EMBO/soda-nel huggingface dataset:
Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding (2022)
Figure 3

Fig 3. EJC binding is dispensable for NMD activity of UPF3 paralogs.

A. Protein sequence alignment of C-terminal regions of UPF3 proteins from different mammalian species (labeled on the left).

B. Immunoblot showing levels of EJC and UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing different FLAG-tagged proteins indicated above each lane.

C. Bar plots showing isoform specific RT-qPCR-based measurement of relative levels of PTC+ and PTC- isoforms of genes indicated below from wild-type (WT) or 3DKO#2 cells stably expressing the specified proteins. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means. The asterisk (*) represents statistically significant difference (p<0.05, t-test) of indicated samples from EGFP-transfected control cells, which has a value of 1 (n=3 biological replicates).

D. Immunoblot showing EJC/UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing FLAG-tagged proteins given above each lane (hUPF3B = human UPF3B; hUPF3B-h3AC = human UPF3B protein with C-terminal domain from human UPF3A; hUPF3B-m3AC = human UPF3B protein with C-terminal domain from mouse UPF3A).

E. Bar plots of isoform specific RT-qPCR-based analysis of PTC+ and PTC- transcript levels from wild-type (WT) or 3DKO#2 cells stably expressing the proteins specified in the legend. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means. The asterisk (*) represents statistically significant difference (p<0.05, t-test) of indicated samples from EGFP-transfected control cells, which has a value of 1 (n=3 biological replicates).
Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding (2022)
Figure 4

Fig 4. Human UPF3 paralogs differ in NMD activity.

A. Northern blots showing levels of β-globin reporter mRNAs in wild-type HeLa Tet-off cells and UPF3B knockout HeLa Tet-off cells. β39 is a tetracycline (Tet)-inducible reporter with a PTC at codon 39 whose levels are shown at different timepoints after transcriptional shut-off (chase) as indicated above each lane. β-GAP is a stable, constitutively-expressed, longer β-globin mRNA used as transfection control. Proteins overexpressed (OE) in each condition are indicated on top and reporter mRNA half-lives (t1/2) along with standard errors of means are on the bottom.

B. Schematic of human UPF3A, UPF3B and the UPF3A chimeric proteins where UPF3A domains are replaced by the corresponding domains from UPF3B (see material and methods for detailed domain definition). Previously characterized UPF2 binding domain (UPF2-BD) and EJC-binding motif (EBM) are shown.

C. Northern blot showing steady-state levels of β39 NMD reporter and β-GAP control in HeLa Tet-off UPF3B knockout cells upon transient overexpression of wild-type UPF3 proteins or different UPF3A chimeric proteins indicated above each lane. Below each lane, relative fold-change (Rel FC) indicates β39 reporter levels (normalized to β-GAP control) as compared to the normalized β39 reporter levels in UPF3B expressing cells.

D. Immunoblot showing levels of EJC proteins or HNRNPA1 in input or EIF4A3-IP from 3DKO#2 cells stably expressing different UPF3 proteins or EGFP as a control as indicated above each lane. Relative IP of FLAG-tagged proteins are quantified against EIF4A3.
Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding (2022)
Figure 5

Fig 5. NMD activity in human cells in the absence of both UPF3 paralogs.

A, B. CDF plots of (A) PTC+ and PTC-isoforms, (B) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.

C. Two possible models for UPF3A/3B-dependent and -independent NMD. Model 1 predicts that mRNAs (represented by wavy lines) from one set of genes (mRNA 1) undergoes UPF3A/3B-dependent NMD and those from another set of genes (mRNA 2) are targeted by UPF3A/3B-independent NMD. Model 2 posits that same mRNAs are targeted to both UPF3A/3B-dependent and -independent NMD. The number of arrows signify the probability/rate of NMD commitment via each branch. In Model 2, commitment to NMD is higher in the presence of UPF3 paralogs and lower in their absence.

D, E. CDF plots of UPF3A/3B-independent (UPF3A/3B-indep., D) and -dependent (UPF3A/3B-dep., E) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.

F. CDF plots of UPF3A/3B-dependent (UPF3A/3B-dep.), UPF3A/3B-independent (UPF3A/3B-indep.) NMD+ isoforms and NMD- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH (2018)
Figure 1

Figure 1. Liver-specific Sel1L deficiency in mice (Sel1LAlb) causes growth retardation.

(A) Western blot analysis and quantitation of Sel1L-Hrd1 ERAD proteins in the livers of WT mice at 3, 9 and 24 weeks of age (n=3-6 per group, 2 independent repeats).

