EPB41L4A-AS1 long noncoding RNA acts in both cis- and trans-acting transcriptional regulation and controls nucleolar biology
- Department of Immunology and Regenerative Biology, Weizmann Institute of Science, Israel
- Department of Molecular Neuroscience, Weizmann Institute of Science, Israel
Figures
Figure 1 with 3 supplements
EPB41L4A-AS1 is a highly expressed lncRNA with a widespread cellular distribution.
(A) Graphical overview of the screen to identify candidate cis-acting lncRNAs. (B) RT-qPCR to assess the expression of each candidate cis-acting lncRNA and their predicted cis-regulated target genes, following KD with two unique GapmeRs. (C) (Top) Overview of the 5q22.1–2 locus, with the bars highlighting the two TADs. (Bottom) Zoom in on the EPB41L4A-AS1 locus, with tracks for the PhyloP conservation score (UCSC genome browser), H3K4me3 and H3K27ac histone modifications (ENCODE), PolyA+ and Ribo(−) RNA-seq coverage (this study), GENCODE transcripts and CAGE reads (FANTOM5). The location of the GapmeRs used in this study is also reported. (D) A representative single-molecule RNA FISH (smFISH) image shows EPB41L4A-AS1 intracellular localization. GAPDH and DAPI were used to label the cytoplasm and the nucleus, respectively (scale bar = 20 µm, ×100 magnification). (E) RT-qPCR analysis of subcellular fractionation experiments. The percentages of RNA in each compartment were obtained by normalizing the expression in the different fractions to that in whole cells. (F) UMI-4C contact profiles using baits targeting the TSSes of STARD4 (left), EPB41L4A-AS1 (center), or EPB41L4A (right). The dotted line represents the center of the EPB41L4A-AS1 locus. All experiments were performed in n = 3 biological replicates, except UMI-4C with n = 2, with the error bars in the bar plots representing the standard deviation. ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided Student’s t-test).
Figure 1—figure supplement 1
UCSC Genome Browser view of the loci containing the selected lncRNAs and target genes for validation.
View of the EPB41L4A-AS1–EPB41L4A (A), LINC00938–ARID2 (B), MIR9-3HG–POLG (C), and NET1e–NET1–CALML5 (D) loci. In each view, the PhyloP conservation score and GeneHancer (GH) elements are also reported.
Figure 1—figure supplement 2
EPB41L4A-AS1 is dysregulated in several cancer types and correlates with survival.
(A) Expression level of EPB41L4A-AS1 (left) and EPB41L4A (right) across the TCGA cohort. The thick lines and each point represent the median and an individual sample, respectively, and the color on top reflects whether the indicated genes are significantly more expressed in control or tumor conditions. (B) Correspondence between the expression of EPB41L4A-AS1 and EPB41L4A in GTEx v8 (left), TCGA control (center), and tumor (right) samples. The Spearman’s correlation coefficient and p-value are also reported. (C) Kaplan–Meier survival curve stratified into individuals with the lower and higher 50% expression level of EPB41L4A-AS1. (D) Expression level of EPB41L4A-AS1 (left) and EPB41L4A (right) in patients with the different breast cancer subtypes and matched controls. The thick lines, edges of the box, whiskers, and each point represent the median, first and third quartiles, the upper and lower 1.5 interquartile ranges (IQRs), and an individual sample, respectively. (E) Correspondence between the expression of EPB41L4A-AS1 and EPB41L4A in breast cancer (BRCA) samples. (F) Expression level of EPB41L4A-AS1 in patients at different breast cancer stages. The white dot and edges of the box represent the median and the first and third quartiles, respectively. The one-way ANOVA p-value for differential expression is also reported. In all cases, *p < 0.05. Data was accessed using the GEPIA2 portal (Tang et al., 2019).
Figure 1—figure supplement 3
EPB41L4A-AS1 expression is altered upon multiple stimuli.
