Figure 1.
Prolonged cytoplasmic residence of 5′-truncated forms of PTC-hβG mRNA.
A. Actinomycin D (6 µg/ml) was added to DMSO-induced Thal10 cells at time 0 and cytoplasmic RNA recovered at the indicated times was assayed by S1 nuclease protection using a probe for the first 354 nt of β-globin mRNA. Hinf I restriction fragments of φX174 DNA were used as size markers (M, lane 1) and the location of each of the products relative to the 5′ end of β-globin mRNA is indicated on the right side of the autoradiogram. B. The amount of each species was quantified by phosphorimager analysis and plotted on the right as a function of time after addition of Actinomycin D.
Figure 2.
Development of a modified MBRACE assay for quantifying full-length and one of the 5′-truncated β-globin mRNAs.
A. A schematic of the modified MBRACE assay is shown, and the locations of each of the primers are presented in Figure S1. B. RNA from triplicate cultures of MEL cells expressing WT-hβG or PTC-hβG mRNA was added directly to the ligation reaction with the 5′ RNA adapter (–TAP, open bars) or treated with tobacco acid pyrophosphatase (+TAP, grey bars) before ligation. Results obtained without TAP treatment were arbitrarily set to 1 and results obtained with TAP-treated RNA were normalized to the −TAP samples. The results represent the mean ± standard deviation for triplicate samples. *indicates p<0.01 by Student’s two-tailed T-test. C. S1 nuclease protection assay was used to demonstrate the increased accumulation of 5′-truncated RNAs in cells expressing PTC-hβG mRNA (lane 6) compared to WT-hβG mRNA (lane 3). The locations of each of the 5′-truncated RNAs with respect to the cap site are indicated to the right of each autoradiogram. D. The transcript beginning at position 169 nt (Δ169) was selected as a representative truncated RNA. Cytoplasmic RNA from MEL cells expressing WT- or PTC-hβG mRNA was used to demonstrate applicability of the modified MBRACE assay for quantifying changes in full-length mRNA and Δ169 RNA. In each graph the amount of RNA from each cell line was first normalized to β-actin. Shown is the mean ± standard deviation for biological triplicates, *indicates p<0.001 by two-tailed t-test.
Figure 3.
Evidence that NMD is responsible for the accumulation of Δ169 RNA from PTC−hβG mRNA.
Tet-inducible K562 cells in antibiotic-free medium were electroporated with plasmids expressing constitutive wild type or nonsense-containing TCRβ genes, or each of the inducible β-globin genes, and a GFP control. Sixteen hr later they were transfected with Accell SmartPool→ Upf1 or control siRNAs, cultured for an additional 48 hr, then induced with doxycycline for 6 hr. A. Cytoplasmic extracts from WT- and PTC-hβG expressing cells were assayed by Western blotting for efficiency of Upf1 knockdown. B. The effectiveness of Upf1 knockdown in inhibiting NMD was determined by qRT-PCR analysis of changes in WT- versus PTC-TCRβ mRNA. C. The modified MBRACE assay was used to quantify the impact of Upf1 knockdown on full-length WT- and PTC-hβG mRNA. D. Modified MBRACE assay was used to monitor the impact of Upf1 knockdown on the production of Δ169 RNA. The results represent the mean ± standard deviation of triplicate cultures, *indicates p<0.05 by two-tailed Student’s T-test.
Figure 4.
Evidence that 5′-truncated RNAs are decay intermediates.
A. Doxycycline was added at time 0 to tet-inducible K562 cells that were electroporated 16 hr earlier with plasmids bearing inducible WT- or PTC-hβG genes and a GFP control. Cytoplasmic RNA isolated at intervals over 12 hr of induction was analyzed by modified MBRACE assay for changes in full-length and Δ169 forms of hβG mRNA. B. The first 3 hr of induction is shown enlarged. C. The experiment was repeated except that the plasmids that were electroporated into tet-inducible K562 cells expressed a destabilized form of β-globin mRNA (H124 mutation) as a result of disruption of the 3′-UTR nucleolin binding site [12]. Each point represents the mean ± standard deviation for triplicate determinations.
Figure 5.
SMG6 knockdown increases full-length PTC-hβG mRNA and decreases Δ169 RNA.
Tet-inducible K562 cells in antibiotic-free medium were electroporated as in Figure 3, with plasmids expressing constitutive WT- or PTC-TCRβ genes, or each of the inducible hβG genes, and a GFP control. Sixteen hr later they were transfected with Accell SmartPool→ SMG6 or control siRNAs, cultured for an additional 48 hr, then induced with doxycycline for 6 hr. A. Cytoplasmic extracts from cells expressing each form of β-globin mRNA were assayed by Western blotting for efficiency of SMG6 knockdown. B. The effectiveness of SMG6 knockdown in inhibiting NMD was determined by qRT-PCR analysis of changes in WT- versus PTC-TCRβ mRNA. C. The modified MBRACE assay was used to quantify the impact of SMG6 knockdown on full-length WT- and PTC-hβG mRNA. D. Modified MBRACE assay was used to monitor the impact of SMG6 knockdown on the production of Δ169 RNA. The results represent the mean ± standard deviation of triplicate cultures, *indicates p<0.05 by two-tailed Student’s t-test.
Figure 6.
Complementation identifies SMG6 is the endonuclease responsible for generating Δ169 RNA from PTC-hβG mRNA.
Tet-inducible K562 cells in antibiotic-free medium were electroporated as in Figure 3, with control or SMG6 siRNAs. Twenty-four hr later they were electroporated again with the same siRNAs plus empty vector or plasmids expressing siRNA resistant SMG6 or a catalytically-inactive form of SMG6 (SMG6-m4) and plasmids expressing each of the inducible hβG genes. Doxycycline was added 16 hr later to induce each of the hβG genes. A. Cytoplasmic extracts from triplicate cultures were pooled and analyzed by Western blotting with anti-SMG6 antibody or with antibody to GAPDH. B. Cytoplasmic RNA from individual cultures was analyzed by modified MBRACE assay for full-length (upper panel) and Δ169 (lower panel) forms of hβG mRNA. The results represent the mean ± standard deviation of triplicate cultures, *indicates p<0.05 by two-tailed Student’s t-test.
Figure 7.
Upf1 phosphorylation is unaffected by SMG6 knockdown or overexpression of inactive SMG6.
Tet-inducible K562 cells were electroporated as in Figure 6 with control or SMG6 siRNAs and empty vector, or plasmids expressing SMG6 or inactive SMG6-m4. Cytoplasmic extracts recovered 16 hr after the second round of electroporation were analyzed by Western blotting with antibodies to SMG6 (top panel) or to phospho-Upf1 (middle panels). Both light and dark exposures are shown. Ribosomal protein S6 was used as a loading control (bottom panel).