The catalytic activity of Dyskerin is essential for rRNA processing

Inactivation of the catalytic activity of Dyskerin did not affect the accumulation of the tested H/ACA snoRNAs (Figure 3.11). This is different from yeast where the catalytic activity of Dyskerin is required for both H/ACA snoRNP accumulation and rRNA processing (Zebarjadian et al., 1999). This, therefore, provided us with the opportunity to investigate whether the catalytic activity of Dyskerin is important for human ribosome biogenesis. The endogenous protein was replaced by the WT or the D125A FLAG- tagged Dyskerin in HEK293T cells as previously, and HEK293T cells containing the empty pcDNA5 vector were used as a control. RNA was extracted and loaded on a glyoxal-agarose gel and analysed by Northern Blotting using radiolabelled (32_{P) probes} for 5’-ITS1, ITS1 or ITS2 regions (Figure 3.12A).

Figure legend on the next page.

Knockdown of Dyskerin in HEK293T cells resulted in an accumulation of the 32S rRNA precursor, whereas the levels of 41S, 26S, 21S and 18SE rRNA precursors were significantly decreased (Figure 3.12, vector). The levels of the 41S, 21S and 18SE rRNA precursors were restored to normal in HEK293T expressing the RNAi resistant WT FLAG-tagged Dyskerin after Dyskerin knockdown (Figure 3.12B, C, WT). Furthermore, the levels of 30S rRNA remained unaffected after Dyskerin knockdown in these cells as compared to the control, whereas the levels of 26S rRNA precursor were slightly increased (Figure 3.12B, C, WT). Since no significant change was observed in the levels of 21S rRNA, it is unlikely that 26S rRNA accumulation is indicative of rRNA processing defects here. Expression of the RNAi resistant WT FLAG-tagged Dyskerin rescued the levels of the increased levels of 32S rRNA precursor after Dyskerin knockdown as compared to the control, whereas the levels of the 12S rRNA were not significantly altered (Figure 3.12 B, D, WT). These data indicated that expression of the RNAi resistant WT FLAG-tagged Dyskerin rescued the rRNA processing phenotype caused by Dyskerin knockdown in HEK293T cells.

Expression of the RNAi resistant D125A FLAG-tagged Dyskerin resulted in a very slight increase in 41S rRNA precursor levels after Dyskerin knockdown as compared

Figure 3.12. Inactivation of the catalytic activity of Dyskerin results in LSU late processing defects. (A) Schematic representation of the rRNA precursors in humans.

The SSU precursors and mature rRNA are shown in green and the LSU precursors and mature rRNA are shown in blue. The cleavage sites are shown at the top and the 5’ITS1, ITS1 and ITS2 sites recognized by the radiolabeled (32_{P) probed are shown in} red (Adapted from (Sloan et al., 2013c)) (B) Knockdowns using Control or Dyskerin (DKC-2) siRNAs were performed for 48h in HEK293T cells containing the empty pcDNA5 vector or expressing the RNAi resistant FLAG-tagged WT or D125A mutant Dyskerin. 100ng/µl or 1000ng/µl tetracycline was used for 48h in WT-expressing cells or pcDNA5 and D125A-expressing cells respectively. RNA was extracted from the cells and separated on a 1.2% glyoxal-agarose gel. The RNA was transferred on a Hybond N membrane and incubated with radiolabeled-(32_{P)-oligo probes against ITS1, 5’ITS1} (18SE) or ITS2, as indicated on the right of each membrane. A phosphorimager was used for visualization of the rRNAs, and ethidium bromide staining of 28S rRNA (UV) was used as a loading control. (C-D) ImageQuant software was used for quantitation of the northern blots. The graphs represent the relative levels of the rRNAs as averages of three independent experimental repeats, which were normalized to the loading control (28S UV) and the control knockdown in each case. Further normalization of the rRNA precursor levels to the 47/45S rRNA precursor was performed. The error bars represent the standard error (+/-SEM) and statistical analysis took place using an unpaired t-test. Lack of significance values indicates significant differences as compared to the control and the dotted line represents the rRNA precursor levels in control cells. The vector values are the same as the ones presented on Figure 3.6D. *p value < 0.05, **p value < 0.01, ***p value < 0.001.