(A) CPAT (Wang et al., 2013) analysis of the indicated transcripts. As controls for a noncoding and a coding transcript, XIST and ACTB are reported. Changes in gene expression for the indicated genes. (B) during a time course of 7 days in serum starvation conditions, (C) following the release from a double thymidine cell cycle block, (D) in a panel of 250 unique cell type-treatment combinations (the Spearman’s correlation coefficient and trendline are also reported), (E) and after exposing MCF-7 cells to lipopolysaccharide (LPS), hydrogen peroxide (H2O2), thapsigargin, and etoposide. When applicable, all experiments were performed in n = 3 biological replicates, with the error bars in the dot plots and boxplots representing the standard deviation. In all cases, ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided Student’s t-test).
Figure 2 with 3 supplements
EPB41L4A-AS1 is a cis-acting lncRNA affecting genome-wide gene expression.
(A) (Top) In scale view of the EPB41L4A-AS1-EPB41L4A locus, with zoomed areas corresponding to the two TSSes. (Middle) CTCF and H3K27ac coverage across this region. (Bottom) Micro-C data in H1-hESCs (Krietenstein et al., 2020) show continuous contacts throughout the EPB41L4A gene body. (B) RT-qPCR and (C) Western blot for the indicated genes and proteins following EPB41L4A-AS1 KD. (D) H3K27ac CUT&RUN-qPCR following EPB41L4A-AS1 KD with GapmeRs using primers targeting the promoter of EPB41L4A. (E) UMI-4C contact profiles in control and LNA1-transfected cells using baits targeting the TSS of EPB41L4A-AS1. The green area represents the quantified genomic interval, and the p-value was calculated using a Chi-squared test. (F) Changes in gene expression for the genes in the two flanking TADs of the lncRNA in cells transfected with GapmeRs targeting EPB41L4A-AS1. The vertical dotted lines represent the TAD boundaries (as assessed by TADmap; Singh and Berger, 2021), the continuous vertical line the lncRNA locus and inter-TAD boundary, and the horizontal continuous line a log2Fold-change equal to 0. The dots represent individual genes, with the significant ones highlighted. (G) MA plot showing the changes in genome-wide gene expression in cells transfected with GapmeRs targeting EPB41L4A-AS1. (H) Same as (G), but with GapmeRs targeting EPB41L4A. (I) GO enrichment analysis for the upregulated (left) and downregulated (right) genes after EPB41L4A-AS1 KD. All experiments were performed in n = 3 biological replicates, except UMI-4C with n = 2, with the error bars in the bar plots representing the standard deviation. ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided Student’s t-test). A gene was considered to be differentially expressed if both adjusted p < 0.05 and |log2Fold-change| > 0.41 (corresponding to a change of 33%).
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Figure 2—source data 1
Full Western blot gels of EPB41L4A, ACTB and GAPDH after EPB41L4A-AS1 KD with LNA1 and LNA2.
- https://cdn.elifesciences.org/articles/106846/elife-106846-fig2-data1-v1.zip
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Figure 2—source data 2
Full Western blot gels of EPB41L4A, ACTB and GAPDH after EPB41L4A-AS1 KD with LNA1 and LNA2.
- https://cdn.elifesciences.org/articles/106846/elife-106846-fig2-data2-v1.zip
Figure 2—figure supplement 1
EPB41L4A-AS1 unidirectionally facilitates EPB41L4A expression in cis.
RT-qPCR to assess the expression of the reported genes after (A) transfection with siRNAs against EPB41L4A-AS1, (B) CRISPRa with guides targeting the EPB41L4A-AS1 promoter, (C) transfection with plasmid encoding the EPB41L4A-AS1 cDNA, transfection with GapmeRs (D) and siRNAs (E) targeting EPB41L4A, (F) CRISPRa with guides targeting the EPB41L4A promoter, and (G) transfection with plasmid encoding the EPB41L4A cDNA. (H) Changes in EPB41L4A-AS1 expression after rescuing EPB41L4A-AS1 with an ectopic plasmid or CRISPRa following its KD with GapmeRs. In both panels (Ectopic OE and CRISPRa) the ‘-’ samples represent those transfected with the Empty Vector or sgControl. Asterisks indicate significance relative to the –/– control (transfected with both the control GapmeR and vector). (I) Same as in (H), but for changes in EPB41L4A expression. (J) UMI-4C contact profiles in control and LNA2-transfected cells using baits targeting the TSS of EPB41L4A-AS1. The green area represents the quantified genomic interval, and the p-value was calculated using a Chi-squared test. All experiments were performed in n = 3 biological replicates, except UMI-4C with n = 2, with the error bars in the boxplots representing the standard deviation. In all cases, ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided Student’s t-test).
