The 2'-O-ribose methyltransferase for cap 1 of spliced leader RNA and U1 small nuclear RNA in Trypanosoma brucei.
mRNA cap 1 2'-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2'-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2'-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2'-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3'-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.
Zamudio
Jesse R
JR
Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, 609 Charles E. Young Drive East, University of California at Los Angeles, Los Angeles, CA 90095-1489, USA.
Mittra
Bidyottam
B
Foldynová-Trantírková
Silvie
S
Zeiner
Gusti M
GM
Lukes
Julius
J
Bujnicki
Janusz M
JM
Sturm
Nancy R
NR
Campbell
David A
DA
D000818:Animals; D008745:Methylation; D008780:Methyltransferases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D010641:Phenotype; D010802:Phylogeny; D015800:Protozoan Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D012315:RNA Caps; D034622:RNA Interference; D016053:RNA, Protozoan; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D020038:RNA, Spliced Leader / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020040:Trans-Splicing; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism
D015800:Protozoan Proteins; D012315:RNA Caps; D016053:RNA, Protozoan; D012342:RNA, Small Nuclear; D020038:RNA, Spliced Leader; D011993:Recombinant Fusion Proteins; C405483:U1 small nuclear RNA; D008780:Methyltransferases