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to the control, but no change was observed in the levels of 30S or 26S rRNA precursors (Figure 3.12B, C, Mut). Importantly, the levels of 21S and 18SE rRNAs were significantly decreased by approximately 50% and 30% respectively after Dyskerin knockdown in HEK293T cells expressing the RNAi resistant D125A FLAG-tagged Dyskerin as compared to the control (Figure 3.12B, C, Mut), as well as the levels of the 12S rRNA precursors, which were approximately 40% lower (Figure 3.12B, D, Mut panel). No significant difference was observed in the levels of 32S rRNA precursor after Dyskerin knockdown as compared to the control in HEK293T cells expressing the RNAi resistant D125A FLAG-tagged Dyskerin (Figure 3.12B, D, Mut panel). Taken together, these data indicate that inactivation of the catalytic activity of Dyskerin rescues the early rRNA processing defects observed when Dyskerin is knocked-down, but results in late rRNA processing defects.

I next used pulse-chase labelling to further characterize the effects of the catalytically inactive Dyskerin on rRNA production. HEK293T cells containing the empty pcDNA5 vector were used as a control and the endogenous protein was replaced by the WT or D125A mutant FLAG-tagged Dyskerin as previously. The cells were then incubated with phosphate-free media for 1h, followed by the addition of radiolabelled phosphate (32_{P) for 1h. The cells were left to grow in normal media for 3h (chase) for detection of} the newly-synthesized rRNAs. RNA was extracted, separated on an agarose gel and transferred on a Hybond N membrane. As seen previously (Figure 3.5), knockdown of Dyskerin in HEK293T cells containing the empty pcDNA5 vector resulted in a decrease in 47/45S and 32S rRNA precursors as well as a decrease in the newly-synthesized 28S and 18S rRNA after 3h of chase (Figure 3.13A, B, vector). Expression of the RNAi resistant WT FLAG-tagged Dyskerin after Dyskerin knockdown rescued the levels of the 47/45S and 32S rRNA precursors and the levels of the newly-synthesized 28S and 18S rRNAs as compared to the control knockdown in these cells after 3h (Figure 3.13A, B WT). A slight accumulation of the 47/45S rRNA precursor was observed after expression of the RNAi resistant WT FLAG-tagged Dyskerin (Figure 3.13A, B, WT), which might be due to experimental variation. Taken together, these data further support the previous data, showing that expression of the RNAi resistant WT FLAG- tagged Dyskerin rescues the rRNA processing phenotype caused by Dyskerin knockdown. It is worth noting that this experiment was only performed twice due to time limitations. However, since the levels of the H/ACA snoRNAs (Figure 3.11) and rRNA precursors (Figure 3.12) were rescued after expression of the RNAi resistant WT

FLAG-tagged Dyskerin in cells where Dyskerin was knocked down, I am confident that the RNAi rescue system worked as expected.

Knockdown of Dyskerin in HEK293T cells expressing the RNAi resistant D125A mutant FLAG-tagged Dyskerin resulted in approximately 35% and 40% decrease in the levels of the 47/45S and 32S rRNA precursors respectively, and approximately 40% and 45%

Figure 3.13. Inactivation of the catalytic activity of Dyskerin is likely to result in slower accumulation of the newly-synthesized LSU and SSU rRNAs. (A)

Knockdowns using Control and Dyskerin (DKC-2) siRNAs were performed for 48h in HEK293T cells containing the empty pcDNA5 vector or expressing the RNAi resistant FLAG-tagged WT or D125A mutant Dyskerin using 100ng/µl or 1000ng/µl tetracycline respectively for 48h. 1000ng/µl tetracycline was used in pcDNA5-containing cells for 48h. During pulse chase labelling, phosphate depletion was performed followed by addition of radiolabelled phosphate (32_{P) for 1h. The cells were left to grow under} normal conditions for 3h (chase), before RNA was extracted and separated on a glyoxal-agarose gel. The RNA was transferred on a Hybond N membrane and visualized using a Phosphorimager. The 28S rRNA levels after ethidium bromide staining (UV) were used as a loading control. (B) ImageQuant software was used for quantitation of the Northern blots. The graph represents the relative rRNA levels of one experimental repeat after Dyskerin knockdown and normalization against the loading control (28S UV) and the control knockdown for each cell line. The dotted line represents the rRNA precursor levels in control cells. (C) Schematic representation of the rRNA processing precursors and mature rRNAs in humans. The cleavage sites are indicated on the top (Adapted from (Sloan et al., 2013c)).

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decrease in the levels of the newly-synthesized 28S and 18S rRNAs respectively, as compared to the control knockdown after 3h of chase (Figure 3.13A, B, Mut). These data indicate that inactivation of the catalytic activity of Dyskerin causes a slower accumulation of the newly-synthesized rRNAs, which further supports the theory that it is required for late rRNA processing in humans.