Figure 2—figure supplement 2
Most of the transcriptomic changes following EPB41L4A-AS1 downregulation are not explained by EPB41L4A.
(A) RT-qPCR and (B) Western blot for CDKN1A mRNA and protein following EPB41L4A-AS1 KD with LNA1 and LNA2. (C) Quantification of CDKN1A protein levels from (B). (D) Hierarchical clustering (top) and principal component analysis (PCA, bottom) of the polyA+ RNA-seq libraries. The color in the heatmap represents the Euclidean distance between the libraries. (E) Heatmap of the differentially expressed genes shared between EPB41L4A-AS1 and EPB41L4A depletions. Color intensity reflects the changes in gene expression (log2FC). (F) GO enrichment analysis of the genes downregulated following EPB41L4A depletion. (G) Bar plot of the lncRNAs (annotated in GENCODE) detected in MCF-7 cells, ordered by their expression. EPB41L4A-AS1 and other representative cis- or trans-acting lncRNAs are highlighted. All experiments were performed in n = 3 biological replicates, with the error bars in the bar plots representing the standard deviation. ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided Student’s t-test). A gene was considered to be differentially expressed if both adjusted p < 0.05 and |log2Fold-change| > 0.41 (corresponding to a change of 33%).
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Figure 2—figure supplement 2—source data 1
Full Western blot gels of CDKN1A, ACTB and GAPDH after transfection with LNA1 and LNA2.
- https://cdn.elifesciences.org/articles/106846/elife-106846-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
Full Western blot gels of CDKN1A, ACTB and GAPDH after transfection with LNA1 and LNA2.
- https://cdn.elifesciences.org/articles/106846/elife-106846-fig2-figsupp2-data2-v1.zip
Figure 2—figure supplement 3
LNA1 and LNA2 induce a similar transcriptional response.
(A) Correspondence of the gene expression levels (in FPKMs) between each individual replicate of the two control GapmeRs. The values depict the Spearman’s correlation coefficients of each pairwise correlation. (B) Correspondence between changes in gene expression after EPB41L4A-AS1 KD using either LNA1 or LNA2, normalized on either NT1 (top) or NT2 (bottom). (C) Average expression levels (in FPKMs) of EPB41L4A, NPM1, and MTREX in cells transfected with the two GapmeRs targeting EPB41L4A-AS1, with the error bars representing the standard deviation across the n = 3 replicates. DESeq2 adjusted p-values compared to control GapmeR #1 (NT1) are also reported. All experiments were performed in n = 3 biological replicates, with the error bars in the bar plots representing the standard deviation. ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 3 with 1 supplement
SUB1 interacts with EPB41L4A-AS1 and affects gene expression at the DNA and RNA levels.
(A) Schematics of the EPB41L4A-AS1 locus with tracks depicting the eCLIP peaks for both SUB1 and NPM1 (source: ENCODE). (B) Average expression levels (in FPKMs) of SUB1 and NPM1 in cells transfected with GapmeRs targeting either EPB41L4A-AS1 or EPB41L4A, with the error bars representing the standard deviation across the n = 3 replicates. DESeq2 adjusted p-values compared to control GapmeR are also reported. (C) Changes in gene expression upon EPB41L4A-AS1 KD of the genes ranked by the SUB1 eCLIP-binding confidence. (D) Western blot following RIP using either a SUB1 or IgG antibody. TUBULIN was used as a negative control, IN—input, SN— supernatant/unbound, B—bound. (E) Same as (C), but with genes ranked by their enrichment in the RIP data (log2FC RIP/Input). (F) Metagene profile around TSS of the normalized SUB1 CUT&RUN signal, stratified by gene expression levels in MCF-7 cells. (G) Heatmap of SUB1 reads coverage around the TSSs of all human genes, stratified by gene expression levels in MCF-7 cells. (H) Changes in gene expression upon EPB41L4A-AS1 KD with GapmeRs for genes with and without a high-confidence SUB1 peak in their TSS. All experiments were performed in n = 3 biological replicates. In the boxplots, the thick line, edges of the box, and whiskers represent the median, first and third quartiles, and the upper and lower 1.5 interquartile ranges (IQRs), respectively. Outliers (observations outside the 1.5 IQRs) are drawn as single points, the significance of the different comparisons was computed by a Mann–Whitney test, and a global ANOVA p-value is also reported. In all cases, ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.
