RNA shortening upon cellular stress.

a) Schematic of experimental design b) Volcano plot for the differential expression of arsenite-treated and unstressed cells. Green color indicates genes with more than 2-fold difference and red indicates statistical significance higher than 10-5. c) Scatter plots of average transcript length for arsenite-treated and unstressed cells stratified by their differential expression change. Down-regulated: (-Inf, -0.5), Unchanged: (−0.5, 0.5), Up-regulated (0.5, Inf) fold-change. Only transcripts with at least 5 aligned reads are shown. Red dotted indicates the y=x line. Color indicates transcripts below (blue) and above (orange) the diagonal. d) Cumulative distribution of read length for arsenite-treated and unstressed cells. e) Cumulative distribution of transcript length for arsenite-treated and unstressed cells using only reads with adaptor ligated at the 5’ end. f) Schematic of read meta-length calculation. Each annotated transcript is divided into 20 equally sized bins. Each read is then assigned meta-coordinates depending on the bin in which its 5’ and 3’ templated ends align. The read meta-length is calculated as the difference of the meta-coordinates and presented as a percentage of full length. g) Scatter plot of average transcript meta-length as a percentage of full-length for arsenite-treated and unstressed cells. Coloring is the same as (d) h) Scatter plot of average transcript length for heat shock and unstressed cells.

Characterization of stress-induced shortened RNAs identified by NanopLen:

a) Left: Line plot of average length difference estimate for simulated data of varying read depths. True value is depicted by dashed grey line. Right: Percentage of simulated genes that are detected as significantly different in length. The null simulation of no shortening corresponds to simulated shortening proportion 1.0. b) Box plot of average transcript length for statistically significant and non-significantly shortened transcripts identified through differential length analysis in arsenite-treated and unstressed cells using NanopLen. c) Gene Ontology analysis for biological processes of significantly shortened transcripts. d) IGV screenshot of ASCC3 aligned reads for arsenite-treated (red) and unstressed cells (blue). Libraries were randomly downsampled to maximum 50 reads per window and the libraries were overlayed. All reads were used, irrespective of adaptor ligation status. e) Short-read RNA-Seq density at the 5’ half of transcripts for unstressed, AsO2, H2O2 and heat shock -treated cells. Shade indicates standard error of the mean for replicates. f) Scatter plot of Transcription Start Site position for arsenite-treated and unstressed cells

The effect of XRN1 knockdown on RNA shortening.

a) Immunoblot for XRN1, eIF2α-P and eIF2α for cells transfected with a mock (siCTRL) or XRN1-targeting siRNA. ACTB is used as control. b) Cumulative distribution plot of transcript length for XRN1 knockdown (siXRN1) and control (siCTRL) in arsenite-treated cells. c) Same as (b) but only reads with ligated 5’ end adaptor are used. d) Scatter plots of average transcript length for arsenite-treated cells transfected with mock and XRN1-targeting siRNA stratified by their differential expression change. Down-regulated: (-Inf, -0.5), Unchanged: (−0.5, 0.5), Up-regulated (0.5, Inf) fold-change. Only transcripts with at least 5 aligned reads are shown. Red dotted indicates the y=x line. Color indicates transcripts below (blue) and above (orange) the diagonal. e) Box plots of differential transcript length in arsenite-treated versus unstressed cells for significantly and non-significantly shortened transcripts in XRN1 knockdown and control.

Association of poly(A) tail length and cis-regulatory elements with RNA shortening.

a) Nucleotide composition around the 5′ end of reads in arsenite-treated and unstressed cells. All reads were used, irrespective of adaptor ligation status. b) Cumulative distribution of the average poly(A) tail length per transcript for arsenite-treated and unstressed cells. c) Scatter plot of transcript expression against poly(A) tail length differential change in arsenite-treated and unstressed cells. d) Box plots of average transcript poly(A) tail length against average transcript-meta length for arsenite-treated and unstressed cells. e) Box plots of average transcript poly(A) tail length for significantly and non-significantly shortened transcripts in arsenite-treated and unstressed cells. f) Scatter plot of combined average transcript and poly(A) tail length for arsenite-treated and unstressed cells. Only transcripts with at least 5 aligned reads were used. Red dotted indicates the y=x line. Color indicates transcripts below (blue) and above (orange) the diagonal. g-i) Scatter plot of annotated transcript length (g), annotated coding sequence (CDS) length (h) and GC content (i) against transcript differential length in arsenite-treated and unstressed cells. The box plots on the right side summarize the y-axis variable for significant and non-significantly shortened transcripts. The Pearson’s correlation coefficient and the Mann-Whitney-U test p-value are shown.

