Fig 1: A T7 RNAP transcript containing the Ll.LtrB intron is not degraded by nonsense mediated decay in human cells and can be spliced after addition of Mg2+ to the cell culture medium.(A) Diagram of pT7 transcription cassettes expressed from plasmids pLl.LtrB-GFP and pGFP, which contain either the Ll.LtrB intron and short flanking exons or just the ligated-exon sequences that would result from Ll.LtrB intron splicing inserted at the same location within the GFP ORF. Red arrows show the location of primers used for RT-PCR and RT-qPCR of Ll.LtrB intron splicing. (B) RNAi knockdown of UPF1. HEK-293 cells were pretreated for 24 h with either scrambled siRNA (black) or UPF1 siRNA (white) to inhibit NMD and then transfected with the pGFP or pLl.LtrB-GFP. At 48 h after transfection of the plasmids, the number of GFP transcripts per cell was measured by RT-qPCR. The immunoblot below the bar graph confirms knockdown of UPF1 protein by the UPF1 siRNA but not by the control scrambled siRNA in HEK-293 cells at 72 h after transfection of the siRNA corresponding to the time at which GFP transcript levels were measured. Equal amounts of cellular proteins were loaded in each lane. (C) RT-PCR assay for RNA splicing of Ll.LtrB transcripts in HEK-293 cells at 24 h after transfection of the indicated plasmids with or without 80 mM Mg2+ added to the culture medium. The RT-PCR products were analyzed in a 1% agarose gel, which was stained with ethidium bromide. The positions of the PCR products corresponding to unspliced and spliced pLl.LtrB-GFP RNAs are indicated to the right of the gel. Splicing of the Ll.LtrB intron in HEK-293 cells with 80 mM Mg2+ added to the culture medium was confirmed by sequencing across the ligated-exon junction in the PCR product. The experiment was done twice with similar results. (D) GFP fluorescence in HEK-293 cells transfected with plasmids pGFP or pLl.LtrB-GFP + phLtrA with 80 mM MgCl2 added to the culture medium. Images were taken 72 h post-transfection. The experiment was done twice with similar results. Abbreviations: E1 and E2, 5’ and 3’ ltrB exons, respectively; IRES: internal ribosome entry site; pA, polyadenylation signal; PT7: T7 RNAP promoter.
Fig 2: SMG1 effects on α-synuclein expression are independent of its function in the mRNA surveillance pathway.Silencing the UPF1 protein, a key regulator of mRNA surveillance, has no effect on either p-syn or t-syn levels, indicating that disruption of the mRNA surveillance pathway is not the mechanism through which SMG1 affects α-synuclein. The antibodies are the same as in Figure 3, with the addition of anti-UPF1 antibody (α-UPF1). siRNAs used are indicated across the top of the westerns. Results are representative of at least 3 independent replicates.
Fig 3: A Pol II transcript containing the Ll.LtrB intron is subject to nonsense-mediated decay in human cells.(A) Diagram of plasmid-borne PCMV transcription cassettes with or without the Ll.LtrB intron and short flanking exons inserted directly after the start codon of BFP. Red arrows indicate primers used for RT-qPCR analysis of transcript levels. (B) RNAi knockdown of UPF1. HeLa cells were pretreated for 24 h with either a scrambled siRNA (black) or UPF1 siRNA (white) to inhibit NMD, and then transfected with BFP expression plasmids with or without the Ll.LtrB intron and short flanking exons inserted directly after the BFP start codon. At 48 h after transfection of the plasmids, the number of transcripts per cell was measured by RT-qPCR, and normalized to that of the pBFP transcript in the presence of the scrambled control siRNA assayed in parallel. The bar graphs show the average ± the SD for two or three replicates for each condition. (C) Immunoblot showing knockdown of the NMD protein UPF1 at 72 h after transfection of the UPF1 siRNA corresponding to the time at which BFP transcript levels were measured. Equal amounts of cellular proteins were loaded in each lane. This immunoblot control was done twice with similar results. Abbreviations: E1 and E2, 5’ and 3’ ltrB exons, respectively; pA, polyadenylation signal; PCMV, cytomegalovirus immediate-early promoter.
Fig 4: Novel nonsense-mediated decay (NMD) inhibitor KVS0001 is SMG1 specific and induces expression of NMD-targeted genes in vitro and in vivo.(A) Fraction of mutant allele transcripts in genes with heterozygous indels previously established in this study as sensitive to NMD inhibition. Results show mutant levels after siRNA treatment targeting kinases inhibited by LY3023414. RNF43 and DROSHA are common heterozygous single-nucleotide polymorphisms (SNPs) (shaded gray) and serve as negative controls. (B) Fraction of mutant allele transcripts in genes with truncating mutations known to be sensitive to NMD inhibition after siRNA treatment with siUPF1 or non-targeting siRNA. Data from deep-targeted RNA-sequencing. (C) Structure of novel NMD inhibitor KVS0001. (D) Targeted RNA-sequencing on three genes with heterozygous, out-of-frame, indel mutations in LS180 cancer cells treated in a dose–response with KVS0001 or SMG1i-11. RNF43 serves as a control (common heterozygous SNP) and the mutant allele refers to the non-reference genome allele. (E) Western blot of EXOC1 protein in NCI-H358 cells treated with 5 µM novel inhibitor KVS0001, LY3023414, or SMG1i-11 for 24 hr. (F) Western blot of phosphorylated UPF1 on three cell lines treated with 5 µM KVS0001, SMG1i-11, or dimethyl sulfoxide (DMSO). Note that total UPF1 and p-UPF1 were run on different gels, loading controls correspond to indicated gel. (G) Fold change in the number of mutant allele transcripts measured by targeted RNA-seq in genes containing heterozygous out-of-frame indel mutations in NCI-H358 or (H) LS180 subcutaneous xenografts in bilateral flanks of nude mice. Mice were treated once with intraperitoneal (IP) injection of vehicle or 30 mg/kg KVS0001 and tumors harvested 16 hr post IP treatment. All genes shown contain heterozygous out-of-frame truncating mutations except RNF43 and DROSHA which serve as controls (contain heterozygous SNPs).
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