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Figure 3—source data 1
Full Western blot gels for SUB1 and TUBULIN after SUB1 or IgG pulldown.
- https://cdn.elifesciences.org/articles/106846/elife-106846-fig3-data1-v1.zip
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Figure 3—source data 2
Full Western blot gels for SUB1 and TUBULIN after SUB1 or IgG pulldown.
- https://cdn.elifesciences.org/articles/106846/elife-106846-fig3-data2-v1.zip
Figure 3—figure supplement 1
SUB1 is both a chromatin-associated and an RNA-binding protein.
(A) Gene set enrichment analysis (GSEA) using cellular component as ontology. (B) Running score and preranked list of the nucleolus-associated genes in the GSEA analysis. (C) Changes in gene expression upon EPB41L4A-AS1 KD of the genes ranked by the SUB1 eCLIP-binding confidence, for both the actual targets (as in Figure 3C) and expression-matched controls (in gray). (D) RT-qPCR for the indicated genes after RIP with either an anti-SUB1 or control IgG antibody. (E) MA plot showing the relative enrichment in RIP-seq after using either an anti-SUB1 or control IgG antibody. (F) Forest plot for the enrichment of the genes in the ENCODE SUB1 eCLIP in our RIP-seq data. The genes in the eCLIP dataset were ranked according to their enrichment score. (G) GO enrichment analysis of the genes with a SUB1 peak as determined from our CUT&RUN data. All experiments were performed in n = 3 biological replicates, with the error bars representing the standard deviation (C) or the 95% confidence interval (E). In the boxplots, the thick line, edges of the box, and whiskers represent the median, first and third quartiles, and the upper and lower 1.5 interquartile ranges (IQRs), respectively. Outliers (observations outside the 1.5 IQRs) are drawn as single points, and the significance of the different comparisons was computed by a Mann–Whitney test. A gene was considered to be differentially expressed if both adjusted p < 0.05 and |log2Fold-change| > 0.41 (corresponding to a change of 33%). In all cases, ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided Student’s t-test in (C), Fisher’s exact test in (E)).
Figure 4 with 2 supplements
EPB41L4A-AS1 and SUB1 depletion results in a widespread accumulation of mature snoRNAs.
(A) Changes in gene expression upon EPB41L4A-AS1 KD with GapmeRs of the indicated RNA classes. (B) Schematics depicting the different regions which were separately quantified in each SNHG. (C) Changes in RNA-seq read coverage in different regions upon EPB41L4A-AS1 KD with GapmeRs. (D) As in (B) for SUB1 KD with siRNAs of the indicated RNA classes. (E) As in (C) for SUB1 KD. (F) Correspondence between changes in snoRNA expression after EPB41L4A-AS1 and SUB1 KD. The color indicates the different snoRNA classes, and Spearman’s correlation coefficient is shown. All experiments were performed in n = 3 biological replicates. In the boxplots, the thick line, edges of the box, and whiskers represent the median, first and third quartiles, and the upper and lower 1.5 interquartile ranges (IQRs), respectively. Outliers (observations outside the 1.5 IQRs) are drawn as single points, the significance of the different comparisons was computed by a Mann–Whitney test, and a global ANOVA p-value is also reported.
Figure 4—figure supplement 1
EPB41L4A-AS1 and SUB1 depletion affect the expression of different classes of snoRNAs.
