title: The messenger RNA decapping and recapping pathway in Trypanosoma.
abstract: The 5' terminus of trypanosome mRNA is protected by a hypermethylated cap 4 derived from spliced leader (SL) RNA. Trypanosoma brucei nuclear capping enzyme with cap guanylyltransferase and methyltransferase activities (TbCgm1) modifies the 5'-diphosphate RNA (ppRNA) end to generate an m7G SL RNA cap. Here we show that T. brucei cytoplasmic capping enzyme (TbCe1) is a bifunctional 5'-RNA kinase and guanylyltransferase that transfers a γ-phosphate from ATP to pRNA to form ppRNA, which is then capped by transfer of GMP from GTP to the RNA β-phosphate. A Walker A-box motif in the N-terminal domain is essential for the RNA kinase activity and is targeted preferentially to a SL RNA sequence with a 5'-terminal methylated nucleoside. Silencing of TbCe1 leads to accumulation of uncapped mRNAs, consistent with selective capping of mRNA that has undergone trans-splicing and decapping. We identify T. brucei mRNA decapping enzyme (TbDcp2) that cleaves m7GDP from capped RNA to generate pRNA, a substrate for TbCe1. TbDcp2 can also remove GDP from unmethylated capped RNA but is less active at a mature cap 4 end and thus may function in RNA cap quality surveillance. Our results establish the enzymology and relevant protein catalysts of a cytoplasmic recapping pathway that has broad implications for the functional reactivation of processed mRNA ends.
authors.affiliation: Department of Infection Biology, Graduate School of Comprehensive Human Sciences and.
authors.forename: Anna V
authors.initials: AV
authors.lastname: Ignatochkina
authors.affiliation: Departments of Biological Sciences.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Takagi
authors.affiliation: the Human Biology Program, University of Tsukuba, Ibaraki 305-8575, Japan; and.
authors.forename: Yancheng
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Infection Biology, Graduate School of Comprehensive Human Sciences and.
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Nagata
authors.affiliation: Department of Infection Biology, Graduate School of Comprehensive Human Sciences and Departments of Biological Sciences the Human Biology Program, University of Tsukuba, Ibaraki 305-8575, Japan; and Microbiology and Immunology, State University of New York, Buffalo, NY 14260 kiongho@md.tsukuba.ac.jp.
authors.forename: C Kiong
authors.initials: CK
authors.lastname: Ho
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D003001:Cloning, Molecular; D019175:DNA Methylation / Q000502:physiology*; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009713:Nucleotidyltransferases / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics; D015800:Protozoan Proteins / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*
chemical_list: D020121:5' Untranslated Regions; D009841:Oligonucleotides; D015800:Protozoan Proteins; D012315:RNA Caps; D012333:RNA, Messenger; C030052:mRNA decapping enzymes; C526771:CGM1 protein, Trypanosoma brucei; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D004722:Endoribonucleases
keywords: RNA repair; Trypanosoma brucei; cap methylation; mRNA decapping; mRNA recapping