The 2′-O-Ribose Methyltransferase for Cap 1 of Spliced Leader RNA and U1 Small Nuclear RNA in Trypanosoma brucei

Abstract

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.

SUPPLEMENTAL MATERIAL

We thank Jay Bangs for the pKO vectors; Thomas Seebeck for pMOT vectors; Kent Hill for YTAT cells and for use of the Zeiss fluorescence microscope; Murray Schnare for advice on cap analysis; and Miriam Giardini, Robert Hitchcock, Sean Thomas, and Scott Westenberger for stimulating discussions.

This work was supported by NIH grant AI034056 and in part by grants of the Ministry of Education of the Czech Republic 2B06129 and LC07032. J.R.Z. was supported by NSF Louis Stokes Alliance for Minority Participation Award HRD-0115115:3 and USPHS National Research Service Award GM07104.