Fig. 5

The collateral activity of RfxCas13d cleaved 28s rRNA into two fragments, leading to translation attenuation and activation of ZAKα-JNK/p38-IEG pathway. a Quality control of total RNA of HEK293T-RfxCas13d cells transfected with plasmids encoding tdTomato/SIK3-S and corresponding crRNAs (as shown in Additional file 1: Fig. S8a) by Agilent 2200 Bioanalyzer. td: tdTomato; td-cr4/7: tdTomato crRNA 4/7; K3-cr1/8: Sik3-S crRNA 1/8. b Schematic illustration of the cleavage site of RfxCas13d on human 28s rRNA. c SUnSET essay to measure the protein translation rate of HEK293T-RfxCas13d cells 24 h after transfection of plasmids encoding tdTomato/SIK3-S-K37M and corresponding crRNAs. d Statistical diagram of cell cycle distribution in Additional file 1: Fig. S9b. e Schematic illustration of 509 common DEGs (P adj < 0.05 and change fold ≥2) from four sets of comparisons. f Heatmap representing the log2 fold-change values of IEGs in targeting crRNA-transfected groups vs. NT crRNA-transfected groups. g Transcription factor enrichment analysis using ChEA3. There are Top 15 transcription factors. h Western blot to measure the phosphorylation level of p38, JNK, and ERK in HEK293T-RfxCas13d 24 h after transfection of plasmids encoding tdTomato and tdTomato crRNAs or NT crRNA. Red asterisk represents non-specific band. i, j RT-qPCR to measure the RNA level of EGR1 and FOS in HEK293T-RfxCas13d cells 24 h after transfection of plasmids encoding tdTomato and tdTomato crRNAs or NT crRNA. k Western blot to measure the expression level of ZAKα, ZAKβ, and PKR in indicated cells. PKR-KO-1/2#: two strains of PKR knockout HEK293T-RfxCas13d cells; ZAK-KO-1/2#: two strains of ZAK knockout HEK293T-RfxCas13d cells; ZAK-KO-2#-r-ZAKα: re-expression of ZAKα in ZAK-KO-2#; ZAK-KO-2#-r-ZAKβ: re-expression of ZAKβ in ZAK-KO-2#. l Western blot to measure the phosphorylation level of p38, JNK 24 h after transfection of plasmids encoding RfxCas13d and tdTomato crRNA 7 or NT crRNA into indicated cells. Red asterisk represents non-specific band. KO-2#: ZAK-KO-2#; r-ZAKα: ZAK-KO-2#-r-ZAKα; r-ZAKβ: ZAK-KO-2#-r-ZAKβ. m Schematic illustration that the collateral activity of RfxCas13d cleaves 28s rRNA into two fragments, leading to translation attenuation and activation of ZAKα-p38/JNK-IEGs pathway. i, j Two-tail unpaired t test. Significance levels are noted as ***P < 0.001, ****P < 0.0001, or ns (P > 0.05). All values are presented as mean ± SEM