A Metazoan/Plant-like Capping Enzyme and Cap Modified Nucleotides in the Unicellular Eukaryote Trichomonas vaginalis
Figure 7
Evaluation of a cap 2 modified nucleotide in T. vaginalis mRNA by 2D-TLC analysis.
A. Scheme of experimental approach. For simplicity, uridine is shown at position +2 and +3 as this is the most common nucleotide at these positions [28], [29]. The presence of cap 1 modified nucleotides Am or Cm (Figure 4B) predict the release of 3′-phosphate mononucleotides (Np), and 2 of 4 possible cap structures depending on the absence or presence of a cap 2 modified uridine upon RNase T2 digestion. The presence of only a cap 1 structure (top) predicts release of m7GpppAmpUp or m7GpppCmpUp; if a cap 2 is present (bottom) m7GpppAmpUmpUp or m7GpppCmpUmpUp is predicted. Subsequent anti-TMG immunoprecipitation and elution by RNase P1 digestion predicts release of only unmodified ribonucleotides or both unmodified and 2-O-ribose modified ribonucleotides in the absence or presence of a cap 2 ribonucleotide, respectively. B. Detection of unmodified ribonucleotides at +2 of T. vaginalis mRNAs using the scheme shown in A. and 2D-TLC (right). Migration standards are shown in left panel.