Journal: bioRxiv
Article Title: Human PARPs modify RNA nucleobases in vitro and in cells
doi: 10.64898/2026.03.31.715305
Figure Lengend Snippet: (A) Overview of the guanine base modification as introduced by ScARP-type toxins or by mammalian PARPs. (B) LC-MS/MS chromatograms monitoring N1-ribosyl-G in digested total RNA from wild type (WT) and TARG1 KO HeLa cells overexpressing either EGFP alone (MOCK) or PARP15-EGFP (PARP15) fusion proteins. Arrows indicate the appearance of the peak corresponding to N1-ribosyl-G. (C) LC-MS/MS chromatograms monitoring N3-Ribosyl-U in digested total RNA from wild type (WT) and TARG1 KO HeLa cells overexpressing either EGFP alone (MOCK) or PARP15-EGFP (PARP15) fusion proteins. Arrows indicate the expected elution time for N3-ribosyl-U. (D) 5’OH-RNA oligos were ADP-ribosylated using ScARP or PARP15cat; a 5’-pRNA oligo was ADP-ribosylated using TRPT1. After purification ADP-oligos were incubated with indicated hydrolases (100 nM) at RT for 30 min. ADPr was released by incubation with 200 nM NUDT5 and AMP was measured using AMP-Glo. (E) RNA was isolated from HeLa TARG1 knock-out cells transfected with GFP or GFP-PARP15. Extracted RNA was first incubated with 100 nM TARG1 followed by NUDT5 and AMPGlo assay as in (D). (F) ADPr release from total cellular RNA isolated from indicated TARG1 knockout cell lines measured as in (D). (G) ADPr release measured as in (F) with total cellular RNA isolated from different TARG1 knockdown cell lines. TARG1 knockdown was induced in cells stably transfected with Tet-pLKO-puro plasmids containing TARG1 specific shRNA using 200 ng/ml doxycycline (72h). (H) . HEK293T cells were transfected with Gaussia luciferase (Gluc) mRNA modified by PARP15cat or control mRNA and medium was collected at indicated intervals. Translation of control Gluc mRNA (grey) is compared to ADPr-Gluc mRNA (red). Data represent baseline-subtracted translation efficiency (ΔRLU). Points represent the mean of n=4; error bars standard deviation. P-values indicate significance as determined by Welch’s t-test. (I) Endpoint analysis of in vitro translation efficiency measured after 4h incubation with non-modified Gluc mRNA and enriched ADPr-Gluc mRNA. Bars represent the mean translation efficiency (RLU) of n=3; and error bars represent standard deviation. Individual data points are shown as jittered dots. P-value indicates significance as determined by Welch’s t-test.
Article Snippet: To make inducible TARG1 knock-down cells, TARG1 shRNA oligos were annealed and cloned into AgeI-EcoRI site of Tet-plko-Puro plasmid (Addgene 21915).
Techniques: Modification, Liquid Chromatography with Mass Spectroscopy, Purification, Incubation, Isolation, Knock-Out, Transfection, Knockdown, Stable Transfection, shRNA, Luciferase, Control, Standard Deviation, In Vitro
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