PMC

Linear RNA amplification. (A) Various amounts of total RNA isolated from the SUDHL-6 cell line were used for two rounds of linear amplification. With decreasing template concentration below approximately 500 ng, the amplified RNA product shows a significant decline in both quantity and quality (size). Lane 1, RNA ladder; lane 2, 1000 ng starting RNA template; lane 3, 500 ng starting RNA template; lane 4, 100 ng starting RNA template; lane 5, 10 ng starting RNA template; lane 6, 1 ng starting RNA template; lane 7, 0.1 ng starting RNA template; lane 8, DNA ladder. (B) Aliquots of 1000 ng of total RNA from the SUDHL-6 cell line were subjected to two rounds of RNA amplification. One round of amplification resulted in approximately 15-fold amplification and two rounds showed approximately 190-fold amplification. Lane 1, RNA ladder; lane 2, two rounds of amplification; lane 3, one round of amplification; lane 4, DNA ladder. (C) The quality and quantity of the amplified RNA is directly dependent upon the quality of the starting template RNA. Partially degraded RNA results in much lower amounts of amplified product and smaller product size after two rounds of linear amplication compared with amplified product obtained from intact starting material. Lane 1, RNA ladder; lane 2, two rounds of amplification starting from 1000 ng of intact total RNA; lane 3, two rounds of amplification starting from 1000 ng of partially degraded total RNA; lane 4, DNA ladder. (D) The concentration of the oligo-dT(15)-T7 promoter primer used to make the cDNA is a crucial component of the RNA amplification reaction. Template independent products (primer dimers) are produced when higher concentrations of oligo-dT(15)-T7 primer are used in the amplification reaction. Lane 1, RNA ladder; lane 2, 1000 ng oligo-dT(15)-T7 primer; lane 3, 500 ng oligo-dT(15)-T7 primer; lane 4, 100 ng oligo-dT(15)-T7 primer; lane 5, DNA ladder. (E) One round of amplification produced amplified RNA (aRNA) from SUDHL-1 (lanes 1 and 2) and Karpas 299 (lanes 3 and 4) starting from 2 μg total RNA template using the RiboAMP kit; lane 5, DNA ladder.

Partial enzymatic sequence determination of the U5 RNA. The RNA was prepared as described in Fig. B. Band II was subjected to partial digestion with base-specific RNases. Lanes: G, guanine-specific RNase T1 ladder; A, adenine-specific RNase U2 ladder; AU, adenine-uridine specific RNase phyM ladder; C, cytosine-specific RNase CL3 ladder; CU, cytosine-uridine specific RNase Bacillus cereus ladder; L, alkaline partial hydrolysis ladder. RNA sequence is indicated in the right.

(A) α1 RNA specifically inhibits the amplification of α1 from a rat kidney cDNA template. PCR was performed with 1 µM sense and 1 µM antisense degenerate primers and rat kidney cDNA; without RNA (lane 1), with 50 nM α1 RNA (lane 2), with 50 nM β1 RNA (lane 3), with 50 nM β2 RNA (lane 4), with 50 nM α1 RNA and 50 nM β1 RNA (lane 5), with 50 nM α1 RNA and 50 nM β2 RNA (lane 6), with 50 nM β1 RNA and 50 nM β2 RNA (lane 7) or with 50 nM α1 RNA, 50 nM β1 RNA and 50 nM β2 RNA (lane 8). After electrophoresis and Southern blotting, the blot was hybridized with a radiolabeled probe specific for α1, washed at high stringency and subjected to autoradiography. A ladder of molecular weight markers is on the left and the arrow indicates the bands corresponding to the expected 750 bp PCR product. (B) β1 RNA specifically inhibits the amplification of β1 from a rat kidney cDNA template. PCR was performed with 1 µM sense and 1 µM antisense degenerate primers and rat kidney cDNA; without RNA (lane 1), with 50 nM α1 RNA (lane 2), with 50 nM β1 RNA (lane 3), with 50 nM β2 RNA (lane 4), with 50 nM α1 RNA and 50 nM β1 RNA (lane 5), with 50 nM α1 RNA and 50 nM β2 RNA (lane 6), with 50 nM β1 RNA and 50 nM β2 RNA (lane 7) or with 50 nM α1 RNA, 50 nM β1 RNA and 50 nM β2 RNA (lane 8). After electrophoresis and Southern blotting, the blot was hybridized with a radiolabeled probe specific for β1, washed at high stringency and subjected to autoradiography. A ladder of molecular weight markers is on the left and the arrow indicates the bands corresponding to the expected 750 bp PCR product. (C) β2 RNA specifically inhibits the amplification of β2 from a rat kidney cDNA template. PCR was performed with 1 µM sense and 1 µM antisense degenerate primers and rat kidney cDNA; without RNA (lane 1), with 50 nM α1 RNA (lane 2), with 50 nM β1 RNA (lane 3), with 50 nM β2 RNA (lane 4), with 50 nM α1 RNA and 50 nM β1 RNA (lane 5), with 50 nM α1 RNA and 50 nM β2 RNA (lane 6), with 50 nM β1 RNA and 50 nM β2 RNA (lane 7) or with 50 nM α1 RNA, 50 nM β1 RNA and 50 nM β2 RNA (lane 8). After electrophoresis and Southern blotting, the blot was hybridized with a radiolabeled probe specific for β2, washed at high stringency and subjected to autoradiography. A ladder of molecular weight markers is on the left and the arrow indicates the bands corresponding to the expected 750 bp PCR product.

