The image contains three panels labeled A, B, and C. Panel A shows a western blot analysis on the left, depicting the phosphorylation levels of P K R and e I F 2 alpha induced by inverted repeat A l u s with varying sequence composition across lanes 1 through 6. Bands are shown for P-P K R, P K R, P-e I F 2 alpha, e I F 2 alpha, and beta-Actin, with molecular weight markers indicated at 75 and 37 kilodaltons. The right side of Panel A presents a table summarizing the number of double stranded R N A segments in the predicted minimum free energy structure capable of activating 17 base pair (O A S 1), 22 base pair (R I G-I), or 33 base pair (P K R) sensors. Categories listed include 1 no R N A, 2 A l u perfect d s R N A, 3 endogenous A l u d s R N A (mean and median values provided), 4 U S P 8 intron 12 inverted repeat A l u, 5 S N A I 2 intron 1 inverted repeat A l u, and 6 A C 009784.3 intron 1 inverted repeat A l u. Panel B consists of three box plots labeled 17 base pair (O A S 1), 22 base pair (R I G-I), and 33 base pair (P K R). The plots compare double stranded R N A sensor site distribution between introns and 3 prime untranslated regions. Statistical significance is indicated above the comparisons. Panel C features three scatter plots labeled 17 base pair (O A S 1), 22 base pair (R I G-I), and 33 base pair (P K R). These plots show cytoplasmic lariat double stranded R N A sensor sites normalized by total mapped reads (lariat reads per 10 cap 7 total reads) in wild type and D B R 1 knockout cell lines. Individual data points are shown, and statistical significance is indicated for specific comparisons. The data collectively demonstrate that sequence variation within inverted repeat Alus affects the number and length of double stranded R N A segments capable of activating innate immune sensors, influencing P K R activation potential.
Sequence variations in IR Alu decrease the length of dsRNA stretches and reduce PKR activation. (A) Left: Western blot analysis of PKR and eIF2α phosphorylation induced by single (or multiple) IR Alus with different degrees of sequence variation. Data are representative of three independent experiments. Right: Count of dsRNA segments in the predicted MFE structure capable of activating OAS1, RIGI-I, or PKR. (B) For IR Alu–containing introns and 3′ UTRs at most 5000 nt long, the predicted MFE structure was used to quantify the distribution of dsRNA lengths capable of activating OAS1, RIG-I, or PKR. P values were from t test. ****P < 0.0001. (C) Lariat reads were mapped from cytoplasmic RNA-seq samples taken from WT and DBR1 KO cell lines. Structure prediction was performed with RNAfold for each lariat, and the number of lariat-associated dsRNA sites was tallied and normalized by the total mapped reads. P values were from t test. *P < 0.05; **P < 0.01. Source data are available for this figure: SourceData F6.