Translation and RNA shortening.

a, b) Immunoblot for eIF2α and eIF2α-P upon increasing concentration of arsenite (a) and cell treatment with 200 nM of ISRIB at different arsenite concentrations (b). c) SGs visualized by immunofluorescence of HeLa cells treated with indicated concentration of arsenite in the presence or absence of 200 nM ISRIB. A secondary goat anti-rabbit IgG H&L (Alexa Fluor 594) against G3BP1 (SG marker) and DAPI were used for visualization. d) Ribosome sedimentation curve following cell treatment with ISRIB (200 nM) for arsenite-treated and unstressed cells. e) Cumulative density plot of transcript length for arsenite-treated cells in the presence or absence of ISRIB. f) Same as (e) for significantly shortened transcripts only. g) Scatter plots of average transcript length for arsenite-treated cells with and without ISRIB stratified by their differential expression change. Down-regulated: (-Inf, -0.5), Unchanged: (−0.5, 0.5), Up-regulated (0.5, Inf) fold-change. Only transcripts with at least 5 aligned reads are used. Red dotted indicates the y=x line. Color indicates transcripts below (blue) and above (orange) the diagonal. h) Same as (g) for arsenite and ISRIB treated cells against control. i) Box plots of differential transcript length for comparisons indicated on the x-axis. i-k) Same as (e) and (f) for cycloheximide (CHX) instead of ISRIB.

Inhibition of stress-granule formation in cells devoid of G3BP1/2 rescues RNA decay

a) Scatter plot of average gene length for arsenite-treated and unstressed cells stratified by gene SG localization. **** indicates p-value < 10-172. b-c) Cumulative distribution and box plots of average gene length difference in arsenite-treated and unstressed cells stratified by gene SG localization. d) Box plot of GC content percentage for SG enriched and depleted gene transcripts. e) Same as (a) for average poly(A) length per gene. f) Cumulative distribution of poly(A) tail length for arsenite-treated and unstressed cells stratified by gene SG localization. g) Cumulative distribution plot of average gene length difference in arsenite-treated and unstressed cells stratified by gene P-bodies localization. h) Scatter plot of average transcript length for arsenite-treated and unstressed U-2 OS and ΔΔG3BP1/2 U-2 OS cells. Only transcripts with at least 5 aligned reads are used. Red dotted indicates the y=x line. i) Cumulative distribution plot of average transcript length difference in arsenite-treated and unstressed U-2 OS and ΔΔG3BP1/2 U-2 OS cells. j) Scatter plot and box plot of differential transcript expression in arsenite-treated and unstressed ΔΔG3BP1/2 U-2 OS cells against differential shortening in arsenite-treated and unstressed U-2 OS cells, stratified by shortening significance.

RNA shortening upon cellular stress.

a) MTS cell proliferation assay showing the percentage of active cells (y-axis) upon arsenite treatment compared to unstressed at different time points (x-axis). Error bars represent std for biological replicates (n = 2). b) Correlogram of transcript expression for unstressed and arsenite-treated cells. c) Gene ontology for biological processes for differentially expressed genes in arsenite-treated versus unstressed cells. d-e) Cumulative distribution plot of average transcript length for coding (d) and non-coding (e) transcripts. Transcripts with less than 5 reads have been removed. f) Cumulative distribution of read meta-length for arsenite-treated and unstressed cells. g) Cumulative distribution plot of average transcript length for H2O2-treated cells and unstressed. h) Cumulative distribution plot of average transcript length for heat shock cells and unstressed. i) Scatter plots of average transcript length for heat shock against unstressed cells stratified by differential expression change. Down-regulated: (-Inf, -0.5), Unchanged: (−0.5, 0.5), Up-regulated (0.5, Inf) fold-change. Only transcripts with at least 5 aligned reads are shown. Red dotted indicates the y=x line. Color indicates transcripts below (blue) and above (orange) the diagonal.