(A) Bar plot depicting the normalized read density in the Ribo(−) RNA-seq data in EPB41L4A exons and introns. (B) Boxplot of gene expression changes (log2FC) after EPB41L4A-AS1 depletion for the indicated RNA categories. (C) Correspondence of the changes in gene expression after EPB41L4A-AS1 KD between the pre-intronic, post-intronic, and snoRNA regions. Spearman’s correlation coefficients are also reported. (D) Normalized Ribo(−) RNA-seq signal over the intronic regions before (top) and after (bottom) the expressed snoRNAs. RT-qPCR (E) and Western blot (F) upon SUB1 depletion with siRNAs to assess KD efficiency. (G) MA plot showing the genome-wide gene expression changes after SUB1 KD with siRNAs. (H) Same as in (B), but after SUB1 KD with siRNAs. All experiments were performed in n = 3 biological replicates, with the error bars in the bar plots representing the standard deviation. In the boxplots, the thick line, edges of the box, and whiskers represent the median, first and third quartiles, and the upper and lower 1.5 interquartile ranges (IQRs), respectively. Outliers (observations outside the 1.5 IQRs) are drawn as single points, the significance of the different comparisons was computed by a Mann–Whitney test, and a global ANOVA p-value is also reported. A gene was considered to be differentially expressed if both adjusted p < 0.05 and |log2Fold-change| > 0.41 (corresponding to a change of 33%). In all cases, ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided Student’s t-test).
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Figure 4—figure supplement 1—source data 1
Full Western blot gels of SUB1 and TUBULIN after SUB1 KD with siRNAs.
- https://cdn.elifesciences.org/articles/106846/elife-106846-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Full Western blot gels of SUB1 and TUBULIN after SUB1 KD with siRNAs.
- https://cdn.elifesciences.org/articles/106846/elife-106846-fig4-figsupp1-data2-v1.zip
Figure 4—figure supplement 2
GAS5 depletion does not affect snoRNAs expression.
(A) Schematics of the GAS5 locus with tracks depicting the eCLIP peaks for both SUB1 and NPM1 (source: ENCODE), as well as the location of the two GapmeRs that were used. (B) RT-qPCR upon GAS5 depletion with GapmeRs to assess KD efficiency. (C) MA plot showing the genome-wide gene expression changes after GAS5 KD with GapmeRs. (D) Violin/boxplots of gene expression changes (log2FC) after GAS5 depletion for the indicated RNA classes. (E) Boxplot of gene expression changes (log2FC) after GAS5 depletion for the indicated RNA categories. (F) Correspondence between the changes in gene expression of the color-coded snoRNA categories after EPB41L4A-AS1 and GAS5 depletion. The trendline and Spearman correlation coefficient are also reported. All experiments were performed in n = 3 biological replicates, with the error bars in the bar plots representing the standard deviation. In the boxplots, the thick line, edges of the box, and whiskers represent the median, first and third quartiles, and the upper and lower 1.5 interquartile ranges (IQRs), respectively. Outliers (observations outside the 1.5 IQRs) are drawn as single points, the significance of the different comparisons was computed by a Mann–Whitney test, and a global ANOVA p-value is also reported. A gene was considered to be differentially expressed if both adjusted p < 0.05 and |log2Fold-change| > 0.41 (corresponding to a change of 33%). In all cases, ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided Student’s t-test).
Figure 5 with 1 supplement
Loss of EPB41L4A-AS1 results in an altered nuclear patterning of SUB1 and NPM1.
(A) Representative immunofluorescence images for SUB1 after EPB41L4A-AS1 depletion with two distinct GapmeRs (scale bar = 20 µm, ×100 magnification). (B) Same as in (A), but for NPM1. (C) Quantification of the kurtosis of SUB1 nuclear signal in the indicated conditions. (D) Same as in (C), but for NPM1. (E) Representative immunofluorescence images for SUB1 after SUB1 KD with siRNAs (scale bar = 20 µm, ×60 magnification). (F) Same as in (E), but for NPM1. (G) Same as in (D), but after SUB1 depletion. All experiments were performed in n = 3 biological replicates. In the boxplots, the thick line, edges of the box, and whiskers represent the median, first and third quartiles, and the upper and lower 1.5 interquartile ranges (IQRs), respectively. Outliers (observations outside the 1.5 IQRs) are drawn as single points, the significance of the different comparisons was computed by a Mann–Whitney test, and a global ANOVA p-value is also reported. In each boxplot, points represent individual measurements (cell nuclei).
