EMBO/soda-nel huggingface dataset:
m6A-modification of HSATIII lncRNAs regulates temperature-dependent splicing (2021)
Figure 4

Figure 4. Sequestration of YTHDC1 and WTAP within nSBs

A-D (A, C) Nucleoplasmic YTHDC1 (A) and WTAP (B) intensities upon HSATIII knockdown. Control and HSATIII knockdown cells after thermal stress exposure (42°C for 2 h and recovery for 1 h at 37°C) were stained by HSATIII-FISH and IF using anti-YTHDC1 or anti-WTAP antibodies. nSB and whole nuclear areas were defined by binarized images of HSATIII and DAPI, respectively. Box plots represent the average intensities of YTHDC1 and WTAP in the non-nSB nucleoplasm area of each nucleus. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. The mean is indicated with X. P-value (Mann-Whitney U-test) is shown above (n=40). (B, D) Correlation between the intensity of YTHDC1 (B) or WTAP (D) in non-nSB nucleoplasm and the nSB occupancy in individual nuclei. nSB occupancy is the ratio of the nSB area to the whole nuclear area of each nucleus in (A) and (C). Pearson coefficient of correlation (R) and p-value are shown above (n=40).

E Western blot validation of WTAP and YTHDC1 protein levels after HSATIII knockdown. HeLa cells were transfected with HSATIII ASO and control sense oligonucleotide, cultured for 16 h, and exposed to thermal stress (42°C for 2 h and recovery for 1 h at 37°C). Cell extracts were used for western blotting. GAPDH is a control.

F Quantification of E showing mean ± SD values (n=3). P-values (Student's t-test) are shown above.

G, H HSATIII dependent sequestration of nucleoplasmic YTHDC1 (G) and WTAP (H) at four time points during thermal stress and recovery. The experiments same as A and C were performed at the indicated timepoints. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above (n=40).
m6A-modification of HSATIII lncRNAs regulates temperature-dependent splicing (2021)
Figure 6

Figure 6. m6A factor sequestration-mediated splicing regulation

A RT-qPCR analysis of FAM214A mRNA splicing. Intron-retaining isoforms (IR) of the FAM214A pre-mRNA are illustrated above. The retained intron is indicated by a red line. Arrows indicate the positions of the PCR primers used. The graphs show the relative amounts of the IR (left) and spliced (right) forms in control and HSATIII KD cells under three conditions: 37°C, 42°C for 2 h, and 42°C for 2 h followed by recovery at 37°C for 1 h (recovery). Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the levels in control cells under normal conditions (37°C). Data are shown as the mean ± SD (n=3); p-values (p<0.05, Sidak's multiple comparisons tests) are shown above.

B Outline of RT-qPCR analysis of nascent RNAs upon HSATIII and YTHDC1 KD.

C RT-qPCR validation of splicing of nascent RNAs. Splicing efficiency of nascent RNAs was quantified as the ratio of the spliced form to the intron retaining isoform (IR) form and normalized to the control. Control siRNA and HSATIII control oligonucleotide are indicated with (-). Data are shown as the mean ± SD (n=3); p-values (Tukey's multiple comparison test) are shown above.

D m6A-dependent splicing of FAM214A intron 6. HeLa cells were transfected with control (siControl), WTAP (siWTAP) or YTHDC1 (siYTHDC1) siRNA and cultured for 48 h. PCR primers are the same as in A. Splicing efficiency was calculated using the method described in C and normalized to the control (siControl). Data are shown as the mean ± SD (n=3); p-values (Dunnett's multiple comparison test) are shown above.

E Read maps represent RNA-seq data of nuclear poly (A)+ RNAs (upper panel, biological triplicate) and m6A-RIP RNAs (lower panel, biological duplicate) in control and HSATIII KD cells. The exon numbers of the FAM214A gene are indicated below. The retaining intron and the m6A peak are marked by a red line and arrow, respectively. For the entire FAM214A region, see also Figure EV4B.

F RT-qPCR validation of m6A-RIP of FAM214A mRNA. The positions of primers are indicated above. Relative pulldown efficiency of each experiment was calculated as the ratio of pulldown efficiency (pulldown/input) in HSATIII KD cells to that in control cells. Data are shown as the mean ± SD (n=3); p-value (Welch's t-test) is shown above.
Sequestration of LINE-1 in cytosolic aggregates by MOV10 restricts retrotransposition (2022)
Figure 1

Figure 1. L1 RNP accumulate as cytoplasmic foci in Dicer_KO mESCs

A. Maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA (red) combined with immunostaining for L1 ORF1p (green) and nuclei stained with DAPI (blue). The grey square marks position of the inset. White arrow heads point to cytoplasmic foci where L1 RNA and ORF1p co-localize. Scatter plot shows the number of co-localized L1 Tf-ORF1 foci in the cytoplasm per cell (n=275 WT, 304 Dicer_KO1, 311 Dicer_KO2 cells). Red arrow heads point to relatively larger sized L1 RNP foci. Bar graphs are mean values of percentage of cells with large L1 Tf-ORF1 foci co-localizing in the cytoplasm (n=3 technical replicates).

B. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p (red), MOV10 (green) and nuclei stained with DAPI (blue). The grey square marks position of inset in the zoomed image. White arrow heads point to cytoplasmic foci where L1 ORF1p and MOV10 co-localize. Scatter plot shows the number of co-localized ORF1-MOV10 foci in the cytoplasm per cell (n= 293 WT, 295 Dicer_KO1, 295 Dicer_KO2 cells). Red arrow heads point to relatively larger sized L1 ORF1-MOV10 foci. Bar graphs are mean values of percentage of cells with large ORF1-MOV10 foci co-localizing in the cytoplasm (n=3 technical replicates).

Data information: Data are depicted as Scatter plots where circles are single data points representing number of co-localized L1 Tf-ORF1 foci in the cytoplasm per cell (A), or co-localized L1 ORF1-MOV10 foci in the cytoplasm per cell (B) red bar is median. Statistical analysis was performed using Mann-Whitney U test and **** P-value < 0.0001. Bar graphs represent mean ± SD. Scale bar 5 µm.

Source data are available online for this figure.
Sequestration of LINE-1 in cytosolic aggregates by MOV10 restricts retrotransposition (2022)
Figure 2

Figure 2. Cytosolic L1 RNP foci are poised to be stress granules that co-localize with multiple small DDX6 foci

A. WT and Dicer_KO mESCs were treated with 0.5mM Sodium Arsenite (NaAsO2) for 20 minutes or left untreated prior to fixation with formaldehyde. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for G3BP1 (red) and MOV10 (green) with nuclei stained with DAPI (blue).

B. Representative western blots showing low AGO2 protein levels in Dicer_KO as compared to WT mESCs (right side). LAMINB1 served as loading control. On the left, maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA FISH (red) combined with immunostaining for a resident protein of P-bodies, DDX6 (green) and nuclei stained with DAPI (blue). The grey square marks position of the inset. Yellow arrow heads point to cytoplasmic foci where L1 RNA and DDX6 protein co-localize.

Data information: A and B are representative images of 3 independent experiments. Bar graphs represent mean ± SD. 94-150 cells per cell line per experiment were analyzed for each experiment. Scale bar 5 µm.

Source data are available online for this figure.
Sequestration of LINE-1 in cytosolic aggregates by MOV10 restricts retrotransposition (2022)
Figure 3

Figure 3. Endogenous L1 upregulation leads to increased L1 retrotransposition

A. Representative western blots showing L1 ORF1p and MOV10 levels in the indicated cell lines. Immunoblot with antibody recognizing TUBULIN and coomassie stained membranes depict the loading, asterisk marks position of non-specific band in the ORF1 immunoblot (n=3 technical replicates).

B. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p (green) combined with RNA FISH for L1 Tf RNA (red) and nuclei stained with DAPI (blue) (n=3 technical replicates). Scale bar 5 µm.

C. Representative images of BlastR colonies stained with crystal violet blue of indicated cell lines is shown on the left. Cells were transfected with either mutant reporter plasmid (L1N21A) or retrotransposition competent reporter (L1WT) as shown in the scheme with timeline for the experiment on the top. Bar graphs on the right depict the average number of BlastR colonies (n=3 biological replicates).

Data information: In C, bar graphs represent mean ± SD, P-value was determined using ordinary one-way ANOVA test. *** represent p-value < 0.001.

Source data are available online for this figure.
Sequestration of LINE-1 in cytosolic aggregates by MOV10 restricts retrotransposition (2022)
Figure 4

Figure 4. Multiple miRNAs regulate MOV10 expression and L1 retrotransposition in mESCs

A. Scheme and quantification of relative luminescence from luciferase assays validating post-transcriptional regulation of MOV10 by miRNAs in HEK293T cells (n=4 biological replicates).

B. Scheme and quantification of relative luminescence from luciferase assays in Hek293T cells to confirm specificity of post-transcriptional regulation of MOV10 by miRNAs with either wild-type (WT) or Mutant (MUT) reporters where the MicroRNA Response Element (MRE) for the depicted miRNAs in the 3'UTR of MOV10 were mutated (n=3 biological replicates).

