Tbl3 encodes a WD40 nucleolar protein with regulatory roles in ribosome biogenesis

Figure 5 Effects of transducin β-like 3 knockdown on rRNA levels. A: Specific knockdown of transducin β-like 3 (Tbl3) by small hairpin RNAs (shRNAs). MRPO cells were transfected with pMKO (negative control) vs pMKO-Tbl3 shRNA and selected with puromycin to establish stable transfectants. Ten μg of total RNAs of stable transfectants were subjected to Northern analysis using a ³²P-labeled Tbl3 probe. The knockdown effect is 50%-70%. Middle panel: The same blot probed with a β-actin probe. Bottom panel: ethidium bromide-stained gel showing equal loading. The positions of the 28S and 18S rRNA are indicated; B: Tbl3 knockdown increases the level of newly synthesized 47S pre-rRNA but has no discernible effect on rRNA processing. MPRO stably transfected with pMKO (negative control) or pMKO-Tbl3 shRNA were pulse labeled with ³H-uridine for 30 min, washed and chased for 0, 30, 60, 90 and 180 min with fresh medium without ³H-uridine. Total RNAs were purified, electrophoresed and Northern blotted. ³H-uridine-labeled rRNAs were visualized by fluorography. Tbl3 knockdown consistently increases the level of the 47S pre-rRNA by about 2-4 fold. Two negative controls (Neg-1 and Neg-2) are included to show the range of variation in signal strength in the negative control group. Bottom panel: Ethidium bromide-stained gel showing similar steady-state levels of 28S and 18S rRNAs (and the 5.8S rRNA; not shown). Each lane contained total RNAs purified from equal numbers of starting cells. No adjustment was made on the basis of RNA concentration or yield; C: An independent Tbl3 knockdown experiments similar to that described in B but the analysis was performed after 0, 90 and 150 minutes of chase. The 41S and 36S are better visualized in this blot. Bottom panel: Ethidium bromide-stained gel showing similar steady-state levels of 28S and 18S rRNAs (and the 5.8S rRNA; not shown). Each lane contained total RNAs purified from equal numbers of starting cells. No adjustment was made on the basis of RNA concentration or yield; D: Time course of disappearance of ³H-uridine-labeled 47S pre-rRNAs. The fluorograph in B was analyzed and the measured signal volume was plotted against time. Note that ³H-uridine incorporation peaked earlier (30 min vs 60 min) in the shRNA group, consistent with the notion that tbl3 knockdown increased the rate of pre-rRNA synthesis. There is no evidence of delay of processing of the 47S pre-rRNA in the shRNA group during the most relevant (60-90 min) or later (90-180 min) period judging from the slopes of decline.