Present addresses: Koch Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA;
Hypermethylated cap 4 maximizes Trypanosoma brucei translation
Article first published online: 11 MAY 2009
© 2009 Blackwell Publishing Ltd
Volume 72, Issue 5, pages 1100–1110, June 2009
How to Cite
Zamudio, J. R., Mittra, B., Campbell, D. A. and Sturm, N. R. (2009), Hypermethylated cap 4 maximizes Trypanosoma brucei translation. Molecular Microbiology, 72: 1100–1110. doi: 10.1111/j.1365-2958.2009.06696.x
- Issue published online: 22 MAY 2009
- Article first published online: 11 MAY 2009
- Accepted 5 April, 2009.
Through trans-splicing of a 39-nt spliced leader (SL) onto each protein-coding transcript, mature kinetoplastid mRNA acquire a hypermethylated 5′-cap structure, but its function has been unclear. Gene deletions for three Trypanosoma brucei cap 2′-O-ribose methyltransferases, TbMTr1, TbMTr2 and TbMTr3, reveal distinct roles for four 2′-O-methylated nucleotides. Elimination of individual gene pairs yields viable cells; however, attempts at double knock-outs resulted in the generation of a TbMTr2−/−/TbMTr3−/− cell line only. Absence of both kinetoplastid-specific enzymes in TbMTr2−/−/TbMTr3−/− lines yielded substrate SL RNA and mRNA with cap 1. TbMTr1−/− translation is comparable with wildtype, while cap 3 and cap 4 loss reduced translation rates, exacerbated by the additional loss of cap 2. TbMTr1−/− and TbMTr2−/−/TbMTr3−/− lines grow to lower densities under normal culture conditions relative to wildtype cells, with growth rate differences apparent under low serum conditions. Cell viability may not tolerate delays at both the nucleolar Sm-independent and nucleoplasmic Sm-dependent stages of SL RNA maturation combined with reduced rates of translation. A minimal level of mRNA cap ribose methylation is essential for trypanosome viability, providing the first functional role for the cap 4.