title: Guide RNA biogenesis involves a novel RNase III family endoribonuclease in Trypanosoma brucei.
abstract: The mitochondrial genome of kinetoplastids, including species of Trypanosoma and Leishmania, is an unprecedented DNA structure of catenated maxicircles and minicircles. Maxicircles represent the typical mitochondrial genome encoding components of the respiratory complexes and ribosomes. However, most mRNA sequences are cryptic, and their maturation requires a unique U insertion/deletion RNA editing. Minicircles encode hundreds of small guide RNAs (gRNAs) that partially anneal with unedited mRNAs and direct the extensive editing. Trypanosoma brucei gRNAs and mRNAs are transcribed as polycistronic precursors, which undergo processing preceding editing; however, the relevant nucleases are unknown. We report the identification and functional characterization of a close homolog of editing endonucleases, mRPN1 (mitochondrial RNA precursor-processing endonuclease 1), which is involved in gRNA biogenesis. Recombinant mRPN1 is a dimeric dsRNA-dependent endonuclease that requires Mg(2+), a critical catalytic carboxylate, and generates 2-nucleotide 3' overhangs. The cleavage specificity of mRPN1 is reminiscent of bacterial RNase III and thus is fundamentally distinct from editing endonucleases, which target a single scissile bond just 5' of short duplexes. An inducible knockdown of mRPN1 in T. brucei results in loss of gRNA and accumulation of precursor transcripts (pre-gRNAs), consistent with a role of mRPN1 in processing. mRPN1 stably associates with three proteins previously identified in relatively large complexes that do not contain mRPN1, and have been linked with multiple aspects of mitochondrial RNA metabolism. One protein, TbRGG2, directly binds mRPN1 and is thought to modulate gRNA utilization by editing complexes. The proposed participation of mRPN1 in processing of polycistronic RNA and its specific protein interactions in gRNA expression are discussed.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA.
authors.forename: Bhaskara Reddy
authors.initials: BR
authors.lastname: Madina
authors.forename: Gokulan
authors.initials: G
authors.lastname: Kuppan
authors.forename: Ajay A
authors.initials: AA
authors.lastname: Vashisht
authors.forename: Yu-He
authors.initials: YH
authors.lastname: Liang
authors.forename: Kurtis M
authors.initials: KM
authors.lastname: Downey
authors.forename: James A
authors.initials: JA
authors.lastname: Wohlschlegel
authors.forename: Xinhua
authors.initials: X
authors.lastname: Ji
authors.forename: Sing-Hoi
authors.initials: SH
authors.lastname: Sze
authors.forename: James C
authors.initials: JC
authors.lastname: Sacchettini
authors.forename: Laurie K
authors.initials: LK
authors.lastname: Read
authors.forename: Jorge
authors.initials: J
authors.lastname: Cruz-Reyes
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D005786:Gene Expression Regulation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D017394:RNA, Guide, Kinetoplastida / Q000096:biosynthesis*; D043244:Ribonuclease III / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
chemical_list: D043244:Ribonuclease III; D017394:RNA, Guide, Kinetoplastida