title: Complete cap 4 formation is not required for viability in Trypanosoma brucei.
abstract: In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5' ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m(7)G cap, ribose 2'-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2'-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A(2) and is not required for subsequent steps; TbMT511 methylates C(3), without which U(4) methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m(7)G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A(2), C(3), and U(4) methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095-1489, USA.
authors.forename: Jesse R
authors.initials: JR
authors.lastname: Zamudio
authors.forename: Bidyottam
authors.initials: B
authors.lastname: Mittra
authors.forename: Gusti M
authors.initials: GM
authors.lastname: Zeiner
authors.forename: Marcin
authors.initials: M
authors.lastname: Feder
authors.forename: Janusz M
authors.initials: JM
authors.lastname: Bujnicki
authors.forename: Nancy R
authors.initials: NR
authors.lastname: Sturm
authors.forename: David A
authors.initials: DA
authors.lastname: Campbell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012315:RNA Caps / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D016053:RNA, Protozoan / Q000378:metabolism*; D020038:RNA, Spliced Leader / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology
chemical_list: D012315:RNA Caps; D012333:RNA, Messenger; D016053:RNA, Protozoan; D020038:RNA, Spliced Leader; D011993:Recombinant Fusion Proteins; D008780:Methyltransferases; C055099:mRNA (nucleoside-O(2'))-methyltransferase