Fig 1: Deprotection of hTR results in degradation and not accumulation of poly(A) or 3′ extended hTR species. Following siRNA targeting of dyskerin (sidkc1.A and sidkc1.B), qPCR analysis revealed that (A) total and poly(A) hTR levels are both reduced relative to untreated HEK293 cells. The levels of the C/D RNA U3 are not reduced by depletion of dyskerin. This has been performed in experimental replicate n = 3. Additionally, (B) the accumulation of 3′ extended hTR precursors was examined by PCR following knockdown of endogenous dyskerin (sidkc1.B), or the NEXT/hTRAMP complex component MTR4 (siMTR4 - a positive control for defective processing of hTR). The expected 288 nt product observed with cDNA from HEK293 cells depleted of MTR4 (indicated by an asterisk) was not observed in cells depleted of dyskerin, with or without exogenous WT and dyskerin variants (middle panel: F2 + R1). GAPDH amplification was used as a positive control (bottom panel), and no amplification of genomic contamination was observed (top panel: F1 + R1), indicating that the amplified product is a 3′ extended hTR transcript. This was repeated in experimental replicate n = 2, and a representative image is shown. Double depletion of endogenous dyskerin and (C) PARN or (D) RRP40 from HEK293 cells with siRNA (sidkc1.B and siPARN, or sidkc1.B and siRRP40), as well as expression of FLAG-tagged constructs (the higher molecular weight band detected by αDyskerin) was assessed by immunoblotting following 96h of siRNA treatment. Total hTR levels were assessed from HEK293 cells with stable expression of FLAG-tagged wildtype or variants of dyskerin by qPCR following 96 h of siRNA treatment, relative to empty vector HEK293 cells (mock) lacking FLAG-tagged dyskerin and untreated with siRNA. Mock cell hTR level is indicated by the black dashed line, while the red dashed line indicates 50% of mock cell hTR level. Empty vector HEK293 cells treated with scramble siRNA were used to control for possible effects of siRNA transfection. These data represent experimental replicates of n = 3. No statistically significant reductions in RNA enrichment compared to WT + siPARN + sidkc1 were observed. Statistically significant reductions in RNA enrichment compared to WT + siRRP40 + sidkc1 treated cells are indicated by * (P value < 0.01). Error bars represent SEM.