Figure 3.

The nucleotide polymorphisms in pri-miR-BART-21 and 22 from C666-1-EBV. (A) The nucleotide variations in pri-miR-BART21 (AJ507799, 145435:145614; left) and pri-miR-BART22 (147126:147305; right) are shown in shaded color. The mature miRNA sequences are shown in gray boxes. (B) The predicted secondary structures of C666-1-EBV miR-BART22 (EU828629, 8087:8267; left), Raji-EBV miR-BART22 (AJ507799, 147137:147456; middle), and the two nucleotide variations of Raji-EBV, Raji-EBV-2nt mut (147144 A > T and 147146 C > A; right) are shown. The folding energy (dG) with units (kcal/mol) is indicated. (C) In vitro Drosha/DGCR8 processing of the pri-miR-BART22 transcript. C666-1-EBV and Namalwa-EBV pri-miR-BART22 transcripts (pri-RNA) (AJ507799, 147137:147456) were in vitro-transcribed and incubated with bead-bound Flag-tagged Drosha/DGCR8 complex (+) or beads containing empty vector transfected cell extract (-). The digested products were separated on 8% PAGE for analysis (left). The presence of pre-miR-BART22 with a size around 70 nt was confirmed by Northern blot with a miR-BART22 complementary oligonucleotide probe (right). The representative result from at least three individual blots is shown in panel C.