GAP1/2 heterotetramer exhibits accumulation of mRNAs undergoing RNA editing and reduced association with RECC upon MRB1 core disassembly by MRB8620 RNAi. (A) Scheme depicting GAP1/2 IP in MRB8620 RNAi-silenced cells (left). Western blot (right) of GAP1 IPs performed in MRB8620 RNAi-induced (Tet+) and uninduced cells (Tet−) were immunodecorated with antibodies against GAP1 and RECC subunit KREL1. (B) GAP1/2-bound RNAs isolated from the elutions from A were converted to cDNA for quantification by real-time PCR with selected primer sets for minimally edited and pan-edited RNAs. The bar graph indicates fold changes in RNA isolated from MRB8620 RNAi-induced cells compared to that from uninduced cells normalized to never-edited mRNAs CO1 (black bars) and ND4 (striped bars). Whiskers denote range of obtained relative abundances within technical triplicates. (C) Relative abundance of pre-edited and edited mRNAs pulled down in GAP1/2 IPs as in B but when normalized to the levels of the same transcripts from total RNA (Fig. 3A) in MRB8620-depleted and uninduced controls. This normalization is elaborated in Materials and Methods. (D) Relative abundance of total CYB transcripts pulled down with GAP1 in the IP as well as total RNA (Tot. RNA) from MRB8620-depleted and uninduced controls, and depicted as in B. The scheme on the right in D indicates the primers designed to detect the pre-edited (P), edited (E), and total CYB transcripts. All qPCR graphs are representative data from one of two IPs.
