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. 2004 Sep 8;32(16):4776–4785. doi: 10.1093/nar/gkh824

Figure 5.

Figure 5

Inhibition of ExoT or Dicer cleavage of pre-miR-30 by Exp5 in vitro. (A) This assay measures the ability of ExoT to remove the 3′ overhang present on the pre-miR-30(19) RNA probe (see Figure 2) in the presence or absence of Exp5 and Ran-GTP. ExoT cleavage assays were performed according to Materials and Methods. For lanes 1, 5, 6 and 10, RNAs were directly mixed with 2× loading buffer (98% formamide, 10 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol) at the indicated time points, without going through extraction or precipitation. Lower RNA levels in lanes 2–4, 7–9 resulted from sample loss during extraction and precipitation. Shown on the left-hand side of the autoradiograph are DNA size markers. (B) Dicer was incubated with radiolabeled pre-miR-30 in the absence (lanes 3, 5 and 7) or presence (lanes 4, 6 and 8) of Exp5/RanQ69L-GTP. At the indicated time points, reactions were stopped and RNA isolated. Lane 1, pre-miR-30 without Dicer, no incubation at 37°C; lane 2, pre-miR-30 without Dicer, after incubation at 37°C. Positions of DNA markers are shown on the right-hand side of the autoradiograph. After quantification, the fraction of product formation was calculated as product intensity/(substrate intensity + product intensity) and is presented below the autoradiograph. Squares represent reactions in the absence of Exp5/Ran-GTP, while triangles represent reactions in the presence of Exp5/Ran-GTP.