Siwi-depleted BmN4 cells (Siwi KD) and Siwi KD cells where Flag-Siwi KA mutant (red) was expressed. DAPI shows the nuclei (blue). Scale bar: 10 μm. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105). A significant difference was found plus/minus Flag-Siwi expression by the Fisher's exact test (P < 0.05).
Myogenin promoter-associated lncRNA Myoparr is essential for myogenic differentiation (2018)
Figure 1

Figure 1. A promoter-associated lncRNA, Myoparr, is expressed from the upstream region of myogenin locus

A Occupancies of Pol II at the myogenin locus in C2C12 myoblasts (MB, shown in blue) and myotubes (MT, shown in red). Myoparr is located immediately upstream from the myogenin gene.

B Schematic representation of the upstream region of myogenin and amplified regions by RT-PCR (top). RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes (bottom). The presence or absence of reverse transcriptase (RT) is shown by (+) or (-), respectively. The templates (cDNA or genomic DNA) are indicated by C or G, respectively.

C The primers used for RT-PCR (top). Strand-specific RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA (middle and bottom) and poly(A)+ RNA (bottom).

D Relative expression of indicated RNAs in differentiating C2C12 cells. The presence or absence of RT is shown by (+) or (-), respectively. PCR products were verified by sequencing. n = 3, mean ± SD. The nuclear/cytoplasmic fractionation is confirmed by the expression of H2B and tubulin, respectively.

E Chromatin pellet extract (CPE)/soluble nuclear extract (SNE) ratio of SRA1, Malat1, and Myoparr in C2C12 myotubes on a log scale. n = 3, mean ± SD.
Myogenin promoter-associated lncRNA Myoparr is essential for myogenic differentiation (2018)
Figure 2

Figure 2. Myoparr expression is correlated with myogenin expression and regulated by MyoD and TGF-β

A, B Quantitative RT-PCR for myogenin (A) and Myoparr (B) during myogenesis of C2C12 cells, primary mouse myoblasts (growth medium, GM), and mouse embryonic skeletal muscle (S.K.). The x-axis shows days after differentiation induction or embryonic days. n = 3, mean ± SD.

C qRT-PCR showing increased Myoparr expression by MyoD in C3H10T1/2 fibroblasts. n = 4, mean ± SD. **p < 0.01 (unpaired two-tailed Welch's t-test).

D Treatment of recombinant TGF-β for 24 h decreased Myoparr expression in differentiating C2C12 cells. n = 3, mean ± SD. *p < 0.05 (unpaired two-tailed Student's t-test).

E Schematic diagram of the constructs used for luciferase assays.

F Relative luciferase activities of the indicated promoter in differentiating C2C12 cells by exogenous MyoD. n = 3, mean ± SD. **p < 0.01. (unpaired two-tailed Student's t-test).

G Relative luciferase activities of the indicated promoter with/without E-box mutations in differentiating C2C12 cells. n = 3, mean ± SD. **p < 0.01. (unpaired two-tailed Student's t-test).

H Relative luciferase activities of the indicated promoter with/without E-box mutations in differentiating C2C12 cells by TGF-β treatment. n = 3, mean ± SD. *p < 0.05. **p < 0.01. n.s., not significant (unpaired two-tailed Student's t-test).
Myogenin promoter-associated lncRNA Myoparr is essential for myogenic differentiation (2018)
Figure 3

Figure 3. Myoparr is required for the activation of myogenin and C2C12 differentiation

A C2C12 cells were transfected with 50 nM of indicated siRNAs in growth medium. After additional 24-h incubation in growth medium, cells were transferred to differentiation medium. The levels of Myoparr and myogenin expression were quantified by qRT-PCR 24 h after differentiation induction. n = 3, mean ± SD. *p < 0.05. n.s., not significant. Statistical analyses were performed using unpaired two-tailed Student's t-test. In cases of unequal variances, unpaired two-tailed Welch's t-test was used.

B Western blot showing decreased expression of myogenin in differentiating C2C12 cells 48 h after Myoparr KD. Tubulin expression served as an internal control.

C The expression levels of myogenin and Myoparr evaluated by qRT-PCR in myogenin-depleted C2C12 cells. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test).

D ChIP-qPCR detection of Pol II occupancy and histone modification status at the myogenin locus in Myoparr-depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. **p < 0.01 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test).

