Mitochondrial mRNAs in Trypanosoma brucei undergo extensive insertion and deletion of uridylates that are catalyzed by the RNA editing core complex (RECC) and directed by hundreds of small guide RNAs (gRNAs) that base pair with mRNA. RECC is largely RNA-free, and accessory mitochondrial RNAbinding complex 1 (MRB1) variants serve as scaffolds for the assembly of mRNA-gRNA hybrids and RECC. However, the molecular steps that create higher-order holoenzymes ("editosomes") are unknown. Previously, we identified an RNA editing helicase 2-associated subcomplex (REH2C) and showed that REH2 binds RNA. Here we showed that REH2C is an mRNA-associated ribonucleoprotein (mRNP) subcomplex with editing substrates, intermediates, and products. We isolated this mRNP from mitochondria lacking gRNA-bound RNP (gRNP) subcomplexes and identified REH2-associated cofactors 1 and 2 ( H2 F1 and H2 F2). H2 F1 is an octa-zinc finger protein required for mRNP-gRNP docking, pre-mRNA and RECC loading, and RNP formation with a short synthetic RNA duplex. REH2 and other eukaryotic DEAH/RHA-type helicases share a conserved regulatory C-terminal domain cluster that includes an oligonucleotide-binding fold. Recombinant REH2 and H2 F1 constructs associate in a purified complex in vitro. We propose a model of stepwise editosome assembly that entails controlled docking of mRNP and gRNP modules via specific base pairing between their respective mRNA and gRNA cargo and regulatory REH2 and H2 F1 subunits of the novel mRNP that may control specificity checkpoints in the editing pathway.RNA editing by uridylate insertion and deletion in Trypanosoma brucei modifies over 3000 sites in mitochondrial mRNAs in a gradual process directed by hundreds of small guide RNAs (gRNAs) 4 (1-3). The basic regulatory mechanisms of substrate specificity and developmental control in RNA editing remain unknown. The uridylate changes are catalyzed by the RECC enzyme from 3Ј to 5Ј in discrete blocks. Each gRNA directs editing of one mRNA block. Surprisingly, RECC has little or no RNA and lacks the processivity found in vivo, as established in early purifications of this multiprotein enzyme (4 -6). So, accessory components of the editing apparatus must facilitate substrate recruitment and editing catalysis. There are many non-RECC proteins that affect editing. Most of these proteins (Ͼ25 proteins) are components of the MRB1 complex in T. brucei, also termed gRNA-binding complex (GRBC) in Leishmania, that binds and stabilizes gRNA. MRB1 interacts transiently with the RECC enzyme and mitoribosomes (1,7,8). It has also been found that MRB1 contains all three classes of mRNA in editing: unedited pre-mRNAs, partially edited intermediates, and fully edited transcripts. This indicates that MRB1 complexes serve as scaffolds for the assembly of hybrid substrates and the RECC enzyme (9 -11). Transient addition of RECC to these scaffolds would establish higher-order editing holoenzymes or editosomes. MRB1 was first considered a single dynamic complex, but the reason of its variable compos...