title: REH2C Helicase and GRBC Subcomplexes May Base Pair through mRNA and Small Guide RNA in Kinetoplastid Editosomes.
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.
authors.affiliation: From the Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843.
authors.forename: Vikas
authors.initials: V
authors.lastname: Kumar
authors.affiliation: From the Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843.
authors.forename: Bhaskara R
authors.initials: BR
authors.lastname: Madina
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Shelly
authors.initials: S
authors.lastname: Gulati
authors.affiliation: the Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095.
authors.forename: Ajay A
authors.initials: AA
authors.lastname: Vashisht
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Chiedza
authors.initials: C
authors.lastname: Kanyumbu
authors.affiliation: From the Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843.
authors.forename: Brittany
authors.initials: B
authors.lastname: Pieters
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Afzal
authors.initials: A
authors.lastname: Shakir
authors.affiliation: the Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095.
authors.forename: James A
authors.initials: JA
authors.lastname: Wohlschlegel
authors.affiliation: the Department of Microbiology and Immunology, University of Buffalo School of Medicine, Buffalo, New York, and.
authors.forename: Laurie K
authors.initials: LK
authors.lastname: Read
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Blaine H M
authors.initials: BHM
authors.lastname: Mooers
authors.affiliation: From the Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843,. Electronic address: cruzrey@tamu.edu.
authors.forename: Jorge
authors.initials: J
authors.lastname: Cruz-Reyes
mesh_terms: D000818:Animals; D020029:Base Pairing; D002417:Cattle; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D017393:RNA Editing; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D017394:RNA, Guide, Kinetoplastida / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000378:metabolism*; D014354:Trypanosomiasis, Bovine / Q000382:microbiology
chemical_list: D015800:Protozoan Proteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D020365:RNA Helicases; D017394:RNA, Guide, Kinetoplastida
keywords: DEAH/RHA proteins; REH2; RNA editing; RNA helicase; RNA processing; RNP subcomplexes; Trypanosoma brucei; editosome; mitochondria; small guide RNA