C. Scheme and representative images for western blot analysis and corresponding quantification to assess miRNA mediated regulation on MOV10 expression in mESCs (n=3 biological replicates).

D. Scheme and representative images from colony forming assays for plasmid based retrotransposition analysis where miRNA mediated downregulation of MOV10 in Drosha_KO mESCs is accompanied by an increase in L1 retrotransposition (n=3 biological replicates).

Data information: In A-D, bar graphs represent mean ± SD, P-value determined using ordinary one-way ANOVA test **** p-value < 0.0001, *** p-value < 0.001, ** p-value < 0.005, * p-value < 0.05.

Source data are available online for this figure.
Sequestration of LINE-1 in cytosolic aggregates by MOV10 restricts retrotransposition (2022)
Figure 5

Figure 5. MOV10 upregulation is sufficient to create L1 RNP aggregates in L1UP mESCs abrogating L1 retrotransposition

A. Scheme of experiment and representative images along with quantitation showing induction of L1 RNP aggregate formation upon ectopic MOV10 expression. Images are maximum intensity projections across Z stacks from indicated mESCs stained for L1 ORF1p (red) combined with immunostaining for HA (green) to detect ectopically expressed MOV10 tagged with HA, and nuclei stained with DAPI (blue). White arrow heads point to cytoplasmic foci where L1 ORF1p and HA-MOV10 co-localize. Data collected from 289 Ctrl, 275 L1UP Cl1 and 296 L1UP Cl2 mESCs (n=3 biological replicates).

B. Scheme and representative images from colony forming assays for plasmid based retrotransposition analysis in Ctrl and L1UP mESCs comparing rate of retrotransposition upon transfection with Empty Vector (EV) or ectopic expression of HA-MOV10 (n=3 biological replicates).

Data information: In A, scatter plots where circles are single data points representing number of co-localized HA-ORF1 foci in the cytoplasm per cell. Red bar marks median for the distribution. P-value was determined using Mann-Whitney U test and **** represent p-value < 0.0001. In B, bar graphs represent mean ± SD, P-value determined using ordinary one-way ANOVA test **** p-value < 0.0001.

Source data are available online for this figure.
Sequestration of LINE-1 in cytosolic aggregates by MOV10 restricts retrotransposition (2022)
Figure 6

Figure 6. Overexpression of L1 ORF1p and MOV10 is sufficient to induce cytosolic aggregation in WT mESCs

A. Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of full length L1 and HA-MOV10 in WT mESCs. Images are maximum intensity projections across Z stacks stained for L1 ORF1p (red) combined with immunostaining for HA (green) to detect ectopically expressed MOV10, and nuclei stained with DAPI (blue). The bottom panel shows cells where accumulation of ORF1-MOV10 was observed in relatively large foci compared to cells where co-localization of the two proteins was mostly in relatively small foci (top panel), 68-119 transfected cells were analyzed per experiment (n=3 biological replicates).

B. Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of HA-ORF1 or HA-ORF1 in conjunction with T7-MOV10 in WT mESCs. Images are maximum intensity projections across Z stacks stained for HA (green) and T7 (red) to detect ectopically expressed ORF1p and MOV10 respectively, and nuclei stained with DAPI (blue). 100-147 transfected cells were analyzed per experiment (n=3 biological replicates).

Data information: In A and B, bar graph is mean ± SD. Scalebar 5µm.

Source data are available online for this figure.
GEMC1 is a critical regulator of multiciliated cell differentiation (2016)
Figure 1

Figure 1: Gemc1 deficient animals are growth impaired and develop hydrocephaly. A. Example of littermate males of the indicated genotypes 26 days post partum. Additional examples are provided in Appendix Fig S2. B. Weights of mice of the indicated genotypes from 18 to 120 days post partum. Boxplots indicate 5-95 percentile. A minimum of 8 animals is plotted at each age. Asterisks denote statistical significance (NS, not significant, *P value <0.05, **P value <0.01 and ***P value <0.001) determined by the unpaired two-way Wilcoxon rank-sum test. C. Example of dissected brains (upper panel) and coronal sections (left and right panels) stained with H&E of the indicated genotypes. Note the severe hydrocephaly and ventricular dilation of the lateral ventricle (LV) and third ventricle (TV) in a Gemc1-/- mouse (bottom right panel) compared to a Gemc1+/+ littermate animal (bottom left panel). Periventricular malacia (asterisks) and hippocampal atrophy (arrow) is also evident in the Gemc1-/- sample. Scale bars = 500 µm. D. Kaplan Meyer survival curve of a cohort of mice of the indicated genotypes and numbers (n). The study was terminated at 16 months and mice were examined for histology. 97% (31/32) of the Gemc1-/- mice examined to date exhibited hydrocephaly (additional histological examples in Appendix Fig S2). E. RT-QPCR analysis of Gemc1 expression in murine tissues from Gemc1+/+ mice using a Taqman probe. Data is compiled from duplicate samples of 3 male mice per tissue and mean and standard deviation are plotted. F. RT-QPCR analysis of Gemc1 in trachea, oviduct and ovary tissue from Gemc1+/+ and Gemc1-/- mice shows that the knockout reduces Gemc1 mRNA to undetectable levels. Mean and standard deviation of duplicate samples from 2 female mice are plotted. Additional examples from other Gemc1-/- tissues are shown in Appendix Fig S1.
GEMC1 is a critical regulator of multiciliated cell differentiation (2016)
Figure 2