E Retrieval rate of Myoparr using Myoparr-ChIRP probes from C2C12 cells quantified by qPCR is shown as percent of input values. GAPDH was amplified as a negative control. n = 3, mean ± SD. ND, not detected.

F ChIRP-qPCR detection of the interaction between endogenous Myoparr and the myogenin promoter. The myogenin 3´UTR and GAPDH promoter were amplified as negative controls. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. ** p < 0.01 (unpaired two-tailed Welch's t-test). ND, not detected.

G, H Immunocytochemistry for the detection of myogenin at 24 h (G) or MHC at 72 h (H) in Myoparr-depleted C2C12 cells after differentiation induction. Nuclei were counterstained with DAPI. Bar, 100 μm. The percentage of myogenin-positive cells or fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test).

I The intersection of genes regulated by Myoparr KD and myogenin KD shows a significant (Fisher's exact test) overlap 25.6 times as large as expected by chance alone.

J Genes significantly differentially expressed in both Myoparr- and myogenin-depleted cells show correlated expression (R = 0.63, log 2 ratio scale).

K-N Enrichment of GO categories in genes up-regulated by Myoparr KD (K), down-regulated by Myoparr KD (L), down-regulated by myogenin KD (M), and commonly regulated by both Myoparr KD and myogenin KD (N).
Myogenin promoter-associated lncRNA Myoparr is essential for myogenic differentiation (2018)
Figure 4

Figure 4. Myoparr interacts with DEAD box protein Ddx17

A Scheme of the identification of Myoparr-interacting proteins using RiboTrap and differential proteomics analysis.

B Following immunoprecipitation, the interaction between Myoparr and endogenous Ddx17 was confirmed by immunoblotting using a Ddx17-specific antibody. Two different Ddx17 isoforms (p72 and p82) were observed.

C Determination of the Ddx17-binding region of Myoparr by RNA pull-down analyses. Schematic diagram of full-length or truncated Myoparr used for RNA pull-down (top). In vitro transcribed/translated Ddx17 protein was pulled down by indicated Myoparr and then detected by western blot using a Ddx17 antibody (bottom).

D Schematic diagram of the constructs used for luciferase assays. The details of all constructs are described in Materials and Methods. p(A) indicates poly(A) site.

E-G Relative luciferase activities of indicated constructs in differentiating C2C12 cells.

H Relative luciferase activities of indicated constructs in differentiating C2C12 cells transfected with control or Myoparr shRNA (Myoparr KD).

Data information: In (E-H), bars indicate the average of two independent experiments, and open circles represent the values of each experiment.
Myogenin promoter-associated lncRNA Myoparr is essential for myogenic differentiation (2018)
Figure 5

Figure 5. Myoparr promotes the protein-protein interaction between Ddx17 and PCAF

A Significantly decreased MHC expression in Ddx17-depleted C2C12 cells is shown by immunocytochemistry. Nuclei were counterstained with DAPI. Bar, 100 μm. Fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test).

B Immunocytochemistry for myogenin in Ddx17-depleted C2C12 cells. Bar, 100 μm. Myogenin-positive cells are shown as percent of the control. n = 4, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test).

C Relative luciferase activities of the -1650-Luc by the combination of MyoD and Ddx17 or Ddx17 mutant (K142R). Bars indicate the average of two independent experiments, and open circles represent the values of each experiment.

D Ddx17 or Ddx17 mutant (K142R) was pulled down by full-length Myoparr and then detected by western blot.

E Reduced interaction between endogenous Ddx17 and PCAF in Myoparr-depleted C2C12 cells. After Myoparr KD, the cell lysates were subjected to immunoprecipitation (IP) with a Ddx17-specific antibody 36 h after differentiation induction (right panel). Each lysate was loaded as an input (left panel).

F Relative quantification of (E) is shown as IP/input ratio. n = 3, mean ± SD. *p < 0.05. n.s., not significant (unpaired two-tailed Student's t-test).

G Increased interaction between Ddx17 and PCAF by Myoparr. PCAF protein was pulled down by 3×Flag-Ddx17 in the presence or absence of Myoparr using a Flag antibody and then detected by western blot.

H, I ChIP-qPCR detection of Ddx17 (H) and PCAF (I) occupancies at the myogenin locus in Myoparr-depleted differentiating C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05 (unpaired two-tailed Student's t-test).
Myogenin promoter-associated lncRNA Myoparr is essential for myogenic differentiation (2018)
Figure 6

Figure 6. Myoparr and Ddx17 regulate myoblast cell cycle withdrawal in a myogenin-independent manner.

