Telomerase RNA level limits telomere maintenance in X-linked dyskeratosis congenita - PubMed (original) (raw)
Telomerase RNA level limits telomere maintenance in X-linked dyskeratosis congenita
Judy M Y Wong et al. Genes Dev. 2006.
Abstract
Dyskeratosis congenita (DC) patients suffer a progressive and ultimately fatal loss of hematopoietic renewal correlating with critically short telomeres. The predominant X-linked form of DC results from substitutions in dyskerin, a protein required both for ribosomal RNA (rRNA) pseudouridine modification and for cellular accumulation of telomerase RNA (TER). Accordingly, alternative models have posited that the exhaustion of cellular renewal in X-linked DC arises as a primary consequence of ribosome deficiency or telomerase deficiency. Here we test, for the first time, whether X-linked DC patient cells are compromised for telomerase function at telomeres. We show that telomerase activation in family-matched control cells allows telomere elongation and telomere length maintenance, while telomerase activation in X-linked DC patient cells fails to prevent telomere erosion with proliferation. Furthermore, we demonstrate by phenotypic rescue that telomere defects in X-linked DC patient cells arise solely from reduced accumulation of TER. We also show that X-linked DC patient cells averted from premature senescence support normal levels of rRNA pseudouridine modification and normal kinetics of rRNA precursor processing, in contrast with phenotypes reported for a proposed mouse model of the human disease. These findings support the significance of telomerase deficiency in the pathology of X-linked DC.
Figures
Figure 1.
TERT expression activates telomerase in DC patient and control cell cultures. (A) Whole-cell extracts were assayed for telomerase activity by TRAP. Whole-cell extracts were prepared from control cells expressing wild-type dyskerin (WT) or DC patient cells expressing ΔL37 dyskerin, each with or without an integrated retrovirus expressing TERT. Extracts were assayed using a series of 2.5-fold steps in amount of total protein (5–0.13 μg). (B,C) Genomic DNA was isolated from successive harvests of the parental primary cell cultures prior to their senescence (−TERT) and from the primary cells expressing TERT over ~50 PDL of continuous growth (+TERT). Digested DNA was analyzed by in-gel hybridization with a telomeric-repeat probe. The migration of standards is indicated in kilobases.
Figure 2.
Mature, functional TER can be produced from a retroviral expression vector. (A) Schematic showing the pBABE-U3-TER expression construct. An SV40 promoter (pSV40) drives expression of the selectable marker. The U3-TER cassette was cloned into the 3′ long terminal repeat (LTR) and becomes duplicated during retroviral integration. (B) Northern blot showing the accumulation of mature TER. Each lane contains nuclear RNA isolated from 3 × 106 patient cells expressing TERT and either TER expression vector or empty vector. The TER doublet derives from differential folding of mature RNA. LC denotes an internal control for RNA loading. (C) Whole-cell extracts were assayed for telomerase activity by TRAP. Extracts were prepared from DC patient or control cells expressing TERT, either with or without additional TER. Extracts were assayed using a series of 2.5-fold steps in amount of total protein (2–0.05 μg).
Figure 3.
TER expression rescues the DC telomere maintenance defect. (A,C) Genomic DNA was isolated from DC patient or control cells expressing TERT, either with or without additional TER. Digested DNA from cells at the indicated PDL of continuous culture was analyzed by in-gel hybridization with a telomeric-repeat probe. The migration of standards is indicated in kilobases. (B,D) Growth rates of the DC patient or control cell cultures expressing TERT with or without additional TER are compared by cumulative PDL with time.
Figure 4.
Rescue of telomere length can be obtained using a single retrovirus. (A) Schematic of the dual-expression vector, pBABE-TERT/TER, for expression of both TERT and TER. (B) Whole-cell extracts were assayed for telomerase activity by TRAP. Extracts were prepared from DC patient cells expressing TERT with or without additional TER. Extracts were assayed using a series of fivefold steps in amount of total protein (5–0.008 μg). (C) Genomic DNA was isolated from DC patient cells expressing TERT with or without additional TER. Digested DNA from cells at the indicated PDL of continuous culture was analyzed by in-gel hybridization with a telomeric-repeat probe. The migration of standards is indicated in kilobases.
Figure 5.
TERT and TER expression rescue telomere length in other X-linked DC patient cells. (A) Whole-cell extracts were assayed for telomerase activity by TRAP. Extracts were prepared from DC patient cells (with A386T dyskerin) expressing TERT with or without additional TER. Extracts were assayed using a series of fivefold steps of total protein (5–0.008 μg). (B) Genomic DNA was isolated from DC patient cells (with A386T dyskerin) expressing TERT with or without additional TER. Digested DNA from cells at the indicated PDL of continuous culture was analyzed by in-gel hybridization with a telomeric-repeat probe. The migration of standards is indicated in kilobases.
Figure 6.
X-linked DC patient cells do not have altered rRNA pseudouridine content. (A) Radiolabeled 18S rRNA (shown) and 28S rRNA (not shown) were purified and hydrolyzed to mononucleotides. Mononucleotides were resolved by thin-layer chromatography, and their relative amounts were quantified using PhosphorImager analysis. Note that pseudouridine monophosphate (Ψp) is present at ~10% the level of uridine monophosphate (Up). (B) Values were averaged across repeated trials of the experiment described in A. Each calculation used ratios determined in the four to seven independent experiments that generated well-resolved spots of pseudouridine and uridine. Values for 18S rRNA from left to right were WT + TERT = 0.101 + 0.020, ΔL37DC + TERT = 0.098 + 0.016, ΔL37DC + TERT + TER = 0.096 + 0.016, and A386TDC + TERT = 0.092 + 0.018. Values for 28S rRNA from left to right were WT + TERT = 0.085 + 0.010, ΔL37DC + TERT = 0.077 + 0.017, ΔL37DC + TERT + TER = 0.089 + 0.020, and A386TDC + TERT = 0.093 + 0.022.
Figure 7.
X-linked DC patient cells do not show altered kinetics of rRNA precursor processing. Pulse-chase analysis of rRNA precursor processing was performed by isolating total RNA from parallel plates of a cell culture starting at 0 min of chase and taking additional chase time points each 15 min. Total RNA was resolved by denaturing gel electrophoresis. The 45S precursor and 41S intermediate contain the 28S, 5.8S, and 18S rRNAs. The 32S processing intermediate contains the 28S and 5.8S rRNAs only. To normalize for differential recovery of RNA from each independently harvested plate of cells, the kinetics of processing must be evaluated as a change in ratio of the signal intensities in a lane over time.
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