(B) Western blot analysis and quantitation of Sel1L-Hrd1 ERAD proteins in the livers of 10-week-old WT mice under fasted (overnight) or ad libitum fed conditions (n=3-6 per group, 2 independent repeats).

(C) Western blot analysis and quantitation of ERAD proteins in Sel1Lf/f and Sel1LAlb livers (n=4 per group, 3 independent repeats).

(D) Growth curves of male (n=10 each) and female (n=7 each) mice.

(E-F) Representative images (E) and nose-to-anus length (F) of male mice at 6 weeks of age (n=6-10 per group).

(G) Organ-to-body-weight ratios of liver and kidney in 6-week-old male mice (n=6 per group, 3 independent repeats).

(H) Daily food intake (g/d) normalized to gram of body weight (gbw) (n=3 per group, measured over 3 days).

(I) Representative estrus cycle mapping in 2-4-month old females (n=6 per group).

(J) H&E images of paraffin-embedded liver sections from 9-week-old mice (n=4 per group, 3 independent repeats).

(K) Western blot analysis of cell death (cleaved and pro-caspase-3) in livers of 9-week-old mice, with WT ileum as positive control (n=3 per group, 3 independent repeats).

(L) Representative transmission electron microscope (TEM) images obtained from 9-week-old female mice livers (n=10-12 cells from one mouse each). N, nucleus; mito, mitochondria; ER, endoplasmic reticulum.

Data information: Hsp90 and α-tubulin, loading controls for Western blot analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001, n.s., non-significant by Student's t test.
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH (2018)
Figure 2

Figure 2. Elevated Fgf21 expression in the liver as well as circulating Fgf21 in the absence of Sel1L.

(A) Volcano plot depicting transcriptomics data from the livers of 9-week-old Sel1Lf/f and Sel1LAlb mice (n=3 per group); dotted line marks p=0.05; black dots represent fold change>2.

(B-C) qPCR (B) and Western blot (C) analyses of Fgf21 expression in 9-week-old livers (n=3-6 per group, 3 independent repeats).

(D) ELISA analysis of Fgf21 in serum from 8-9-week-old mice (n=6-7 per group).

(E) Protein levels of Sel1L (left panel) and mRNA levels of Fgf21 (right panel) in primary mouse hepatocytes isolated from the tamoxifen-inducible Sel1L-knockout Sel1LERCre mice (2 independent repeats).

(F-G) Acute loss-of-function model where 8-week-old Sel1Lf/f mice were injected i.v. with either AAV8-Cre or control AAV8-GFP: (F) Western blot analysis of hepatic and control adipose Sel1L protein (n=3 per group); and (G) qPCR analysis of hepatic Fgf21 expression and ELISA analysis of serum Fgf21 (n=3 per group, 2 independent repeats).

(H) Heatmaps of top 15 significantly upregulated and downregulated genes in Fgf21 Tg livers and their expression levels in Sel1LAlb livers (n=3 per group).

(I) Scatter plot depicting the logarithmic fold-change (FC) for 16,402 genes in Sel1LAlb and Fgf21 transgenic (Tg) livers (n=3 per group); genes that are highly upregulated or downregulated in both datasets are marked in red and blue, respectively; genes that are upregulated unique to each data set (e.g. Derl3 for Sel1L-Hrd1 ERAD-deficient liver) are marked in green.

(J-L) Data from rescue experiments where 5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. with AAV8-shFgf21 or control AAV8-shLuc: (J-K) qPCR analysis of Fgf21 mRNA (J) and ELISA analysis of Fgf21 in serum (K) 3 weeks after injection (n=4 per group). (L) Weight gain curve after injection (n=7 per group).

Data information: Hsp90 and α-tubulin, loading control for Western blot analysis. Ribosomal L32, loading control for qPCR analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001, n.s., non-significant by Student's t test (B-G) or 2-way ANOVA analysis (J-L).
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH (2018)
Figure 3

Figure 3. Sel1LAlb mice phenocopy Fgf21-gain-of-function mice.

(A) Western blot analysis of p-Stat5 in livers of 9-week-old mice (n=3 per group, 3 independent repeats), with quantitation of the ratio of p- to total Stat5 shown below the blot.

(B) qPCR analysis of p-Stat5-associated growth genes in the livers of 9-week-old mice (n=6 per group, 2 independent repeats).

(C) Z ambulatory activity of 9-week-old male mice as measured over 24 hr (n=4 males per group).

