Table 1.
Expressions of Frameshift Mutant mRNAs in MMR-Deficient Colorectal Cancer Cell Lines
Figure 1.
Measurement of Degraded Premature Termination Codon-Containing mRNAs by RPA Analysis
No loss of TGFBR2 mutant transcripts was noted after puromycin treatment. In contrast, the amount of hRad50 and hMSH6 mRNA doubled after puromycin treatment. GAPDH was used for standardization. The asterisk indicates mutation status of each gene: (−1) denotes a 1-bp deletion in the cMNR, (w) denotes no mutation in the cMNR, (+1) denotes a 1-bp insertion in the cMNR, and (−2) denotes a 2-bp deletion in the cMNR.
Figure 2.
Western Blotting Analysis of NMD-Sensitive and NMD-Escape Proteins
Truncated proteins were not detected in NMD-sensitive (hRad50 and hMSH6) genes, whereas wild-type proteins were detected in the cell lines containing the wild-type allele. In the NMD-escape hMSH3 gene, wild-type hMSH3 proteins were detected in the cell lines containing the wild-type allele. However, no truncated proteins of hMSH3 were detected in the cell lines with frameshift mutation. GAPDH was used as a loading control. The single asterisk indicates mutation status of each gene: (−1) denotes a 1-bp deletion in the cMNR, (w) denotes no mutation in the cMNR, and (+1) denotes 1-bp insertion in the cMNR. The double asterisk indicates frameshift mutations of the other coding regions present in both alleles of DLD-1 [38]. The dagger indicates that the expected size of the truncated protein is indicated by the arrow.
Figure 3.
Transfection Assay of the TGFBR2 Vector
(A) Schematic diagram of construct K (TGFBR2 wild-type cDNA), construct L (TGFBR2(−1)-deleted cDNA), construct M (TGFBR2 wild-type genomic DNA), construct N (TGFBR2(−1)-deleted genomic DNA), and construct O (TGFBR2(−1)-deleted genomic DNA with a PTC artificially located in the last exon).
(B) Analysis of TGFBR2 mRNA by Northern blotting. The abundant expression of TGFBR2 mRNAs from the transfected constructs is shown. β-Actin was used as a RNA loading control. Con. denotes the control vector pSecTag2B.
(C) Western blotting using anti-FLAG. Protein expression of transfected TGFBR2 constructs. Cell lines transfected with the constructs K and M expressed wild-type TGFBR2. Cell lines transfected with the constructs L and O expressed truncated TGFBR2. However, cell lines transfected with construct N did not express truncated TGFBR2, indicating translational repression after normal splicing. Con. denotes the control vector pSecTag2B. Enhanced green fluorescent protein (EGFP) was used as a transfection control.
(D) Sucrose gradient fractionation of cytoplasmic extracts from cells expressing TGFBR2(WT)-splicing mRNA.
(E) Sucrose gradient fractionation of cytoplasmic extracts from cells expressing TGFBR2(−1)-splicing mRNA.
(F) TGFBR2(WT)-splicing transfected cells were treated with puromycin prior to lysis and fractionation.
(G) TGFBR2(−1)-splicing transfected cells were treated with puromycin prior to lysis and fractionation.
In (D, E, F, and G), RNA extracted from each fraction was subjected to RT-PCR. Endogenous GAPDH mRNA was used as a control. TGFBR2(WT) mRNA was found in the polysome-containing fractions similar to endogenous GAPDH mRNA. However, a greater percentage of PTC-containing TGFBR2(−1) mRNA was found in the fractions containing ribosomal subunits and monosomes. The plots denote quantitative representation of TGFBR2 mRNA distribution in polysome gradients. Relative mRNA levels in each fraction were calculated as a percentage of the total. Results from two independently performed experiments did not vary, and the plot represents the average of the two independent experiments.
Figure 4.
Both 3′ UTR and Splicing Are Important for Translational Suppression of PTC-Containing TGFBR2 mRNA
(A) Schematic diagram of constructs P (NMD-irrelevant-type TGFBR2(−1)-deleted construct containing a full coding sequence), O, and N.
(B) Western blotting using anti-UPF1, anti-UPF2, or as a loading control, anti-GAPDH.
(C) Analysis of TGFBR2 mRNA by Northern blotting. The abundant expression of TGFBR2 mRNAs from the transfected constructs is shown. β-Actin was used as a RNA loading control.
(D) Protein expression of transfected TGFBR2 constructs. Truncated proteins are not expressed in the cell lines transfected with TGFBR2(−1)-splicing, whereas, truncated proteins are expressed by cell lines transfected with TGFBR2(−1)-irrelevant and TGFBR2(−1)-irrelevant-F. The amount of truncated protein from the TGFBR2(−1)-irrelevant-F is approximately 35% of TGFBR2(−1)-irrelevant, indicating that the 3′ UTR length or cis-element(s) affect translational efficiency. Truncated proteins are not detected from cell lines transfected with TGFBR2(−1)-splicing when treated with MG132, a proteasome inhibitor. Truncated proteins are not generated from cell lines transfected with TGFBR2(−1)-splicing when UPF1 and UPF2 are knocked down by siRNA, indicating that UPF1 and UPF2 do not have an important role in the translational repression of TGFBR2(−1)-splicing mRNA. Enhanced green fluorescent protein (EGFP) was used for transfection control. Con. denotes the control vector pSecTag2B. Luci denotes luciferase siRNA.
Figure 5.
Truncated Protein Expression from Mutant NMD-Irrelevant MARCKS mRNA
(A) Schematic diagram of construct Q (MARCKS wild-type genomic DNA) and construct R (MARCKS(−2) deleted genomic DNA).
(B) Western blotting (WB) analysis of total protein (30 μg) isolated from transiently transfected cell lines with constructs Q or R. An antibody against FLAG was used as the primary antibody.
(C) Western blotting (WB) analysis of total protein (30 μg) isolated from transiently transfected cell lines with the constructs Q or R was performed using an antibody against MARCKS. Enhanced green fluorescent protein (EGFP) was used as a control for transfection efficiency. Comparable results were obtained in at least two independent experiments. Asterisks (*) denote endogenous MARCKS reacted with anti-MARCKS antibody; Con. denotes the control vector pcDNA3.1(+). The dagger indicates uncharacterized protein that did not interfere with experimental interpretations.
Figure 6.
Schematic Model for the Functional Consequences of the Three Classes of PTC-Containing mRNAs
NMD-sensitive mRNAs are degraded by the NMD system. NMD-escape mRNAs are not degraded by the NMD system, but experience repression of protein expression. NMD-irrelevant mRNAs are not recognized by the NMD system and generate truncated proteins.