A Heatmap displaying expression changes of 754 genes significantly altered either in Myoparr- or Ddx17-depleted cells (log 2 ratio scale).

B The intersection of genes regulated by Myoparr KD and Ddx17 KD shows a significant (Fisher's exact test) overlap 26.9 times as large as that expected by chance alone.

C Genes significantly differentially expressed in both Myoparr- and Ddx17-depleted cells show correlated expression (R = 0.94, log 2 ratio scale).

D-F Enrichment analysis of GO categories in genes up-regulated by Ddx17 KD (D), down-regulated by Ddx17 KD (E), and commonly regulated by both Myoparr KD and Ddx17 KD (F).

G Myoparr and Ddx17 are required for C2C12 cell cycle withdrawal. C2C12 cells transfected with each siRNA were cultured in growth medium for 24 h. After differentiation induction, cells were maintained in differentiation medium for 40 h and then treated with EdU for 6 h. EdU-positive cells are shown as percent of the control. Nuclei were counterstained with Hoechst 33342. n = 3, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test). Bar, 100 μm.
Myogenin promoter-associated lncRNA Myoparr is essential for myogenic differentiation (2018)
Figure 7

Figure 7. Myoparr regulates myoblast cell cycle withdrawal through the activation of miR-133b, miR-206, and H19 expression

A qRT-PCR for pri-miR-133b, pri-miR-206, and H19 expression in differentiating C2C12 cells transfected with indicated siRNAs. n = 3, mean ± SD. *p < 0.05. **p < 0.01.

B ChIP-qPCR detection of Pol II occupancy at the indicated promoters in Myoparr-depleted C2C12 cells. The data were normalized to input values. n = 3, mean ± SD. *p < 0.05. Myoparr KD decreased Pol II occupancy at the miR-133b promoter with a marginal trend toward significance (p = 0.052).

C, D ChIP-qPCR detection of Ddx17 (C) and PCAF (D) occupancies at the indicated promoters in Myoparr-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01.

E qRT-PCR showing decreased expression of miR-133b and miR-206 in both Myoparr- and Ddx17-depleted C2C12 cells. n = 3, mean ± SD. *p < 0.05. **p < 0.01.

F Western blots showing increased ERK1/2 activity (pERK1/2) and Cdc6 expression in differentiating C2C12 cells 48 h after Myoparr and Ddx17 KD. Tubulin expression served as an internal control.

G Increased Pola1 expression detected by qRT-PCR. n = 3, mean ± SD. **p < 0.01.

Data information: Statistical analyses were performed using unpaired two-tailed Student's t-test. In cases of unequal variances, unpaired two-tailed Welch's t-test was used.
Myogenin promoter-associated lncRNA Myoparr is essential for myogenic differentiation (2018)
Figure 8

Figure 8. Knockdown of Myoparr blocks skeletal muscle atrophy caused by denervation in mice

A Seven days after denervation, weights of innervated (-) and denervated (+) tibialis anterior (TA) muscles were measured. n = 4, mean ± SEM. *p < 0.05 (unpaired two-tailed Student's t-test).

B Expression of myogenin in innervated and denervated TA muscles detected by qRT-PCR 7 d after denervation. n = 4, mean ± SD. ***p < 0.001 (unpaired two-tailed Welch's t-test).

C qRT-PCR showing increased Myoparr expression in denervated TA muscles 7 d after denervation. n = 3, mean ± SD. ***p < 0.001 (unpaired two-tailed Student's t-test).

D Immunoblot showing decreased expression of myogenin by Myoparr depletion in denervated TA muscle 7 d after denervation. Expression of tubulin served as an internal control.

E Weights of innervated and denervated TA muscles electroporated either with control or Myoparr shRNA. Muscle weights were measured 7 d after denervation. n = 5 for each group, mean ± SEM. ***p < 0.001. n.s., not significant (unpaired two-tailed Student's t-test).

F Representative immunostaining images for laminin (red) and EmGFP (green) of innervated or denervated TA muscles electroporated with control or Myoparr shRNA, both containing EmGFP. Bar, 100 μm.

G Cross-sectional area (CSA) of electroporated EmGFP-positive myofibers (250 fibers per sample) was analyzed 7 d after denervation. n = 5 for each group, mean ± SEM. **p < 0.01. n.s., not significant (unpaired two-tailed Student's t-test).