Figure 5.

TLC analysis of uridine-to-Ψ conversion at the PTC within the TRM4 mRNA transcript. The PTC-containing TRM4 mRNA, coexpressed with a PTC-specific guide RNA, is purified by oligonucleotide affinity chromatography. The RNA is cleaved by RNase H (directed by a specific 2′-O-methyl RNA–DNA chimera) at the site 5′ of the PTC (UAA). The resulting 3′-half fragment is 5′ radiolabeled with ³²P through dephosphorylation and rephosphorylation (see text). The labeled RNA is digested with nuclease P1 to completion. The digested sample is mixed with an equal amount of 5′-³²P-adenosine-monophosphate, 5′–³²P-cytidine-monophosphate, and 5′–³²P-guanosine-monophosphate, and analyzed by two-dimensional TLC. The first and second dimensions are indicated. The origin and the positions of each 5′-phosphorylated nucleotide are also indicated.