(A) Fluorescence images of HOE-1::GFP in day 1 adult wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi. Scale bar 200 μm. (B) Fluorescence intensity quantification of intestinal nuclei relative to extranuclear signal in day 1 adult wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi (n = 40 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (C) Western blot for HOE-1::GFP and actin from day 1 adult wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi. (D) Quantification of HOE-1::GFP western blot band intensity from day 1 adult wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi normalized to total protein (n = 4 for each condition, mean and SD shown, ordinary two-way ANOVA with Tukey’s multiple comparisons test). (E) Fluorescence images of HOE-1::GFP in day 1 adult wildtype and atfs-1(et15) animals. Scale bar 200 μm. (F) Fluorescence intensity quantification of intestinal nuclei relative to extranuclear signal in day 1 adult wildtype and atfs-1(et15) animals (n = 40 for each condition, mean and SD shown, unpaired t-test). (G) Western blot for HOE-1::GFP and actin from day 1 adult wildtype and atfs-1(et15) animals. (H) Quantification of HOE-1::GFP western blot band intensity from day 1 adult wildtype and atfs-1(et15) animals normalized to total protein (n = 4 for each condition, mean and SD shown, unpaired t-test). (I) Mitochondrial stress triggers activation of HOE-1 resulting in altered RNA processing that facilitates UPR^mt via ATFS-1. Activation of UPR^mt negatively regulates HOE-1.
Figure 8—source data 1.Blots for wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi ().All panels are the same membrane. (A) Image of stain-free blot for total protein from day 1 adult wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi. Two biological replicates of each condition: Lane # 1&11 BR Spectra Protein Ladder – ladder bands in kDa denoted. Lane # 2&7 wildtype on control RNAi, 3&8 wildtype on atfs-1 RNAi, 4&9 nuo-6(qm200) on control RNAi, and 5&10 nuo-6(qm200) on atfs-1 RNAi. Lane # 6 empty. (B) Chemiluminescence image of blot for HOE-1::GFP using GFP primary antibody. (C) Composite image of chemiluminescence and colorimetric images of blot for HOE-1::GFP to show bands relative to ladder. (D) Chemiluminescence image of blot for actin using β-actin primary antibody. (E) Composite image of chemiluminescence and colorimetric images of blot for actin to show bands relative to ladder.
Figure 8—source data 2.Blots for wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi ().Samples were loaded and ran on two separate membranes simultaneously (Membrane A and Membrane B). All panels in each column are the same membrane. (A) Image of stain-free blots for total protein from day 1 adult wildtype and nuo-6(qm200) animals on control and atfs-1 RNAi. Two biological replicates on each blot of each condition: Lane # 1&10 BR Spectra Protein Ladder – ladder bands in kDa denoted. Lane # 2&6 wildtype on control RNAi, 3&7 wildtype on atfs-1 RNAi, 4&8 nuo-6(qm200) on control RNAi, and 5&9 nuo-6(qm200) on atfs-1 RNAi. (B) Chemiluminescence image of blots for HOE-1::GFP using GFP primary antibody. (C) Composite images of chemiluminescence and colorimetric images of blots for HOE-1::GFP to show bands relative to ladder. (D) Chemiluminescence images of blots for actin using β-actin primary antibody. (E) Composite images of chemiluminescence and colorimetric images of blots for actin to show bands relative to ladder.
Figure 8—source data 3.Blots for wildtype and atfs-1(et15) animals ().All panels are the same membrane. (A) Image of stain-free blot for total protein from day 1 adult wildtype and atfs-1(et15) animals. Two biological replicates of each condition: Lane # 1&6 BR Spectra Protein Ladder – ladder bands in kDa denoted. Lane #2&4 wildtype and #3&5 atfs-1(et15). (B) Chemiluminescence image of blot for HOE-1::GFP using GFP primary antibody. (C) Composite image of chemiluminescence and colorimetric images of blot for HOE-1::GFP to show bands relative to ladder. (D) Chemiluminescence image of blot for actin using β-actin primary antibody. (E) Composite image of chemiluminescence and colorimetric images of blot for actin to show bands relative to ladder.
Figure 8—source data 4.Blots for wildtype and atfs-1(et15) animals ().All panels are the same membrane. (A), Image of stain-free blot for total protein from day 1 adult wildtype and atfs-1(et15) animals. Four biological replicates of each condition: Lane # 1 BR Spectra Protein Ladder – ladder bands in kDa denoted. Lanes #2,4,6,8 wildtype and #3,5,7,9 atfs-1(et15). (B) Chemiluminescence image of blot for HOE-1::GFP using GFP primary antibody. (C) Composite image of chemiluminescence and colorimetric images of blot for HOE-1::GFP to show bands relative to ladder. (D) Chemiluminescence image of blot for actin using β-actin primary antibody. (E) Composite image of chemiluminescence and colorimetric images of blot for actin to show bands relative to ladder.