Figure 2: GEMC1 is required for spermatogenesis. A. Example of dissected testes from littermate mice of the indicated genotypes at 2 months of age. B. Testes from Gemc1-/- mice have strongly reduced cellularity compared to Gemc1+/+ or Gemc1+/- animals, as measured by disaggregation of the tissue and cell counting with a Neubauer Chamber (n=3). Mean and standard deviation are indicated. C. Histological analysis of tubules from 6-week-old Gemc1-/- mice revealed thinning of the spermatogenic cell layer and decreased numbers of elongated spermatids. Scale bars = 100 µm (left panels) and 50 µm (right panels). D. Sperm counts from the cauda epididymis of wild type and Gemc1-/- mice revealed no sperm in mice lacking GEMC1 (n=3). Mean and standard deviation are indicated. E. Examples of sperm morphology from spermatocyte spreads from 2 month old mice. Scale bars = 10 µm. F and G. Histological evaluation of the caput epididymis in sagittal (F) and coronal (G) orientation from 2 and 4 month old animals, respectively. Note the absence of luminal spermatozoa in the Gemc1-/- mice compared to wild type. Scale bars = 100 µm (F, left panels) and 50 µm (F, right panels and G).
GEMC1 is a critical regulator of multiciliated cell differentiation (2016)
Figure 3

Figure 3: GEMC1 is required for female fertility and multiciliogenesis. A. Examples of ovaries from Gemc1+/+ (top panels) and Gemc1-/- mice (bottom panels) at 6 weeks of age. Ovaries were smaller in Gemc1-/- mice and contained primarily degenerated antral follicles. A histologically normal antral follicle is indicated in the Gemc1+/+ ovary (top right, black arrow) and degenerated antral follicles with high levels of cell death are indicated in the Gemc1-/- ovaries (bottom right, red arrows). Scale bars = 200 µm (left panels)) and 100 µm (right top panels and right bottom panel respectively). B. Morphological abnormalities in the oviduct of 3-week-old Gemc1-/- mice (right panels) compared to Gemc1+/+ (left panels). A clear lack of cilia (bottom panels) in Gemc1-/- oviducts is observed by H&E staining. Scale bars = 50 µm (top panels). C. Staining of frozen sections of oviduct with antibodies for Ac-tubulin (cilia) and γ-tubulin (basal bodies) reveals normal cilia organization in the wild type oviduct (left) while basal bodies and cilia are not detectable in Gemc1-/- oviducts (right). D. FOXJ1 and E. RFX3 staining that is characteristic of MCCs was clearly evident in Gemc1+/+ oviducts and absent in Gemc1-/- mice. Scale bars = 50 µm (C-E).
GEMC1 is a critical regulator of multiciliated cell differentiation (2016)
Figure 4

Figure 4: GEMC1 is required for the generation of multiciliated tissues

A. H&E staining of the nasal mucosa of E17.5 embryos of the indicated genotype. Normal respiratory pseudostratified columnar ciliated epithelium is shown in Gemc1+/+ embryos compared to the attenuated, flat columnar to squamous epithelium of Gemc1-/- embryos. Scale bars = 20 µm. B. PAS staining (purple) of the nasal mucosa of E18.5 embryos of the indicated genotype. Normal PAS positive goblet cells (inset, red arrows) are interspersed between MCCs (inset, black arrows) with their characteristic goblet or cup-like shape in Gemc1+/+ embryos. The Gemc1-/- mucosa has abundant PAS positive materials that accumulate in the apical borders of almost every columnar epithelial cell (inset, red arrows) and lacks typical goblet cells. Scale bars = 20 µm. C. Transmission electron micrographs of cross-sections of adult trachea from Gemc1+/+ animals revealed the presence of secretory cells (SC) and multiciliated cells (MCC). The apical membrane of MCCs accumulated basal bodies (BB) nucleating multiple cilia (C) and microvilli (MV). In the Gemc1-/- airway epithelium, no MCCs are identifiable and only secretory-like cells with MV at the apical surface are detectable. Scale bars = 5µm. Higher magnifications of the boxed regions are shown below, scale bars = 0.5µm. D. Lack of Ac-tubulin (top panel) and RFX3 (second panel from top) staining in the trachea of 1 month old mice. E. Cilia (Ac-tubulin staining) are not detectable in the ependymal cells of 2 month old Gemc1-/- mice (right panel). Choroid plexus (CP) and lateral ventricles (LV) are indicated. Scale bars = 20 µm
GEMC1 is a critical regulator of multiciliated cell differentiation (2016)
Figure 5

