Fig 1.
Heat shock induces nuclear foci detectable with dsRNA-specific antibody J2.
Mid-animal intestinal region of 4th larval stage drSI13 worm fixed 40 minutes after heat shock at 35° C. (A) Nuclear J2 foci (red arrows). (B) HSF-1 foci (green arrows). (C) Overlap of J2 foci and HSF-1 foci (orange arrows). White size bar in bottom right corner (20 microns across). DNA stained with DAPI (blue). (D) Quantification of occurrence of HSF-1 and J2 foci over time. 19–20 worms scored per time point with 4 intestinal nuclei scored per worm.
Fig 2.
Quantification of genes with changes in antisense/sense ratios after heat shock or deletion of tdp-1 in input RNA.
Violin plots of the ratio of read counts over gene regions for each condition compared to wild type (WT) [mean >20 for pooled counts (sense and antisense, condition and wild type), n = 1]. (A) With heat shock 5513/11091 genes have a higher antisense/sense ratio compared to wild type (log2FC > 0, area of red violin plot above the black line). With tdp-1(ok803) 7551/10831 genes have a higher antisense/sense ratio compared to wild type (log2FC > 0, area of purple violin plot above the black line). (B) With heat shock, 4125/11091 sense and 3679/11091 antisense transcripts are upregulated compared to wild type (log2FC > 0, area of violin plot above the black line). With tdp-1(ok803) 419/10831 sense and 3301/10831 antisense transcripts are upregulated compared to wild type.
Fig 3.
Comparison of J2 enriched sense and antisense transcripts in heat shock and tdp-1(ok803) worms.
MA plots [“M” (log2FC) on y-axis and “A” (log2Mean) on x-axis] of significant (FDR <0.05) dsRNA enrichment for sense and antisense transcripts (analyzed independently) along with Venn diagrams of enrichment for enriched sense and antisense [n = 2 for heat shock J2 samples, n = 3 for wild type, n = 3 for tdp-1(ok803)]. (A) Heat shock over wild type J2 enriched sense transcripts with 4774 enriched (red) and 1669 depleted (blue). (B) Heat shock over wild type J2 enriched antisense transcripts with 650 enriched (red) and 477 depleted (blue). (A-B) enriched (180) and depleted (48) heat shock vs wild type transcripts found in both sense and antisense (green triangles). C: tdp-1(ok803) over wild type significant J2 enriched sense transcripts with 418 enriched (purple) and 59 depleted (blue). (D) tdp-1(ok803) over wild type significant J2 enriched antisense transcripts with 245 enriched (purple) and 14 depleted (blue). (C-D) enriched (6) and depleted (1) tdp-1(ok803) vs wild type transcripts found in both sense and antisense (green triangles). (E) Overlap of genes with significantly J2 enriched sense transcripts in both conditions compared to wild type worms. (F) Overlap of genes with significantly J2 enriched antisense transcripts in both conditions compared to wild type worms.
Fig 4.
Aberrant transcription past the end of heat shock family genes showed enrichment in heat shock J2.
Normalized histogram from the Integrative Genomics Viewer (IGV). On each track, the sense strand is on the top part of the histogram and antisense is on the bottom (Max read depth +/- 200). Wild type (WT) sense (dark blue) and antisense (light blue), heat shock (HS) sense (red) and antisense (orange). Gene transcription continues past the 3’ end of gene (blue arrow) in heat shock, leading to an annotated downstream of gene transcript (DoG) (green arrow).
Fig 5.
J2 enrichment of DoGs and ADoGs in heat shock and tdp-1(ok803) worms.
MA plots of significant (FDR <0.05) dsRNA enrichment for DoGs and ADoGs. Annotated genes that were not significantly changing were added in with DoGs or ADoGs for DESeq2 normalization but were taken out of the plots for clarity [n = 2 for heat shock J2 samples, n = 3 for wild type, n = 3 for tdp-1(ok803)]. (A) Heat shock over wild type J2 enriched read through sense transcripts with 84 enriched (red) and 25 depleted (blue) out of 421 scored DoGs. (B) tdp-1(ok803) over wild type significant J2 enriched read through sense transcripts with 3 enriched (purple) out of 265 scored DoGs. (C) Heat shock over wild type J2 enriched read through antisense transcripts with 2 enriched (red) out of 70 scored ADoGs. (D) No significant tdp-1(ok803) over wild type J2 enriched read through antisense transcripts out of 43 scored ADoGs.
Fig 6.
Heat shock induces transcripts antisense to the eif-3B locus.
IGV view of eif-3.B. Normalized tracks with the sense strand on the top part of the histogram and antisense on the bottom with a max read depth of 200 for sense or antisense. Wild type (WT) sense (dark blue), WT antisense (light blue), heat shock (HS) sense (red), heat shock antisense (orange). Horizontal blue arrows indicated genes and gene direction 5’ to 3’. Horizontal red arrows on the right show a cluster of ncRNAs including W01D2.8 and transcription downstream of W01D2.8 (doW01D2.8) into eif-3.B (green arrow going left). Arrows on the bottom correspond to locations of probes for FISH (brown: 5’ Intergenic, purple: Second exon, black: 3’ UTR).
Fig 7.
Fluorescence in situ Hybridization (FISH) of eif-3.B regions.
100x oil immersion images of worm hypodermal and neuronal cells. Heat shock panels are in the three columns to the left (merged channel in the middle column). Control panels show exposure from every channel (right column). Row (A) Immunohistochemistry with J2 antibody (green) along with FISH of doW01D2.8 antisense to the last exon and 3’ UTR (LE 3’ UTR) (red) of eif-3.B. dsRNA and the antisense LE 3’UTR transcript aggregate into nuclear foci with heat shock and do not appear to colocalize. Row (B) FISH of doW01D2.8 in two regions antisense to the 5’ intergenic region (5’ INT) (green) and last exon and 3’ UTR (LE 3’UTR) (red) of eif-3.B. Row (C) FISH of doW01D2.8 antisense to the last exon and 3’ UTR (LE 3’UTR) (red) of eif-3.B and sense probe of ncRNA C3DE1.9 (green). Probing of C3DE1.9 is not affected by heat shock and C3DE1.9 is not induced by heat shock. C3DE1.9 and LE 3’UTR show no overlap. (D) Diagram of eif-3.b gene with FISH probe locations and orientation. (E) Heat shock of tdp-1(ok803) induces nuclear foci from probes antisense to the last exon and 3’ UTR (LE 3’UTR) of eif-3.B (left panel) and is not visible with no heat shock (right).