Figure 2. Knockdown of NIR resulted in inhibition of 18S, 28S and 5.8S rRNA processing.

A. Schematic representation of rRNA processing pathways in HeLa cells. B. U2OS cells were transfected with a NIR-specific siRNA (siNIR-1) or luciferase siRNA (siLC) as a control. At 72 hrs post-transfection, cell lysates were prepared. Proteins from the lysates were separated on SDS-PAGE, transferred onto a PVDF membrane. Blot was probed for detection of NIR protein (upper panel). NS, non-specific band. Above siRNA transfected U2OS cells were labeled with L-[methyl-³H] methionine and RNA was extracted at indicated time points. Equal amount of RNA from each sample was resolved on a 1% agarose gel. The gel was stained with ethidium bromide (EB) for photography (lower left panel) and transferred to a membrane for autoradiography (lower right panel). C. U2OS cells were transfected with siRNAs as in B. At 72 hrs post-transfection, cells were labeled with 3 µCi/ml [5, 6-³H] uridine for 30 min. RNA was extracted at indicated time points. Equal amount of RNA from each sample was resolved on a 10% polyacrylamide/7.5 M urea gel. The gel was stained with ethidium bromide (EB) for photography (left panel) and transferred to a membrane for autoradiography (right panel). D. U2OS cells were transfected with a second NIR-specific siRNA (siNIR-2) or luciferase siRNA (siLC) as a control. At 72 hrs post-transfection, cell lysates were prepared and subjected to Western blotting for detection of NIR protein (upper panel). Beta-actin was used as a loading control. Above siRNAs (siNIR-2 and siLC) transfected U2OS cells were labeled with L-[methyl-³H] methionine and RNA was extracted at indicated time points. Equal amount of RNA from each sample was resolved on a 1% agarose gel. The gel was stained with ethidium bromide (EB) for photography (lower left panel) and transferred to a membrane for autoradiography (lower right panel).