Characterization of stress-induced shortened RNAs identified by NanopLen.

a) IGV screenshot of aligned reads in arsenite-treated (red) and unstressed cells (blue) for significantly shortened transcripts FASTKD2, HNRNPM, DSG2 and MTRR. Libraries were randomly downsampled to maximum 50 reads per window and the libraries were overlayed. All reads were used, irrespective of adaptor ligation status. b) Same as (a) but for non-significantly shortened transcripts AGPAT2, EPHX1 and TBCA. c) Density plot of transcript shortening at the 5’ and 3’ templated end. Positive values on X-axis indicate that the 5’ or 3’ of transcripts in arsenite-treated cells are correspondingly downstream or upstream of their unstressed counterparts. d) Meta-plot of short-read RNA-Seq density on all transcripts for unstressed, AsO2, H2O2 and heat shock -treated cells. Shade indicates standard error of the mean for replicates.

The effect of XRN1 knockdown on RNA shortening.

a) Immunoblot for XRN1 for cells transfected with mock and XRN1-targeting siRNA at varying concentrations. ACTB is used as control. b) Scatter plot of average transcript length for XRN1 knockdown and control in arsenite-treated cells. Only transcripts with at least 5 aligned reads are shown. Red dotted indicates the y=x line. Color indicates transcripts below (blue) and above (orange) the diagonal.

Association of poly(A) tail length and cis-regulatory elements with RNA shortening.

a) Nucleotide composition around the 5′ end of reads in arsenite-treated and unstressed cells upon XRN1 silencing. All reads were used, irrespective of adaptor ligation status. b) Scatter plots of average poly(A) tail length for arsenite-treated and unstressed cells stratified by differential expression. Down-regulated: (-Inf, -0.5), Unchanged: (−0.5, 0.5), Up-regulated (0.5, Inf) fold-change. Only transcripts with at least 5 aligned reads are shown. Red dotted indicates the y=x line. Color indicates transcripts below (blue) and above (orange) the diagonal. c-d) Violin plots of average transcript poly(A) length and 5′ or 3′ meta-coordinates for arsenite-treated and unstressed cells. e-f) Scatter plot for 5′ (e) and 3’UTR (f) length of annotated transcripts against transcript differential length in arsenite-treated and unstressed cells. The box plots on the right side summarize the y-axis variable for significant and non-significantly shortened transcripts. The Pearson’s correlation coefficient and the Mann-Whitney-U test p-value are shown.

Translation and RNA shortening.

a-b) Scatter plot of ribosome profiling footprint (a) and translational efficiency (b) levels against differential length in arsenite-treated and unstressed cells. Transcripts are stratified by significance of shortening c) Ribosome sedimentation curve following cell treatment with cycloheximide (CHX) (25 µg/ml) for arsenite-treated and unstressed cells. d, e) Scatter plots of average transcript length for arsenite plus cycloheximide treated cells against arsenite plus DMSO (d) and unstressed cells (e) stratified by their differential expression change. Down-regulated: (-Inf, -0.5), Unchanged: (−0.5, 0.5), Up-regulated (0.5, Inf) fold-change. Only transcripts with at least 5 aligned reads are shown. Red dotted indicates the y=x line. Color indicates transcripts below (blue) and above (orange) the diagonal.

Inhibition of stress-granule formation in cells devoid of G3BP1/2 rescues RNA decay.

a) Box plots of annotated total, 5′ UTR, coding sequence and 3′ UTR length for SG enriched and depleted transcripts. b) Scatter plot of upper 90th quantile normalized gene expression for arsenite-treated and unstressed cells stratified by gene SG localization. **** indicates p-value < 10-172. c) Cumulative density plot of transcript differential length for SG enriched and depleted gene transcripts in U-2 OS cells. Only transcripts of coding genes are used. d) Box plots of differential gene length in arsenite-treated and unstressed cells stratified by SG enrichment in U-2 OS cels. e) Scatter plot of average gene length for arsenite-treated and unstressed cells stratified by gene P-bodies localization. ** indicates p-value < 0.01. f-i) Scatter plot of average transcript length for arsenite-treated and unstressed U-2 OS and ΔΔG3BP1/2 cells. Only transcripts with at least 5 aligned reads are used. Red dotted indicates the y=x line. Color indicates transcripts below (blue) and above (orange) the diagonal.