Figure 4

PRL3 Restrains Transcriptional Elongation of Endomembrane Vesicle Genes

(A) Schematic of 4sU protocol to detect nascent RNA transcripts.

(B) 4sU transcriptional profile for long genes in EV (empty vector) versus PRL3 over-expressing cells (PRL3). TSS, transcription start site; TES, transcription end site. Arrows indicate accumulation of transcripts to the 5′ end and loss of transcripts in the 3′ end of PRL3-expressing cells

(C) 4sU RNA-seq transcript coverage for ATP6V1C1 and ATP1A1 nascent transcripts. In control EV cells (black) most transcripts align with exons (black arrows), whereas transcripts in PRL3 expressing cells (red) accumulate are enriched at the 5′ end of the gene (red arrows).

(D) 4sU transcriptional profile for 5′-enriched genes in control and PRL3-expressing cells. Accumulation of transcripts to the 5′ end and a decrease at the 3′ end of the gene body (red arrows) in PRL3-expressing versus control EV cells.

(E) Volcano plot of differentially expressed genes in PRL3 versus EV control A375 cells by DESeq2-analysis using 4sU nascent RNA-seq data. See also Table S3.

(F) Bubble plot of GO cellular components in the PRL3-induced 5′ enriched genes. Endomembrane vesicle components highlighted in red.

(G) A375 melanoma cells expressing EV, PRL3, and PRL3 (C104S) stained with LysoTracker (red) and hoechst (blue). Scale bars: 50 μm.

(H) Quantification of the area of LysoTracker-positive vesicles per cell. ANOVA using Tukey’s analysis, ^∗∗p = 0.0015; ^∗∗∗∗p < 0.0001. Line and error bars, mean ± SEM.

(I) Quantification of the proportional mean intensity of LysoTracker-positive vesicles per cell. ANOVA using Tukey’s analysis, ^∗∗∗p = 0.0005; ^∗∗∗∗p < 0.0001. Line and error bars, mean ± SEM.

See also Figure S4.