REH2C Helicase and GRBC Subcomplexes May Base Pair through mRNA and Small Guide RNA in Kinetoplastid Editosomes

Vikas Kumar,Bhaskara R Madina,Shelly Gulati,Ajay A Vashisht,Chiedza Kanyumbu,Brittany Pieters,Afzal Shakir,James A Wohlschlegel,Laurie K Read,Blaine H.M Mooers,Jorge Cruz-Reyes

doi:10.1074/jbc.m115.708164

Abstract

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 RNA-binding 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.

Highlights

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 RNAs4 [1,2,3]
We propose that assembly of stable mRNA-guide RNAs (gRNAs) hybrids in editing scaffolds requires docking of mRNA-associated ribonucleoprotein subcomplex (mRNP) and gRNA-bound RNP (gRNP) modules by base pairing of mRNA and gRNA
Because mitochondrial RNAbinding complex 1 (MRB1) complexes contain pre-mRNA substrates and products of editing, we and others proposed that these complexes serve as scaffolds for specific assembly of mRNA-gRNA hybrid substrates and their processing by RNA editing core complex (RECC) [9, 10]

Summary

Introduction

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 [1,2,3]. The regulatory REH2 helicase and its eight-zinc finger H2F1 cofactor enable studies of mRNA-gRNA hybrid assembly and substrate access to RECC on molecular scaffolds. QRT-PCR of unedited and fully edited transcripts in IPs, normalized to both a mock pulldown and input (loaded extract) values, showed REH2C retention (ϳ50% or higher) of the tested transcripts in mitochondria depleted of GAP1 and gRNA (Fig. 4F).

Results

Conclusion