Figure 5—figure supplement 1
Nucleolar stress induced by CX-5461 treatment affects SUB1 and NPM1 nuclear patterns.
(A) Representative immunofluorescence images for NPM1 after treating cells with the indicated CX-5461 concentrations and time (scale bar = 20 µm). (B) Boxplot depicting the changes of the kurtosis of the nuclear NPM1 signal after treating MCF-7 cells with the indicated CX-5461 concentrations and time. (C) Same as in (A), but for SUB1. (D) Same as in (B), but for SUB1. All experiments were performed in n = 3 biological replicates. In the boxplots, the thick line, edges of the box, and whiskers represent the median, first and third quartiles, and the upper and lower 1.5 interquartile ranges (IQRs), respectively. Outliers (observations outside the 1.5 IQRs) are drawn as single points, and the significance of the different comparisons was computed by a Mann–Whitney test.
Figure 6
EPB41L4A-AS1, but not the expression of SNORA13, is required for proper snoRNAs expression.
(A) Average expression levels (in FPKM) of SNORA13 in cells transfected with GapmeRs targeting EPB41L4A-AS1 (rRNA-depleted RNA-seq), with the error bars representing the standard deviation across the three replicates. DESeq2 adjusted p-values compared to control GapmeR are also reported. (B) Northern blot for SNORA13 in cells transfected with GapmeRs targeting EPB41L4A-AS1. U6 was used as a loading control, and total RNA stain is also shown on top. (C) Schematics of the expected fragment sizes following HinfI digestion in the aRT-PCR assay (left), and agarose gel following HinfI digestion of the PCR-amplified 18S rRNA in cells transfected with GapmeRs targeting either EPB41L4A-AS1 or EPB41L4A (right). No enzyme and genomic DNA (gDNA) were used as negative controls. (D) Allele frequency at 18S:1248 in the polyA+ RNA-seq dataset, using rRNA reads. (E) UCSC Genome Browser view of the EPB41L4A-AS1 locus, with tracks showing the rRNA-depleted RNA-seq coverage of EPB41L4A-AS1 OE and control cells. (F) RT-qPCR for the indicated genes upon EPB41L4A-AS1 KD with GapmeRs (right) or overexpression with a full-length unspliced vector (left) over the course of 3 days post-transfection. (G) RT-qPCR for the indicated snoRNAs upon EPB41L4A-AS1 KD with GapmeRs over the course of 3 days post-transfection. (H) Schematics of the different EPB41L4A-AS1 and SNORA13 overexpressing vectors used in the rescue experiments. (I) The ratio between changes in gene expression detected by RT-qPCR in cells transfected with an EPB41L4A-AS1 or SNORA13 overexpressing vector vs control cells. All experiments were performed in n = 3 biological replicates, with the error bars in the bar plots and forest plots representing the standard deviation. In all cases, ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided Student’s t-test).
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Figure 6—source data 1
Full agarose gels of the MACP quantification and full northern blot gels for total RNA, SNORA13 and U6 after EPB41L4A KD with LNA2.
- https://cdn.elifesciences.org/articles/106846/elife-106846-fig6-data1-v1.zip
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Figure 6—source data 2
Full agarose gels of the MACP quantification and full northern blot gels for total RNA, SNORA13 and U6 after EPB41L4A KD with LNA2.
- https://cdn.elifesciences.org/articles/106846/elife-106846-fig6-data2-v1.zip
Figure 7 with 1 supplement
The increased abundance of snoRNAs is primarily due to their hosts' increased transcription and stability.