(D-G) Blood glucose (D), serum insulin (E), serum triglyceride (TG) (F), serum cholesterol (CHOL) (G) levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).

(H) H&E images of inguinal white adipose tissue (iWAT) from 8-week-old mice (n=3 per group, 2 independent repeats).

(I) qPCR analysis of browning-related genes in iWAT (n=3-6 per group).

(J) Western blot analysis of Ucp1 in iWAT of 8-week-old mice (n=3-4 per group, 3 independent repeats).

(K) Weight gain curve of male mice after 60% high fat diet (HFD) starting at 5 weeks of age (n=4 per group, 2 independent repeats).

(L) Adipose tissue weight normalized to body weight in male mice following 9 weeks of HFD feeding (n=4 per group).

Data information: Hsp90, loading control for Western blot analysis. Ribosomal L32, loading control for qPCR analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001 by Student's t test or 2-way ANOVA, as needed.
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH (2018)
Figure 4

Figure 4. Sel1L deficiency leads to the accumulation of Crebh in the liver.

(A-C) Western blot analysis of Crebh, Crebh-N, Pparα and Xbp1s in whole cell lysates (A) and nuclear extracts (B) in WT and Sel1LAlb livers (n=3 per group, 3 independent repeats) with quantitation shown in (C).

(D) qPCR analysis of Crebh, Ppara and Xbp1s in WT and Sel1LAlb livers (n=4-6 per group, 2 independent repeats).

(E) Western blot analysis of Crebh in acute Sel1L loss of function model as described in Figure 2F-G (n=3 per group).

(F) Western blot analysis of Crebh protein in 9-week-old mice (n=3 per group, 2 independent repeats). WT mice injected i.p. with tunicamycin (Tm, 1.5 µg/g body weight) for 72 hours were included as a control.

(G) Western blot analysis of Crebh in the livers of 9-week-old mice after NP40-detergent fractionation into NP40 soluble (NP40S) and pellet (NP40P) (n=3 per group, 2 independent repeats).

(H) Representative confocal images of Crebh in the liver cryosections of 8-week-old mice (zoomed out versions in Figure EV3F). Note that a fraction of hepatocytes is binucleated. Yellow arrows represent Crebh staining inside in the hepatocyte nucleus.

(I) ChIP analysis of Crebh binding onto the Fgf21 promoter in the livers of 9-week-old mice, normalized first to 5% input group and then to no-antibody ChIP samples (n=3 pooled per group, 2 independent repeats).

(J-K) Western blot analysis of SEL1L, HRD1 and CREBH proteins (J) and qPCR analysis of SEL1L, HRD1 and FGF21 (K) in human Hep3B hepatocytes upon CRISPR deletion of SEL1L with two different guides.

Data information: Hsp90, H2A, β-actin and lamin, loading controls for Western blot analysis. Ribosomal L32, loading control for qPCR analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001; n.s., non-significant by Student's t test.
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH (2018)
Figure 5

Figure 5. Crebh is an Sel1L-Hrd1 ERAD substrate.

(A) Western blot analysis of Crebh protein half-life in transfected WT and HRD1-/- HEK293T cells treated with cycloheximide (CHX) for indicated times. The decay of protein from one experiment is shown below.

(B-C) Western blot analysis and quantitation of Crebh in Crebh-transfected WT (B) and HRD1-/- (C) HEK293T cells pre-treated with the proteasomal inhibitor bortezomib (BTZ) or lysosomal inhibitor chloroquine (CHQ) for 2 hours and then with CHX for additional 1 hour (n=2 per group, 2 independent repeats).

(D) Western blot analysis of Crebh ubiquitination following immunoprecipitation (IP) of Crebh-Flag and Crebh-N-Flag in HEK293T cells transfected with indicated plasmids. Samples were boiled with SDS before IP for denaturing IP and not so for native IP. These cells were treated with proteasomal inhibitor BTZ for the last 6 hr prior to immunoprecipitation.

(E) Western blot analysis of endoglycosidase H (endoH)-sensitivity of Crebh in the livers of 9-week-old mice; 'r' refers to endoH-cleavage-resistant species and 's' refers to endoH-cleavage-sensitive species (n=3 per group, 2 independent repeats).

Data information: Hsp90, loading control for Western blot analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; n.s., non-significant by 2-way ANOVA analysis.
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH (2018)
Figure 6

Figure 6. Crebh links hepatic Sel1L-Hrd1 ERAD to Fgf21.

