Fig 1.
The IL-6-SRE broadly protects against multiple viral endonucleases.
(A) 293T cells were transfected with empty vector (mock) or a plasmid expressing the indicated endonucleases along with a GFP reporter. After 24 h, total RNA was harvested and subjected RT-qPCR to measure GFP mRNA levels. Graphs here and afterwards display individual replicates as dots, together with the mean values (±SEM). Statistical significance was determined by the Student t test (* p<0.1; ** p<0.05; *** p<0.01). (B) 293T cells were co-transfected with the indicated endonuclease-expressing plasmid expressing together with a GFP-SRE or GFP-ΔSRE reporter. After 24 h, total RNA was harvested and subjected RT-qPCR to measure GFP mRNA levels. (C) Top: diagram showing the structure of the reporter mRNA containing MS2 repeats upstream of the SRE or ΔSRE fragment of the IL-6 3’UTR. Red lines denote the region detected by the MS2 probe. Bottom: 293T cells were transfected with the indicated MS2 reporters. 24h later, cells were fixed and processed for RNA FISH staining. Signals from the MS2 probes (red) and DAPI stained nuclei (blue) were detected by confocal microscopy.
Fig 2.
The IL-6-SRE does not protect against cellular endonucleases.
(A) 293T cells were transfected with the indicated dsRed2 reporters. After 24 h, total RNA was harvested and subjected RT-qPCR to measure dsRed mRNA levels. (B) 293T cells were treated with siRNAs targeting Smg6 or control non-targeting siRNAs. 48h later, cells were transfected with the indicated dsRed reporters. After 24h, total RNA was harvested and subjected RT-qPCR to measure dsRed mRNA levels (left). The efficiency of SMG6 knockdown was measured by western blotting, with actin serving as a loading control (right). (C) 293T cells were transfected with the indicated dsRed2 reporters. After 24 h, total RNA was harvested and subjected to RT-qPCR to measure dsRed mRNA levels. (D) 293T cells were transfected with an empty vector (mock) or a plasmid expressing SCoV nsp1 along with the indicated GFP reporters. After 24 h, total RNA was harvested and subjected RT-qPCR to measure GFP mRNA levels.
Fig 3.
GADD45B mRNA is protected against SOX degradation.
(A, B) Total RNA was extracted from unreactivated or reactivated KSHV-positive TREX-BCBL-1 (A) cells or iSLK.219 cells (B) and subjected to RT-qPCR to measure endogenous levels of the GADD45B or GAPDH transcript. (C) 293T cells were transfected with an empty vector or a plasmid expressing SOX or vhs. After 24 h, total RNA was harvested and subjected to RT-qPCR to measure endogenous levels of GADD45B or GAPDH transcripts. (D) Diagram of the reporter constructs where fragments of GADD45B transcript were fused to GFP. (E) 293T cells were transfected with the indicated GFP-GADD45B fusion constructs in the presence or absence of a plasmid expressing SOX. After 24 h, total RNA was harvested and subjected to RT-qPCR to measure GFP levels.
Fig 4.
The SREs contain a long hairpin required for protection against SOX.
(A) 293T cells were transfected with the indicated GFP reporter along with a control empty vector (mock) or a plasmid expressing SOX. After 24 h, total RNA was harvested and subjected to RT-qPCR to measure GFP mRNA levels. (B) Diagram of the structure prediction obtained with RNAfold for IL-6 and GADD45B SREs. The color scale represents the confidence score of the structure as calculated by RNAfold, with red representing the highest confidence. Asterisks denote the location of mutations that were introduced in the structure for the following assays. The insets are RNAfold predictions of the stem loops of interest in isolation. (C) 293T cells were transfected with the indicated GFP reporters containing mutations within IL-6 SRE at the residues marked by a * in (B) along with a control empty vector (mock) or a plasmid expressing SOX. After 24 h, total RNA was harvested and subjected RT-qPCR to measure GFP mRNA levels. (D) 293T cells were transfected with the indicated GFP reporters mutated within G- SRE at the residues marked by a * in (B) along with a control empty vector (mock) or a plasmid expressing SOX. After 24 h, total RNA was harvested and subjected to RT-qPCR to measure GFP mRNA levels.
Fig 5.
The IL-6 and GADD45B SRE bind a partially overlapping set of cellular proteins.
(A) Diagram outlining the ChIRP assay. (B) Cytoscape network showing the proteins reported to interact with the IL-6 SRE from a previous screen [27] (gray nodes) overlaid with the set of proteins that were also recovered by ChIRP-MS for the IL-6 and GADD45B 3’ UTRs (purple nodes) (C) 293T were transfected with the GFP-GADD45B 3’UTR reporter, then 24h later they were subjected to ChIRP analysis and protein samples were western blotted. (D) Crosslinked lysates of 293T cells were subjected to RNA immunoprecipitation (RIP) with control IgG, anti-NCL, or anti-HuR antibodies and the level of co-purifying endogenous GADD45B mRNA was quantified by RT-qPCR. Bars represent the fold enrichment over the mock IP. (E) 293T cells were transfected with either the GFP-GADD45B 5’UTR, GFP-GADD45B 3’UTR, or GFP-GADD45B 3’UTR_GG reporter for 24 h. Lysates were then subjected to RIP as described in (C). Bars represent the fold enrichment over mock IP.
Fig 6.
The GADD45B SRE function requires HuR, but is only protective against SOX and vhs.
(A, B) 293T cells were treated with siRNAs targeting HuR (A) or NCL (B) or control non targeting siRNAs. 48h later, cells were transfected with the GFP-GADD45B-3’UTR reporter along with a control empty vector or a plasmid expressing SOX. After 24 h, total RNA was harvested and subjected to RT-qPCR to measure GFP mRNA levels. Protein levels of HuR and NCL after siRNA-mediated depletion are shown under the bar graphs (~30% and 45% of total protein left respectively) (C) 293T cells were transfected with the GFP-GADD45B-3’UTR reporter along with a control empty vector or a plasmid expressing the indicated viral endonucleases. After 24 h, total RNA was harvested and subjected to RT-qPCR to measure GFP mRNA levels.