Premature translation termination mediated non-ER stress induced ATF6 activation by a ligand-dependent ribosomal frameshifting circuit

Figures

• 

  Figure 1.. Ligand structure and design principle of ligand-dependent −1 PRF stimulating pseudoknots. (A) Design of 5′CGG3′/3′GGC5′ GG-mismatch motif mediated −1 PRF stimulator using DU177-PK as a template. The sequences and secondary structure of DU177-PK are shown with the filled and open circles representing Watson-crick and reversed Hoogsteen base pairs, respectively. A dashed line indicates a tertiary base-triple interaction and the double arrow indicates stem1–loop2 tertiary interactions identified in DU177-PK. The nucleotide sequences used to replace base-pairs in the stem1 or stem2 of DU177-PK are boxed with the 5′CGG3′/3′GGC5′ motifs typed in bold, while the unchanged nucleotide sequences of DU177-PK are typed in gray. (B) The scheme showing insertion of a −1 PRF signal into the dual-reporter system for −1 PRF activity measurement. As the −1 PRF signal is followed by a 0 frame UAA stop codon, the occurrence of −1 PRF will lead to the translation of the second reporter gene as a fusion protein containing the translated first reporter gene. By contrast, only the first reporter gene is translated without −1 PRF. (C) Chemical structure of NCT7.

• 

  Figure 2.. Activity analyses of designed NCT7 dependent −1 PRF stimulators in vitro and in vivo. (A) Sequences of designed D1–D7 RNA pseudoknots and spacers. The sequences different from those in DU177 construct are typed in red. The spacer sequences used in designed pseudoknots were different from those used for DU177-PK to reduce the composition of guanosine. Furthermore, the spacer length used in D4-D7 was six nucleotides. This modification restored the reading frame change caused by the deletion of a bridging GC base-pair between the GG-mismatch motif and the minor-groove base-triple in D2-PK that led to the generation of D4-PK and variants D5-D7. A 0 frame stop codon (UGA) was located at the stem2 of DU177, D2, D4 and D6 pseudoknots. By contrast, a 0 frame UAA further downstream in the vector backbone was used as the stop codon (not shown) for D1, D3, D5 and D7 pseudoknots because the original stop codon in stem2 disappeared due to a 5′CGG3′/3′GGC5′ motif (in red) insertion in each of these pseudoknots. The adjustments above led to different sizes of 0 frame translation products in (B). (B) A 12% denaturing gel analysis of 35S radioactive methionine labeled −1 PRF products mediated by…

• 

  Figure 3.. Spontaneous RNA hydrolysis analyses reveal NCT7 dependent conformational changes in 5′CGG3′/3′GGC5′ GG-mismatch motifs of D5 pseudoknot. 12% denaturing gel analyses of in-line probing results of DU177-PK and D5-PK. Labels on the top of lanes in the gel image are experimental conditions described as the following: T1, partial digestion with RNase T1 (cleaves after G residues); OH, partial digestion with alkaline (cleaves at each internucleotide linkage); N, no reaction; 0, without NCT7; and 1, 1 μM NCT7. The assigned sequences for DU177-PK and D5-PK were based on the guanine-specific T1 ribonuclease cleavages, and are listed in the left and right of the image, respectively. * represents the flexible CUC sequences in the loop1 of each pseudoknot. An open circle represents reduced cleavage upon NCT7 treatment, whereas a dot represents enhanced cleavage upon NCT7 treatment. Triangle symbols indicate the enhanced internucleotide cleavages corresponding to the bulged-out cytosines (typed in red) in NCT7-bound 5′CGG3′/3′GGC5′ GG mismatch motifs.

• 

  Figure 4.. Comparisons of NCT ligands-dependent −1 PRF activity of D58 construct in different human cell-lines. (A)The −1 PRF efficiency of D58 construct with different NCT ligands in different cell-types calculated from dual-luciferase assay results using its own in-frame control. Value for each bar is the mean of six independent experiments with standard deviation of the mean. (B) Fluorescent imaging analyses of HEK293T cells transfected by the D58 −1 PRF signal-containing dual-fluorescent reporter with or without ligand induction. The signals shown in the three horizontal rows were from the detection of: top panel, sfGFP (−1 frame); middle panel, mCherry (0 frame); bottom panel, bright field. (C) Results of Western-blot analysis of the cell lysates of dual-fluorescent reporter transfected cells in (B) using anti-mCherry (in 0 frame) or anti-GFP (in −1 frame) antibody. The cell lysates were collected after the treatment of NCT ligands for 24 h.

• 

  Figure 5.. Functional ATF6N mimic formation by NCT-dependent −1 PRF mediated premature translation termination in human HEK293T cell-lines. (A) Diagram of ER stress-induced processing of ATF6. The positions of bZIP domain, transmembrane (TM) domain, S1P and S2P are indicated. (B) Scheme of FLAG-ATF6-S1P construct derived translation products by engineered NCT7-induced −1 PRF. The positions of FLAG-tag, bZIP domain, transmembrane (TM) domain and S1P cleavage site are also indicated. The −1 PRF signal (FS) capable of mediating NCT7-induced premature termination was inserted between amino acids 373 and 374 of ATF6 (in blue). The −1 frame and 0 frame translation products will lead to the synthesis of FLAG-tagged ATF6N mimic and full-length ATF6 chimera, respectively. (C) Western blot analyses of FLAG-ATF6N mimic formation in HEK293T cells transfected with FLAG-ATF6(2A) -S1P-FS related expression vectors. The cells transfected with plasmid (pFLAG-ATF6(2A)-S1P-FS), containing S1P (R415A/R416A) and −1 PRF signal were treated with NCT7 (0.1 μM) or Z-NCTS (0.3 μM). The proteins extracted from transfected cells were detected by anti-FLAG antibody. The pFLAG-ATF6(2A)-S1P-FS-RT is a readthrough…

• 

  Figure 6.. Activation of ATF6-specific gene expression by ATF6N mimic generated from NCT-dependent −1 PRF circuit in HeLa-FS1 stable cell line. (A) Fluorescence microscopy images of −1 PRF circuit mediated gene expression in HeLa-FS1. The cell line was treated with tetracycline (4 μg/ml) and NCT7 (0.1 μM) or Z-NCTS (0.3 μM) for 48 h or with 2 μg/ml Tm for 1 day. The cells were fixed and stained with DAPI (upper panel), anti-Flag antibody and secondary antibody conjugated Alexa Fluor® Plus 488 sequentially (middle panel). The images of ATF6N-induced mCherry were showed in lower panel. Bar, 10 μm. (B) The qRT-PCR analyses of ATF6 and XBP1s target genes in the HeLa-FS1 in (A) following expression of −1 PRF circuit and treatment with NCT7 (0.1 μM) or Z-NCTS (0.3 μM) for 24 h. The qRT-PCR values are relative to the cell line without ligand treatment, and are presented as mean with standard deviation of the mean. ** means P < 0.001 as determined by student's t-test. (C) The qRT-PCR analyses of ATF6 and XBP1 targeting genes of HeLa-FS1 in (A) following treatment with 10 μg/ml Tm for 6 h. The relative expression levels are calculated by comparing with those without ligand treatment after…