5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc.

(A) Western blot analysis of hepatic Sel1L and Crebh 5 weeks post injection (n=3 mice each, 2 independent repeats).

(B-C) qPCR analysis of Crebh and Fgf21 mRNA (B) and ELISA analysis of circulating Fgf21 (C) 5 weeks post injection (n=3 per group, 2 independent repeats).

(D) qPCR analysis of hepatic growth-associated genes 5 weeks after injection (n=6 per group).

(E) Weight gain 6 weeks post injection (n=10 per group).

(F) Insulin tolerance test (ITT) 5-weeks after injection (n=10 per group).

(G-H) qPCR (G) and Western blot analysis (H) of Ucp1 levels in inguinal white adipose tissue (iWAT) 5 weeks post injection (n=3 per group, 2 independent repeats).

Data information: Hsp90, loading control for Western blot analysis. Ribosomal L32, loading control for qPCR analysis. Values are mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001; n.s., non-significant by 2-way ANOVA analysis (B-G) and Student's t test (A,H).
Hepatic Sel1L-Hrd1 ERAD manages FGF21 levels and systemic metabolism via CREBH (2018)
Figure 7

Figure 7. Sel1L-Hrd1 ERAD represses Crebh and Fgf21 under fasting-feeding and growth. Analysis of correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 during fasting-feeding (A-D) and growth (E-H).

(A) Western blot analysis of hepatic Sel1L-Hrd1 ERAD and Crebh of nuclear (Nuc) and cytosolic (Cyto) fractions from 10-week-old Sel1Lf/f and Sel1LAlb mice under overnight fasted or fed states.

(B) Quantitation of levels of Sel1L/Hrd1/Crebh proteins and Fgf21 mRNA in the livers of 10-week-old Sel1Lf/f mice.

(C) Quantitation of total Crebh (Crebh+Crebh-N) protein levels in the livers of 10-week-old Sel1Lf/f and Sel1LAlb mice.

(D) Serum Fgf21 levels in 10-week-old Sel1Lf/f and Sel1LAlb mice.

(E-H) Similar to (A-D) with the exception that these experiments were done with the livers from Sel1Lf/f and Sel1LAlb mice at 3, 9 and 24 weeks of age.

Data information: Hsp90 and lamin, loading controls for cytosolic and nuclear fractions. Values are mean ± SEM; **, p<0.01 by two-way ANOVA analysis. n=3-4 per group, 2 independent repeats.
Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain (2022)
Figure 2

Figure 2. CG2662/Lint-O binds to and functions with L(3)mbt in gene regulation.

(A, B) The mRNA levels of aub (A) and vasa (B) were quantified upon the depletion (RNAi) of L(3)mbt, Lint-1, CoRest, E2F2, Myb, Mip120, and Mip130, and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05, **p: < 0.01. All t-tests were performed against samples with⎾ symbol.

(C) Proteins immunoprecipitated with the anti-L(3)mbt antibody from the OSC lysates before (control) and after L(3)mbt knockdown (KD) were silver-stained. The bands corresponding to L(3)mbt-L and L(3)mbt-S are indicated.

(D) Flow chart of the identification of L(3)mbt-interactors in OSCs. All peptides obtained from LC-MS/MS are listed in Appendix Table S1.

(E) Summary of the sum of the −log posterior error probability (Sum Pep Score: SPS), peptide spectra match values (PSM), and known functions of the proteins in (D). Mass spectrometry analysis was performed on biological duplicates.

(F, G) The mRNA levels of vasa (F) and aub (G) were quantified upon the depletion (RNAi) of L(3)mbt, Atac3, Prod, CG2662/Lint-O, and CG2199, and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. **p: < 0.01. All t-tests were performed against samples with⎾ symbol.
Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain (2022)
Figure 3

Figure 3. The L(3)mbt−Lint-O interaction is crucial for the repression of target genes.

(A) Domain structures of L(3)mbt and Lint-O. L(3)mbt has three MBT repeats (orange), a C2H2-type zinc finger (Zf) (gray), and a SAM domain (blue). Lint-O has two PHD finger domains (green) and a SAM domain (blue).

(B) Immunoprecipitation (IP)/western blotting shows that WT Lint-O, but not its ∆SAM mutant (Fig EV3A), co-immunoprecipitated with L(3)mbt from the OSC lysates. n.i.: non-immune IgG.

(C) Subcellular localization of WT Lint-O and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.

