FIG. 3.

SL RNA is underprocessed in TbMTAP^−/− cells. (A) The SL RNA-modified nucleotides implicated in kinetoplastid trans splicing and translation are shown within the exon sequence. The nomenclature for cap intermediates is listed above the modified nucleotides. An m above the sequence indicates base methylation; an m below the sequence indicates 2′-O-ribose methylation. (B) SL RNA cap phenotype in the wild type (WT) and TbMTAP knockout (MTAP KO) (center) determined using γ-³²P-labeled oligonucleotide specifically targeting substrate SL RNA in primer extensions (left). Quantification of cap intermediates was performed with QuantOne software from Amersham (right) and are listed as percentages of the total. (C) Primer extension with SL RNA intron-specific oligonucleotide detecting CMC attachment to ψ₂₈ in wild-type (WT), TbMTr1 knockout (MTr1 KO), and TbMTAP knockout (MTAP KO) RNA samples. (D) Primer extension analysis of hydrazine-aniline-treated RNA samples as described above for detection of unmodified uridine nucleotides at position 28 of the SL RNA. (E) High-resolution RNA blot probed for substrate SL RNA in wild-type and TbMTAP^−/− cells with radiolabeled oligonucleotide complementary to the intron sequence. 3′ ext., 3′-extended forms. (F) Mature mRNA cap structures are undermethylated in TbMTAP-null mutants. Primer extension assay on poly(A)-purified RNA with [γ-³²P]-labeled oligonucleotide complementary to SL exon sequence for wild type (WT) and TbMTAP knockout (MTAP KO).