Picture from Rousakis A et al. (2014) PLoS One "Diverse functions of mRNA metabolism factors in stress defense and aging of ...."

(A) Representative confocal images of 1-day adults expressing the translational fusion dcap-1::gfp (BRF155 in Table S1) or ife-2::gfp (BRF70 in Table S1) under normal conditions (-SA) or after exposure to 10 uM sodium arsenite for 3h(+SA) at 25C.

Picture from Rousakis A et al. (2014) PLoS One "Diverse functions of mRNA metabolism factors in stress defense and aging of ...."

Figure S2 Monitoring of granule formation under oxidative stress, heat-shock or aging by using other reporter markers. (A) Representative confocal images of 1-day adults expressing the translational fusion dcap-1::gfp (BRF155 in Table S1) or ife-2::gfp (BRF70 in Table S1) under normal conditions (-SA) or after exposure to 10 uM sodium arsenite for 3h(+SA) at 25uC. (B) Representative confocal images of 1-day adults expressing the translational fusion ain-1::gfp (MH2704 in Table S1), the transcriptional fusion of ife-2 putative promoter to gfp (BRF68 in Table S1) or the translational fusion ife-2::gfp (BRF70 in Table S1) under normal conditions (-HS) or after heatshock at 35C for 3 h (+HS). The same transgenic animals are shown at the day 5 of adulthood, grown under normal conditions, at 25C. (C) Representative confocal images of 1-day adults coexpressing ife-2::gfp and dcap-1::rfp (BRF313 in Table S1). Arrows point to induced granules. Scale bar: 25 mm. (D) Expression levels of ain-1 gene in N2, as 1-day or 5-day adults grown under normal conditions (-HS) or as 1-day adults exposed to heat-shock at 35uC for 3 h (+HS), measured by quantitative RT-PCR and normalized to ama-1(mRNA) levels. Error bars show the SD of the means of two independent experiments.

Picture from Ofenbauer, Andreas et al. (2021) MicroPubl Biol

Figure 1. sspt-16 is a novel ncRNA: (A) Models of FACT subunits in H. sapiens (top) and C. elegans (middle). Conserved protein domains according to Pfam (pfam.xfam.org) and InterPro (ebi.ac.uk/interpro) are indicated. The pseudogene sspt-16 and its mother gene spt-16 are less than 2 kb apart from each other (bottom). The ok1829 deletion allele of sspt-16 is indicated in red. RNA image adapted from https://commons.wikimedia.org (CC BY-SA 3.0, free to use and change). (B) After broad induction of the ASE neuron specifying TF CHE-1 in L4 animals, no ectopic expression of the ASE neuron fate reporter gcy-5p::GFP can be detected 24 h later (top and middle left). However, in the sspt-16(ok1829) deletion background, ectopic gcy-5p::GFP expression can be detected in the germline (top and middle right). Some reprogrammed germ cells show axo-dendridic like projections (bottom right, indicated by white arrows) and some also show the pan-neuronal marker rab-3p::NLS::tagRFP (bottom left and middle, indicated by orange arrows). Scale bars = 20 µm. (C) We could verify sspt-16 RNA transcripts through RT-PCR (top; blue primers result in 143 bp for spt-16, while green primers result in 764 bp for spt-16 and 289 bp for sspt-16). All primers were designed at intron/exon borders, so that the expected sizes correspond to spliced mRNA or ncRNA, but not DNA. Additionally, we could detect sspt-16 ncRNA transcripts through long-read (Nanopore) and short-read (Illumina) RNA-seq (bottom). (D) Using CRISPR/Cas9 gene-editing, a 3xFLAG-tag was introduced at the C-terminal end of endogenous sspt-16 / F55A3.7 (top). SSPT-16::3xFLAG worm lysates did not show any immunofluorescent FLAG staining, while the LMN-1::2xFLAG positive control did (bottom left). N2 (negative control), HIS-71::2xFLAG (positive control) and SSPT-16::3xFLAG worm lysates were used in a western blot. A positive band for SSPT-16::3xFLAG would have been expected at 24.3 kDa, if a tagged protein was generated from the edited sspt-16 locus. (E) We checked the sspt-16(ok1829) deletion background for gene expression changes of the germline FACT members spt-16 and hmg-3, as well as of the histone chaperone and reprogramming barrier lin-53, by using qPCR (left) and western blots (right), but could not detect any significant difference neither on mRNA nor on protein level for the tested genes. Error bars represent SD, n.s.: not significant according to a one-way ANOVA. (F) Using the CRISPR/Cas system, we generated an additional sspt-16 deficient strain (bar37) by knocking in an aptamer-ribozyme sequence that depletes transcribed sspt-16 ncRNA molecules and confirmed its functionality by qPCR using two different primer pairs which target sspt-16 ncRNA. ***: p<0.001 according to a one-way ANOVA. (G) The phenotype penetrance (ectopic GFP expression in the germline after broad CHE-1 induction) was assessed for WT control, the Δsspt-16(ok1829) deletion strain and the Δsspt-16(apta) aptamer-ribozyme strain. n = 147-252; 4 biological repeats. ***: p<0.001 according to a one-way ANOVA. Error bars represent SEM. (H) Germline specific ATAC-seq of N2 and Δsspt-16(ok1829) strains revealed that there are basically no significant differences in chromatin accessibilities. Two loci that were identified as different (turquoise) were the ok1829 deletion itself, as well as another undocumented deletion that is present in the original ok1829 strain, so represent deletions on DNA level rather than differentially accessible chromatin. (I) Genomic location of sspt-16 and spt-16 loci showing active and repressive chromatin marks. A screenshot of JBrowse implemented in wormbase.org (WS280) is shown with publicly available tracks for repressive H3 modifications: H3K27me3 (emb/L3/adult), H3K9me2/3 (emb/L3/adult); active: H3K36me3 (emb/L3), H3K9ac (L3), H3K77ac (adult). The loci are located at the right arm of chromosome I. The red line indicates the sspt-16 / spt-16 location, which appears to lie within a putative chromosomal compartment border region.