(A) Workflow of the SLAM-seq experiment. MCF-7 cells were transfected with the indicated GapmeRs for 48 hr, after which the media was replaced with media containing 4sU and the cells were harvested at different time points. (B) Fitted model depicting the synthesis and decay rates of the EPB41L4A mRNA upon EPB41L4A-AS1 KD with GapmeRs. (C) Changes in synthesis rate upon EPB41L4A-AS1 KD with GapmeRs for the indicated group of genes. (D) Same as in (C), but for changes in half-lives. (E) Half-lives in control conditions of the indicated SNHG regions as described in Figure 4B. p-values refer to the comparison with the unrelated genes. (F) Same as in (E), but after EPB41L4A-AS1 KD with LNA2. (G) Changes in synthesis rate upon EPB41L4A-AS1 KD with GapmeRs for the indicated SNHG regions. (H) Same as in (G), but for changes in half-lives. All experiments were performed in n = 3 biological replicates. In the boxplots, the thick line, edges of the box, and whiskers represent the median, first and third quartiles, and the upper and lower 1.5 interquartile ranges (IQRs), respectively. Outliers (observations outside the 1.5 IQRs) are drawn as single points, the significance of the different comparisons was computed by a Mann–Whitney test, and a global ANOVA p-value is also reported. In all cases, ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 7—figure supplement 1
EPB41L4A-AS1 affects local RNA metabolism.
(A) Changes in synthesis rate in KD cells of the genes in the two flanking TADs of EPB41L4A-AS1. The vertical dotted lines represent the TAD boundaries (as assessed by TADmap), the continuous vertical line the lncRNA locus and inter-TAD boundary, and the horizontal continuous line a log2Fold-change equal to 0. The dots represent individual genes, with the significant (adjusted p < 0.05 and |log2Fold-change| > 0.41) ones highlighted in red. (B) Same as in (A), but for RNA half-lives. (C) Same as in (A) and (B), but for CTCF binding as assessed by CUT&RUN. Each dot and name represent a single CTCF peak and the closest gene, respectively. (D) Changes in gene expression of the p53 signature genes (from MSigDB) after KD with GapmeRs targeting either EPB41L4A-AS1 or EPB41L4A (polyA+ RNA-seq data), and SNORA13 KO cells (Cheng et al., 2024). The points in the boxplot represent individual genes, and their color indicates whether they were found to be significantly dysregulated (red) or not (black). All experiments were performed in n=3 biological replicates. In the boxplots, the thick line, edges of the box, and whiskers represent the median, first and third quartiles, and the upper and lower 1.5 interquartile ranges (IQRs), respectively. Outliers (observations outside the 1.5 IQRs) are drawn as single points, and the significance of the different comparisons was computed by a Mann–Whitney test.
Figure 8
Cells with reduced EPB41L4A-AS1 expression display reduced proliferation and increased invasion capacity.
(A) Representative brightfield images of the wound at the indicated time points and conditions. (B) Wound area (top-left), normalized wound area (top-right), closure percentage (bottom-left) and migration rate (bottom-right) at the indicated time points and conditions. The significance was calculated by a two-sided Student’s t-test. (C) Growth curve of the cells in the indicated conditions over the course of 3 days. The significance was calculated by a two-sided Student’s t-test. (D) Changes in gene expression of the epithelial-to-mesenchymal transition (EMT) signature genes (from MSigDB) after KD with GapmeRs targeting either EPB41L4A-AS1 or EPB41L4A (polyA+ RNA-seq data). The significance of the comparison was computed by a Mann–Whitney test. (E) Average expression levels (in FPKMs) following EPB41L4A-AS1 KD with GapmeRs of selected genes that have been previously linked to EMT, with the error bars representing the standard deviation across the n = 3 replicates. DESeq2 adjusted p-values compared to control GapmeR are also reported. All experiments were performed in n = 3 biological replicates. The error bars in the bar plots represent the standard deviation. In the boxplot, the thick line, edges of the box, and whiskers represent the median, first and third quartiles, and the upper and lower 1.5 interquartile ranges (IQRs), respectively. Outliers (observations outside the 1.5 IQRs) are drawn as single points. The points in the boxplot represent individual genes, and their color indicates whether they were found to be significantly (adjusted p < 0.05 and |log2Fold-change| > 0.41) dysregulated (red) or not (black). In all cases, ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided Student’s t-test).
Figure 9 with 1 supplement
Proposed model of the cis and trans functions of the EPB41L4A-AS1 lncRNA.
Under normal conditions, the EPB41L4A-AS1 locus is transcribed and ensures the proper expression of several genes located in cis. In trans, it interacts with SUB1, which eventually is evenly distributed throughout the nucleus. When there are reduced levels of EPB41L4A-AS1, the expression of the genes in cis is reduced, and SUB1 accumulates in the nucleolus. As a consequence, snoRNA abundance increases, likely due to their increased stability, and MCF-7 cells acquire a more invasive in vitro phenotype. Created with Biorender.com.
