Native immunopurified subcomplexes of MRB1 with either H2 or 3010 subunits. (A) Western analysis of H2 and 3010 in IPs by the indicated antibodies, and in mitochondrial extract (ME). H2 (∼240 kDa) is often fragmented, and 3010 (57.5 kDa) migrates slightly below IgG in the IPs. A fainter band below 3010 is a breakage product of this protein in ME (*). The blot was split into halves treated with either anti-H2 or anti-3010 antibodies. (B) Western analysis of the RECC subunit REL1 ligase in IPs and ME. (C) [32P]G-capping of gRNA 5′ triphosphate with guanylyltransferase on 15% UREA-PAGE to concentrate gRNA in a discrete band. (D) Western analysis of the MRB1 subunit GAP1 in test and mock IPs. Mock IPs (Mk) used an irrelevant affinity-purified antibody. (E) Site-specific crosslinking of H2 and 3010 IPs with a pre-edited mRNA construct whose first editing site contains 32P in its phosphodiester bond and 4-thioU in the flanking 5′ nucleotide. After RNase trimming, the protein-RNA adducts were resolved on 10% SDS-PAGE. (F) Crosslinks as in E, but on a high-resolution 6% SDS-PAGE. Native (wt) and ectopic (tagged) H2 differ in mobility due to an ∼20 kDa tag. Tagged H2 was affinity-purified (AP). Intact and fragmented H2 are marked. Molecular markers are in kDa. All IPs (200 mM KCl, 5% BSA) used precleared extract.
