Figure 1.

The formation of 48S complexes on capped mRNA with the rabbit beta-globin leader sequence. Toeprinting electropherograms (denaturing 6% PAGE, GelDoc Systems (BioRad, USA) visualization) are presented. Toeprints were detected by fluorescence; the fluorescent dye (FAM) was within the reverse transcription primer. Full-length cDNA and the 48S complex are indicated. The addition of components is denoted as +, their absence as 0. Red lanes, capped unmodified mRNA; green lanes, capped m⁶A-modified mRNA; azure lanes, uncapped m⁶A-modified mRNA. Detailed examination of toeprinting electropherograms (lanes 3, 10, 11, 12, and 15) revealed additional bands between the 5′ and 3′ ends of modified mRNA. These stops in reverse transcription were observed previously and associated with the presence of a stretch of adjacent modified nucleotides m⁶A(4) [24], which were poorly recognized by the reverse transcriptase and caused the termination of reverse-transcription elongation.