Figure 9—figure supplement 1
EPB41L4A-AS1 depletion consistently represses MTREX.
(A) RT-qPCR and (B) Western blot for MTREX mRNA and protein following EPB41L4A-AS1 KD with LNA1 and LNA2. (C) Quantification of MTREX protein levels from (B). All experiments were performed in n = 3 biological replicates, with the error bars in the bar plots representing the standard deviation. ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided Student’s t-test).
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Figure 9—figure supplement 1—source data 1
Full Western blot gels of MTREX and GAODH after EPB41L4A-AS1 KD with LNA1 and LNA2.
- https://cdn.elifesciences.org/articles/106846/elife-106846-fig9-figsupp1-data1-v1.zip
Author response image 1
The EPB41L4A transcript is nuclear-enriched in the MCF-7 ENCODE subcellular RNA-seq dataset.
Scatterplot of gene length versus cytoplasm/nucleus ratio (as computed by DESeq2) in MCF-7 cells. Each dot represents an unique gene, color-coded reflecting if their DESeq2 adjusted p-value < 0.05 and absolute log2FC > .41 (33% enrichment or depletion).GAPDH and MALAT1 are shown as representative cytoplasmic and nuclear transcripts, respectively. Data from ENCODE.
Author response image 2
Most EPB41L4A-AS1 transcripts end before the 3’ end of EPB41L4A.
UCSC genome browser view showing tracks from 3P-seq data in different cell lines and neural crest (top, with numbers representing the read counts, i.e. how many times that 3’ end has been detected), and stranded ENCODE subcellular RNA-seq (bottom).
Author response image 3
EPB41L4A-AS1 KD has only marginal effects on the levels of nucleolar proteins.
(A) Western Blots for the indicated proteins after the transfection for 3 days of the control and targeting GapmeRs. (B) Quantification of the protein levels from (A). All experiments were performed in n=3 biological replicates, with the error bars in the barplots representing the standard deviation. ns - P>0.05; * - P<0.05; ** - P<0.01; *** - P<0.001 (two-sided Student’s t-test).
Author response image 4
Western blot for SUB1 following RIP using either a SUB1 or IgG antibody.
IN - input, SN - supernatant/unbound, B - bound.
Author response image 5
UMI-4C coverage over the TSS of the genes located on chromosome 5.
(A) Correlation between the normalized UMI-4C coverage over the TSS (± 5kb) of chromosome 5 genes and the absolute distance (in megabases, Mb) from EPB41L4A-AS1. (B) Same as in (A), but with the x axis showing the relative distance from EPB41L4A-AS1. In both cases, the genes in the two flanking TADs are colored in red and their names are reported.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/106846/elife-106846-mdarchecklist1-v1.docx
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Supplementary file 1
Table containing the results of the in silico screening combining transcriptomics and GeneHancer data.
- https://cdn.elifesciences.org/articles/106846/elife-106846-supp1-v1.xlsx
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Supplementary file 2
Table containing DESeq2 results of the different RNA-seq in this study.
- https://cdn.elifesciences.org/articles/106846/elife-106846-supp2-v1.xlsx
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Supplementary file 3
MACS output for the peaks of SUB1 and CTCF obtained by CUT&RUN.
- https://cdn.elifesciences.org/articles/106846/elife-106846-supp3-v1.xlsx
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Supplementary file 4
GRAND-SLAM/grandR kinetic results of the SLAM-seq experiment.
- https://cdn.elifesciences.org/articles/106846/elife-106846-supp4-v1.xlsx
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Supplementary file 5
List of reagents (GapmeRs, siRNAs, CRISPR guide RNAs, RT-qPCR primers, northern blot probes, antibodies, UMI-4C baits) used in this study.
- https://cdn.elifesciences.org/articles/106846/elife-106846-supp5-v1.xlsx
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EPB41L4A-AS1 long noncoding RNA acts in both cis- and trans-acting transcriptional regulation and controls nucleolar biology
eLife 14:RP106846.
https://doi.org/10.7554/eLife.106846.3
