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Hypermethylated cap 4 maximizes Trypanosoma brucei translation
Author(s) -
Zamudio Jesse R.,
Mittra Bidyottam,
Campbell David A.,
Sturm Nancy R.
Publication year - 2009
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2009.06696.x
Subject(s) - biology , trypanosoma brucei , translation (biology) , messenger rna , microbiology and biotechnology , rna , rna splicing , methylation , translational efficiency , gene expression , eukaryotic translation , wild type , primary transcript , methyltransferase , gene , protein biosynthesis , biochemistry , mutant
Summary 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.