Journal: Molecular cell

Article Title: Circular RNAs trigger nonsense-mediated mRNA decay.

doi: 10.1016/j.molcel.2024.11.022

Figure Lengend Snippet: Figure 3. The EJC loaded onto circRNA is recruited into the 30 UTR of mRNA through an interaction between circRNA and mRNA (A and B) MBP pull-down experiments. As performed in Figures 1E and 1F, except that HeLa cells expressing circG-AS30, the indicated linear RbG variant depicted in Figure 2A, and either MS2-HA or MS2-HA-MBP were either treated or not treated with CHX for 6 h before cell harvest. (A) Relative co-enrichment of circG-AS30 RNA under CHX-untreated conditions. The levels of circG-AS30 RNA after the pull-down were normalized to those before the pull-down and adjusted based on the relatively enriched levels of linear reporter mRNA to calculate the abundance of co-enriched circG-AS30 RNA per enriched linear reporter mRNA. The normalized value of circG-AS30 RNA in the MBP pull-down of cells expressing RbG mRNA was arbitrarily set to 1.0. n = 4; **, p < 0.01; #, not significant. (B) Relative co-enrichment of circG-AS30 RNA under CHX-treated conditions. n = 4; **, p < 0.01. (C) Global enrichment of endogenous circRNAs in IP of eIF4A3. Total-cell circRNAs before and after IPs of eIF4A3 were analyzed using circRNA microarray. The changes in the levels of each circRNA type were calculated using Pearson correlation coefficients (R). (D) Schematic diagram illustrating the experimental approaches for MBP pull-down and IP experiments to demonstrate spatial recruitment of EJC through an association between circRNA and mRNA. (E) MBP pull-down experiments using the extracts of HEK293T expressing RbG-S mRNA and effector circRNA. (F and G) Relative enrichment of linear reporter mRNA and effector circRNAs after IPs of eIF4A3 (F) or Y14 (G). The relatively enriched levels of RbG-S mRNA (upper) and effector circRNA (lower) after IP in circG-expressing cells were arbitrarily set to 1.0, respectively. n = 3; **, p < 0.01; *, p < 0.05; #, not significant. (H) Schematic diagram for circG variants containing the first intron of b-globin gene and their expected products. (I) An effect of circG-In(+) variants depicted in (H) on the levels of RbG-S mRNA. n = 3; **, p < 0.01. See also Figure S3.

Article Snippet: Primary antibodies against the following proteins were purchased and used for IP, RNA-IP, or western blotting [listed in the format ‘‘protein name (catalog number, supplier)’’]: HA (11867431001, Roche), b-actin (A5441, Sigma-Aldrich), eIF4A3 (sc-365549, Santa Cruz Biotechnology), Y14 (MAB2484, Abnova), MAGOH (ab38768, Abcam), MLN51 (ab90651, Abcam), GAPDH (LF-PA0212, AbFrontier), SMG5 (ab33033, Abcam), STAU1 (A303-956A, Bethyl Laboratories), ATG5 (12994, Cell Signaling Technology), LC3B (3868, Cell Signaling Technology), cleaved-PARP (9541, Cell Signaling Technology), BCL2L11 (2933, Cell Signaling Technology), eIF4E (610269, BD Biosciences; for western blotting), HRSP12 (PA5-31352, Thermo Fisher Scientific), POP1 (12029-1-AP, Proteintech), ADAR1 (ab168809, Abcam), Drosha (ab245398, Abcam), Dicer (PA5-17021, Invitrogen), AGO1 (5053, Cell Signaling Technology), AGO2 (07-590, Merck Millipore), AGO3 (SAB4200112, Sigma-Aldrich), PACT (sc-377103, Santa Cruz Biotechnology), and TRBP (15753-1-AP, Proteintech).

Techniques: Expressing, Variant Assay, Microarray