Hypermethylated cap 4 maximizes Trypanosoma brucei translation

Abstract

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.

Fig. 1

Establishment of cap ribose MTase double knockout cells. A. Schematic for Southern blot detection of gene disruption for TbMTr2 and TbMTr3. Restriction enzymes and probe location are noted for each. Int = processing sequences for drug markers. B. Southern blot for attempts to establish TbMTr3 knockout cells in wildtype (Lanes 1–3) and TbMTr1^−/− genetic background (Lane 4). Resistance markers used: P = puromycin; F = phleomycin; N = neomycin; H = hygromycin. Blot of SacI/XbaI-digested genomic DNA probed for detection of the TbMTr3 5′ UTR. Arrowhead marks wildtype alleles in each blot. L = 1-kb molecular weight ladder (New England Biolabs). C. Detection of TbMTr2 knockouts in the TbMTr3^−/− genetic background by probing with the TbMTr2 5′ UTR. Genomic DNA was digested with BbvCI and XbaI. * marks an extraneous, weakly cross-hybridizing band. D. Verification of loss of TbMTr2 in TbMTr2^−/−/TbMTr3^−/− clonal lines (C2, C3, Lanes 4–5) by probing for absence of TbMTr2 coding region (CR). Genomic DNA was digested with BbvCI and XbaI. Ethidium bromide staining of the gel served as a loading control. E. Southern blot of blasticidin resistant cell line established by a TbMTr1 blasticidin knockout vector in TbMTr2^−/−/TbMTr3^−/− double knockout cells. The blot was probed with 5′-UTR of TbMTr1 (left panel) and blasticidin (right panel).

Fig. 2

TbMTr2^−/−/TbMTr3^−/− cells contain only cap 1 ribose methylated mRNA. A. Diagram of cap 4 structure with methylations circled and kinetoplasid-specific methylation noted with filled circles. The cap ribose methyltransferase for each nucleotide is indicated; ‘?’ Indicates the possibility of dual function for TbMTr3. B. Wildtype cap 4 profile on substrate SL RNA determined using radiolabeled TbSL40 oligonucleotides in total cell RNA samples with Superscript™ II (SSRT II) or Moloney murine leukemia virus (MMLV) RT. An asterisk (*) marks cap 3 intermediate not detected by primer extension; an arrow marks the spurious −1 extension produced by SS RT II. C. Cap 4 profile of SL RNA in cap ribose MTase knockout lines using TbSL40 on total cell RNA. r1KO = TbMTr^−/−cells; r2KO = TbMTr2^−/− cells; r3KO = TbMTr3^−/−cells. D. Mature mRNA cap structures in cap ribose MTase knockout cells determined on poly(A)+ RNA using SL exon-specific primer. The contrast of the DNA sequencing ladder was adjusted in Photoshop.

Fig. 3

TbMTr2^−/−/TbMTr3^−/− substrate SL RNA is not 3′-extended. High-resolution RNA blots of total cell RNA from cap MTase knockout cells were hybridized with TbSL40. See Experimental Procedures for details. M = mature length.

Fig. 4

Decreased translational efficiency due to loss of kinetoplastid-specific ribose cap methylation. A. Graph of [³⁵S]-methionine incorporation into proteins in TbMTr1 knockout cells. B. Graph of [³⁵S]-methionine incorporation in TbMTr3 knockout cells. C. Graph of [³⁵S]-methionine incorporation in TbMTr2^−/−/TbMTr3^−/− double knockout cells. The bars represent the range of results from two independent experiments.

Fig. 5

mRNA levels are not affected in MTase knockout lines on a per-cell basis. To compare endogenous mRNA levels, quantitative RT-PCR was used to measure RPS23 and GAPDH mRNA in wildtype (WT), TbMTr3^−/− (r3KO) and TbMTr2^−/−/TbMTr3^−/− (r2/r3KO) cells. The bars represent the range of results from three independent reactions.

Fig. 6

Growth of TbMTr1^−/− and TbMTr2^−/−/TbMTr3^−/− cells is compromised under serum starvation conditions. A. Wildtype (YTAT), TbMTr1^−/−, and TbMTr2^−/−/TbMTr3^−/− growth curves in SM medium supplemented with 10% Fetal Bovine Serum (FBS). B. YTAT and TbMTr1^−/−growth curves in SM medium supplemented with 5% FBS. C. YTAT and TbMTr2^−/−/TbMTr3^−/−growth curves in SM medium supplemented with 5% FBS.