Figure 5: GEMC1 controls the MCC transcriptional program in vivo

A. Gene ontology (GO) analysis of microarray data from the trachea and oviduct. GO scores (negative enrichment) of differentially expressed genes from the trachea and oviduct of Gemc1-/- mice (additional information in Dataset EV2). B. Geneset enrichment analysis of the microarray data using standard and custom genesets (additional information in Appendix Fig S4 and Appendix Table S1). Gene rank lists from the individual tissues were analyzed against genesets for gene ontology (GOSlim, top row), a custom geneset containing all known or suspected ciliopathy genes (second row), the Ciliome database (DB), the SYSCILIA consortium gold standard cilia gene list version 1 (SYSCILIA GS)(Roosing et al, 2015; van Dam et al, 2013), E24F target genes(Lee et al, 2011; Ma et al, 2014) and FoxJ1 target genes(Choksi et al, 2014a). Several additional genesets generated from analysis of ciliopathy patient data(Geremek et al, 2011; Li et al, 2015) or multiciliated cells(Hoh et al, 2012; Treutlein et al, 2014) were also queried (detailed information in Appendix Table S1). Negative enrichment scores are shown for genes downregulated in Gemc1-/- tissues. C. The most differentially expressed genes related to cilia in the trachea (Tr) and oviduct (Ov) of Gemc1-/- mice are plotted in a heatmap. Normalized, centered and scaled Affymetrix probeset intensities are plotted. D. Heatmap of differential expression of known human ciliopathy genes in the trachea and oviduct of Gemc1-/- mice compared to wild type littermates. E and F. RT-QPCR analysis of individual genes related to MCC formation in the oviduct (E) and trachea (F) of Gemc1-/- mice compared to wild type littermates, using 18S rRNA as a reference (n=2). Genes examined generally fall into 3 classes: centriole duplication (Cep152, Plk4, Cetn2), Deuterosome mediated centriole expansion (Deup1, Ccdc78, Ccno) and transcription of genes required for MCC differentiation (FoxJ1, Myb, Mcidas).
GEMC1 is a critical regulator of multiciliated cell differentiation (2016)
Figure 6

Figure 6: GEMC1 interacts with E2F4/5 and Multicilin through distinct domains. A. GEMC1 interacts with E2F4 and DP1. Western blot of HA or FLAG tagged protein expression in the input lysates are shown in the top panel. FLAG immunoprecipiation (IP) of GEMC1 co-IPs DP1 (α-HA, lane 5) and E2F4 (α-HA, lane 6). The interaction is enhanced upon co-expression of HA tagged E2F4 and DP1 (α-HA, lane 7). B. GEMC1 interacts with E2F5 but not E2F1. Co-IP of E2F5 (α-HA, lane 3) and E2F5-DP1 (α-HA, lane 6) but not E2F1 or E2F1-DP1 (α-HA, lanes 2 and 5). In the FLAG-IP panels, a short (top) and long (second from top) exposure of the Western are shown. C. Identification of the domain required for E2F4 interaction. GFP tagged GEMC1 and mutant forms were expressed and IPs were performed against GFP. Wild type GEMC1 (lane 5) or GEMC1 lacking the CC domain (lane 6) pulled down E2F4-DP1. A GEMC1 protein lacking the TIRT domain or GFP alone did not IP E2F4-DP1 (lanes 7, 8). D. Mutation of the single amino acid analogous that identified in MCIDAS in human RGMC patients (G313D) impairs the interaction with E2F4-DP1 (compare land 5 and 6). E. GEMC1 interacts with Multicilin through its CC domain. Wild type GEMC1 or a mutant lacking the TIRT domain pull down Myc-tagged Multicilin (anti- Myc blot, lanes 4 and 6) while a CC domain mutant does not (anti-Myc blot, lane 5). F. Schematic summary of the GEMC1 mutants used in A-E and their effect on EDG complex or Multicilin interactions.