1. TED-Seq Identifies the Dynamics of Poly(A) Length during ER Stress
Yu Mi Woo, Yeonui Kwak, Sim Namkoong, Katla Kristjánsdóttir, Seung Ha Lee, Jun Hee Lee, Hojoong Kwak 
Cell Reports 
Volume 24, Issue 13, Pages e7 (September 2018)
DOI: /j.celrep
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2. Cell Reports 2018 24, 3630-3641.e7DOI: (10.1016/j.celrep.2018.08.084)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 Measuring Poly(A) Lengths Using TED-Seq
(A) Schematics of tail-end displacement sequencing (TED-seq). Poly(A)-tail length (PAL) estimation depends on precise size selection of the inserts, mapping of the 5′ ends of size selected fragments and 3′ cleavage-polyadenylation sites (3′ CPS). PAL = (insert size) – (distance from the 5′ ends to 3′ CPS).
(B) Distribution of PALs in individual transcripts (human embryonic kidney cell line: HEK293).
(C) Representative profiles of PAL in individual genes. The red arrows indicate average PAL in each gene. The y axis indicates relative TED-seq read counts.
(D) PALs of ACTB and GAPDH mRNAs measured by PCR-based method (Hire-PAT). Gene-specific forward and reverse primers (Sp) amplify the region just upstream of poly(A) tails of target genes. The green dotted line indicates the expected baseline of PAL = 0 for each transcript.
(E) The scatterplot showing the correlation between PAL and the lengths of 5′ UTR, coding DNA sequence (CDS), and the 3′ UTR (n = 15,939).
(F) Cumulative proportion of the variances in PAL explained by number of hexamer frequencies in 5′ UTR (blue solid line), 3′ UTR (yellow solid line), and CDS (green solid line) region, respectively. Background random hexamer models are plotted black dashed lines (n = 2).
See also Figure S1 and Tables S1, S2, and S3.
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4. Figure 2 Poly(A) Lengths of ER-Stress-Induced mRNAs Are Increased
(A) Heatmaps of poly(A) length (PAL) distributions and the changes in mRNA expression levels upon ER stress. The mRNAs (n = 15,762) are ordered by ΔPAL between control and stress conditions on the y axis.
(B) A cumulative fraction (CDF) plot showing ΔPAL between groups of mRNAs with different expression level changes upon ER stress. Kolmogorov-Smirnov (KS) test was used to calculate the p values of the ΔPAL difference between the quantile 0–0.5 and 0.95–1 of ΔmRNA.
(C) Network representations of enriched KEGG pathways for genes with the most increase in ΔPAL (top 25th percentile of ΔPAL, n = 3,942) during ER stress. Representations generated by ClueGO of functionally grouped networks of enriched KEGG pathways.
(D) Density plots showing PAL distribution obtained from TED-seq data of representative ER stress-induced genes: XBP1, DDIT3, and HSPA5.
(E) Validation of TED-seq data by Hire-PAT-PCR. The green dotted line indicates the baseline of PAL = 0 in each transcript. Sp, gene-specific PCR; ∗, nonspecific bands.
(F) mRNA expression levels of representative genes in ER stress conditions from the RNA-seq data and qRT-PCR validation. Error bars of RNA-seq data represent ± SD from biological replicates (n = 4). Error bars of qRT-PCR result represent ±SD from biological (n = 3) and technical replicates (n = 3 × 4). ∗∗p < 0.01; ∗∗∗p <
See also Figure S2 and Tables S2, S3, and S4.
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5. Figure 3
Translational De-repression of mRNAs with Increased Poly(A) Length
(A) Distribution of translation efficiencies (TEs) in all transcripts in control and under ER stress. TEs are calculated from the ratio between ribosome profiling and RNA-seq, normalized to rRNA content.
(B) Changes in TE between ER stress and control conditions of representative transcripts: ACTB, GAPDH, XBP1, DDIT3, and HSPA5. Error bars indicate mean ± SD from biological replicates (n = 4). ∗p < 0.05; ∗∗∗p <
(C) Cumulative distribution (CDF) of log2-fold change of TE classified by ΔPAL quantiles. Total number of genes: 8,360. Kolmogorov-Smirnov (KS) test was used to calculate the p value comparing quantiles 0–0.5 and 0.95–1 groups.
(D) Explained variance of poly(A) length (PAL), ΔPAL, and ΔTE/ΔPAL by individual hexamer frequencies in the 5′/3′ UTRs and the CDS. The black dashed lines indicate random hexamer frequencies as a background control.
(E and F) Top three sequence motifs enriched in de-repressed mRNAs with PAL increase, and CDFs of ΔTE comparisons in ΔPAL quantiles between mRNAs with and without the motif enrichments. (E) mRNAs are split into 3′ UTR motif enriched (top 25th percentile, middle panel) and depleted (bottom 25th percentile, right panel) classes. CDFs of ΔTEs classified by ΔPAL quantiles are plotted for each mRNA class. (F) In 5′ UTR motifs and the split CDFs.
See also Figure S3 and Tables S2, S3, and S4.
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6. Figure 4
Association among Transcription, Stability, and Poly(A) Lengths
(A) Schematics of calculating mRNA-polymerase density ratio (R/P ratio) using transcription (PRO-seq) and mRNA expression (RNA-seq) measurements.
(B) Cumulative distribution of log2-fold change of R/P ratio classified by ΔPAL quantiles in genes with constant PRO-seq (n = 16,110). Statistical tests were performed using Kolmogorov-Smirnov (KS) test comparing the quantiles 0–0.5 and 0.95–1.
(C) Genes categorized by profiling PRO-seq, RNA-seq, and R/P ratios. Heatmap shows the differential expression patterns that are clustered according to each log2 fold change level (from DESeq2) of PRO-seq and RNA-seq between control and ER stress conditions. Scale bar shows log2 fold change (ER stress/Control).
(D–G) Four cumulative fraction (CDF) plots show ΔPAL comparisons in four categories of which the R/P ratio can reflect the stability divided according to PRO-seq level and R/P ratio: (D) cluster 1, which includes transcripts with increased PRO-seq level and R/P ratio; (E) cluster 2, which includes transcripts with unchanged PRO-seq level and increased R/P ratio; (F) cluster 3, which includes unchanged PRO-seq level and decreased R/P ratio; (G) cluster 4, which includes decreased PRO-seq level and RP ratio. Each p value was calculated by KS-tests compared to unchanged (n/c) mRNAs.
See also Figure S4 and Tables S2, S3, and S4.
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7. Figure 5 Poly(A) Lengths in Stress-Induced RNA Granules
(A) Schematics of cell fractionation to obtain cytosol (CYT) RNA and stress-induced RNA granule (RG) RNA. Sup, supernatant.
(B) Heatmap showing poly(A) length (PAL) ordered by the difference of normalized PAL (ΔPAL) between CYT and RG RNAs in ER stress (n = 21,040).
(C) Cumulative fraction (CDFs) of the normalized PAL distribution of all TED-seq reads from CYT (blue) and stress-induced RG (yellow) fractions of thapsigargin-treated cells.
(D) CDFs of PAL differences between CYT and RG (ΔPAL) dependent on the frequency of pumilio response element (PRE) (UGUANAUA), which was normalized by 3′ UTR length, indicated by PRE frequency.
See also Figure S5 and Tables S2, S3, and S4.
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