title: Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition.
abstract: Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3'-to-5' in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3' ends and strain-specific alternative 3' editing within 3' UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.
authors.forename: Bhaskara R
authors.initials: BR
authors.lastname: Madina
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.
authors.forename: Vikas
authors.initials: V
authors.lastname: Kumar
authors.affiliation: Texas AgriLife Genomics and Bioinformatics Service, Texas A&M University, College Station, Texas 77845, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Metz
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
authors.forename: Blaine H M
authors.initials: BH
authors.lastname: Mooers
authors.affiliation: Department of Physics, Department of Chemistry & Biochemistry, Division of Hematology, Center of RNA Biology, The Ohio State University, Columbus, Ohio 43210-1117, USA.
authors.forename: Ralf
authors.initials: R
authors.lastname: Bundschuh
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.
authors.forename: Jorge
authors.initials: J
authors.lastname: Cruz-Reyes
mesh_terms: D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D002478:Cells, Cultured; D016830:Kinetoplastida / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D017393:RNA Editing; D017394:RNA, Guide, Kinetoplastida / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000737:chemistry / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; C029305:mitochondrial messenger RNA; D012313:RNA; D017394:RNA, Guide, Kinetoplastida
keywords: RNA editing; Trypanosoma brucei; deep RNA sequencing; guide RNA; mitochondrial RNA-binding complex MRB1