(D) IP/western blotting shows that WT L(3)mbt-L, but not its ∆SAM mutant (Fig EV3B), co-immunoprecipitated with Lint-O from the OSC lysates. n.i.: non-immune IgG.

(E) Subcellular localization of WT L(3)mbt-L and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.

(F, G) The mRNA levels of vasa (F) and aub (G) were quantified upon ectopic expression of EGFP, as well as WT Lint-O and its ∆SAM and 8CA mutants (Fig EV3A), in Lint-O-lacking OSCs (Lint-O RNAi) and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05. All t-tests were performed against samples with⎾ symbol.

(H) IP/western blotting shows that L(3)mbt co-immunoprecipitated with WT Lint-O and its 8CA mutant, but not with ∆PHD mutant (Fig EV3A). An asterisk shows background.

(I) Subcellular localization of WT Lint-O and its 8CA and ∆PHD mutants (green). All proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.

(J) ChIP-qPCR shows that L(3)mbt binding to the vasa promoter was weakened after the loss of Lint-O. Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. **p: < 0.01. All t-tests were performed against samples with⎾ symbol.

(K) Western blotting showing the amounts of L(3)mbt, Lint-O, and histone H3 (H3) in normal (control), L(3)mbt-depleted (RNAi), and Lint-O-depleted (RNAi) OSCs. Asterisks show background signals as in Fig EV3E.

(L) The lint-O mRNA levels in normal (control), L(3)mbt-depleted (RNAi), and Lint-O-depleted (RNAi) OSCs are shown by fragments per kilobase million (FPKM). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05, **p: < 0.01. All t-tests were performed against samples with⎾ symbol.
Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain (2022)
Figure 5

Figure 5. The lack of Lint-O in flies leads to abnormal ovarian morphology and female sterility.

(A) CRISPR-mediated generation of lint-O knockout (KO). Genomic structure of lint-O is shown with gRNA targeting lint-O exon 1 (triangle). Sequences of lint-O DNA in WT (y w) (complementary to the gRNA sequence) and the KO mutant, Lint-OKO, are shown. PAM: protospacer adjacent motif (underlined). E1-3: Exons 1−3. CDS: protein-coding sequence. UTR: untranslated region.

(B) RT-qPCR analysis shows the mRNA levels of lint-O in y w and Lint-OKO ovaries. Data are expressed as mean and error bars represent SD. n = 2 biological replicates.

(C) The numbers of progeny in y w and Lint-OKO are shown. Ten independent crosses were performed. Boxplot central bands, upper edges of boxes, lower edges of boxes, upper whiskers and lower whiskers show median, third quartile, first quartile, maxima, and minima, respectively.

(D) Confocal images of y w and Lint-OKO ovaries immunostained for Vasa. Scale bar: 50 μm.

(E) Confocal images of y w and Lint-OKO ovarioles immunostained for Vasa. Vasa was ectopically expressed in follicle cells (white arrowheads). Scale bar: 50 μm.
Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain (2022)
Figure 6

Figure 6. L(3)mbt and Lint-O cooperatively control target genes in the brain.

(A) Western blotting shows that Vasa, Piwi, and Aub were ectopically expressed in Lint-OKO and L(3)mbtts1 larval brains at 29ºC. Anti-Lint-O, anti-L(3)mbt, anti-Vasa, andi-Piwi, anti-Aub, and anti-Tubulin antibodies were used.

(B) Confocal images of y w, Lint-OKO, and L(3)mbtts1 immunostained for Vasa (magenta), MIRA (green), and ELAV (green). Scale bar: 100 μm.

(C) Quantification of brain lobe volume for the following genotypes: Lint-OKO -/+ (n = 42 biological replicates), Lint-OKO -/- (n = 36 biological replicates), and L(3)mbtts1 -/- (n = 34 biological replicates). Boxplot central bands, upper edges of boxes, lower edges of boxes, upper whiskers and lower whiskers show median, third quartile, first quartile, maxima, and minima, respectively. P-values were calculated by the Student's t-test. (***P-value < 0.001).

(D, E) The genomic regions harboring the vasa (D) and aub (E) genes. The RNA-seq reads in y w, L(3)mbtts1, and Lint-OKO brains are shown. The shading in gray corresponds to exons. The y-axis shows the number of RPM. RNA-seq samples were biological triplicates.

(F) Venn diagram shows that 620 protein-coding genes are shared with the l(3)mbt and lint-O libraries.

(G) High-ranking GO terms for the 620 protein-coding genes in (F).

Expanded View Figure legends