Cell 27, 339C352 [PubMed] [Google Scholar] 46

Cell 27, 339C352 [PubMed] [Google Scholar] 46. and a functional helicase motif I of REH2. REH2 complexes and recently identified related particles share a multiprotein core but are distinguished by several differential polypeptides. Finally, REH2 associates transiently, via RNA, with editing complexes, mitochondrial ribosomes, and several ancillary factors ND-646 that control editing and RNA stability. We propose that these putative higher order structures coordinate mitochondrial gene expression. Introduction Unique gene expression mechanisms in kinetoplastid flagellates include U-insertion/deletion RNA editing by concerted cycles of cleavage, U-addition/removal, and ligation that can create hundreds of amino acid codons in most mitochondrial mRNAs (1, 2). The RNA editing core complex (RECC)4 contains 18C20 subunits (3,C6), although a few subunits seem to exchange in substrate-specific variants of this complex (7). The RECC acronym was recently introduced by Simpson (55). Editing complexes recognize partial helices between pre-mRNA and complementary guide RNAs (gRNAs) initially stabilized by a short anchor duplex (6, 8, 9). Substrate determinants for duplex binding and nuclease specificity (6, 10, 11) and substrate structure in solution (12,C14) have been characterized. Several accessory factors, mostly in multisubunit arrays, have been proposed to modulate RNA editing during catalysis, substrate production, or RNA turnover. The MRP complex ND-646 has RNA annealing activity and may promote mRNA and gRNA pairing (15, 16). Post-transcriptional mRNA terminal 3-poly(A)/(U) and gRNA 3-poly(U) maturation are mediated by KPAP1 and RET1 complexes (17, 18). MRB1, TbRGG1, and GRBC complexes proposed to contain between 14 and 24 proteins (termed here MRB-related complexes) share several components, but their functional relationship remains unclear. Repression of a few common subunits inhibited RNA editing and in some cases also decreased the level of total gRNA. GRBC1 and GRBC2 co-purified with RECC subunits (18,C24). MERS1, MRP, and RBP16 proteins were associated with mRNA stability (23, 25). RBP16 also stimulated RNA insertion (26, 27). DEAD-box mHel61 (also termed REH1) is the only predicted helicase known to impact RNA editing (28). Most of these proteins are likely to have additional roles outside editing. RNA helicases are common across species and typically multifunctional; however, only a few examples have been studied in mitochondria. This work characterized the protein and RNA interactions of a factor REH2 (Tb927.4.1500) that we initially found in native editing complexes of mitochondria, we detected multiple ND-646 unique peptides of most RECC subunits and the accessory MRP factors (6). However, we also found a single peptide for a 241-kDa protein, termed REH2, with highly conserved DExH-helicase domains, a dsRBD, and an N-terminal mitochondrial import sequence (Fig. 1REH2 has 2167 amino acids, including a conserved mitochondrial import signal ((56). is shown at increased (and or and (23) recently reported that GRBC complexes, which co-purified with REH2, bind gRNA. We determined whether REH2 immunopurified complexes associate with gRNA, and we further examined the importance of conserved domains of this protein. To this end, we analyzed IgG-Dynabead pulldowns of ectopically expressed REH2 wild type and mutant dsRBD- or motif I (GK-to-AQ) (Fig. 1and (22), RNAi down-regulation of REH2 decreased the steady-state levels of gRNA (Fig. 5, and and and and 250-kDa protein in REH2 pulldowns photocross-links Rabbit Polyclonal to KNTC2 with RNA. and and are repeats of but after a treatment with 0.1% SDS at 70 and 90 C, respectively, that enriches a cross-link at 250 kDa (and 7C10), the latter before or after RNase/MN treatment. A large number of REH2 unique peptides were found in all pulldowns reflecting the relatively large size of this protein. The 20C30 S fractions contained 22 proteins (besides REH2) previously found in one or more of the reported MRB1, TbRGG1, and GRBC complexes (21, 23, 24). These complexes significantly overlap but also exhibit important compositional differences (Table 1 and supplemental Fig. S4 show RNase-resistant and RNase-sensitive REH2 interactions, respectively). Seven RNase-resistant proteins (out of 13) were common to the known MRB-related complexes, namely REH2, GRBC2, ribosomal.