Telomerase RNA level limits telomere
maintenance in X-linked
Judy M.Y. Wong1and Kathleen Collins2
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA
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.
[Keywords: Telomerase RNA; telomere length; dyskeratosis congenita; dyskerin; ribosomal RNA;
Supplemental material is available at http://www.genesdev.org.
Received July 31, 2006; revised version accepted August 28, 2006.
Limits on the renewal capacity of the human hemato-
poietic system can have negative consequences for
health and longevity (Chen 2005). Despite recent im-
provements in the success of stem cell transplantation as
a bone marrow failure therapy, attaining clinical rescue
of hematopoietic proliferative capacity is far from rou-
tine (MacMillan and Wagner 2005; Steward and Jarisch
2005). Some insights about the requirements for long-
term cellular renewal have come from studies of inher-
ited bone marrow failure syndromes such as dyskeratosis
congenita (DC) and Fanconi anemia (Federman and Saka-
moto 2005). Early studies revealed that cultured cells
from Fanconi anemia patients have enhanced sensitivity
to DNA damaging agents, foreshadowing roles for the
proteins altered in Fanconi anemia in DNA damage re-
pair (Niedernhofer et al. 2005; Kennedy and D’Andrea
2005). In comparison, despite their elevated frequency of
chromosome aberrations, cultured cells from DC pa-
tients have apparently normal capacity for repair of in-
duced DNA damage (Dokal 2000). This distinct cellular
phenotype of DC implied a novel molecular defect asso-
ciated with hematopoietic failure.
DC has X-linked, autosomal dominant (AD), and au-
tosomal recessive patterns of inheritance (Mason et al.
2005; Walne et al. 2005). X-linked DC arises from amino
acid substitutions in a protein termed dyskerin (Heiss et
al. 1998), while AD DC arises from mutations in the
locus encoding telomerase RNA (Vulliamy et al. 2001a).
Dyskerin, also known as Cbf5p and NAP57, is one of
several enzymes responsible for post-transcriptional
modification of uridine to pseudouridine in noncoding
RNA (Ferre-D’Amare 2003). DC-linked dyskerin iso-
forms typically have single amino acid substitutions not
predicted to influence catalysis (Mason et al. 2005;
Walne et al. 2005). Target sites of dyskerin-mediated
pseudouridine modification are determined by its assem-
bly as a larger ribonucleoprotein complex (RNP) with
three other proteins, NOP10, NHP2, and GAR1, and one
member of a family of small RNAs harboring the hair-
pin/Hinge/hairpin/ACA (H/ACA) motif (Henras et al.
2004; Meier 2005). In RNP form, H/ACA-motif small
1Present address: Faculty of Pharmaceutical Sciences, University of Brit-
ish Columbia, Vancouver B.C., Canada V6T 1Z3.
E-MAIL firstname.lastname@example.org; FAX (510) 643-6334.
Article published online ahead of print. Article and publication date are at
2848GENES & DEVELOPMENT 20:2848–2858 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org
nucleolar RNAs (snoRNAs) hybridize to ∼100 sites on
ribosomal RNAs (rRNAs), and other H/ACA-motif
RNAs hybridize to some sites on spliceosomal small
nuclear RNAs (Ofengand et al. 2001; Kiss et al. 2004).
Dyskerin has another function evolved only in verte-
brate species. The telomerase RNP elongates chromo-
some ends by addition of telomeric simple sequence re-
peats, counteracting the repeat loss inherent in genome
replication (Chan and Blackburn 2004; Autexier and Lue
2006; Collins 2006). Vertebrate telomerase RNAs (TERs)
incorporate an H/ACA motif that is crucial for process-
ing and accumulation of mature TER from a precursor
transcript (Mitchell et al. 1999; Chen et al. 2000). The
human TER H/ACA motif assembles with dyskerin, the
other H/ACA-motif-binding proteins, and unknown fac-
tor(s) not shared with other H/ACA-motif RNPs
(Mitchell et al. 1999; Dragon et al. 2000; Mitchell and
Collins 2000; Pogacic et al. 2000; Fu and Collins 2003).
Regions of TER flanking the H/ACA motif then recruit
telomerase reverse transcriptase (TERT) to generate an
active enzyme (Mitchell and Collins 2000; Chen and
Greider 2004). Most human somatic cells accumulate
TER but not TERT and correspondingly lose telomere
length with proliferation, until a defect in telomere
structure triggers apoptosis or cellular senescence. Only
a few human cell types are thought to activate telomer-
ase for telomere elongation, including cells in the germ-
line, rapidly dividing epithelial and lymphoid progenitor
cells and most cancers (Collins and Mitchell 2002; For-
syth et al. 2002; Wong and Collins 2003). Normal human
somatic cells only transiently activate telomerase, but
studies in cultured cells indicate that even transient
telomerase activation can dramatically extend prolifera-
tive capacity (Steinert et al. 2000).
The defective cellular process underlying bone mar-
row failure in X-linked DC remains controversial.
X-linked DC patient lymphoblasts and fibroblasts harbor
much shorter telomeres and lower TER levels than the
same cell types isolated from unaffected carriers of dis-
ease (Mitchell et al. 1999). Additional studies using pe-
ripheral blood mononuclear cells support the generality
of short telomeres and reduced TER as hallmarks of
X-linked DC (Vulliamy et al. 2001b; Wong et al. 2004).
Also, heterozygous expression of truncated or altered
TER can lead to AD inheritance of DC (Vulliamy et al.
2001a, 2006). These observations suggest that DC arises
from a partial loss of telomerase function. However,
previous studies stop short of addressing whether a
modest reduction in TER actually compromises telo-
merase function on telomere substrates. Indeed, TERT
rather than TER is generally considered to be limiting
for telomerase activation in human somatic tissues.
Even in human cancer cells, disruption of one copy of the
TERT gene is sufficient to impose telomere shortening
(Hauguel and Bunz 2003).
X-linked DC has also been attributed to a defect in
ribosome biogenesis (Ruggero et al. 2003; Meier 2005;
Yoon et al. 2006). Support for this hypothesis derives
mainly from a proposed mouse model of X-linked DC
(Ruggero et al. 2003), which shows some similarities and
some disparities of phenotype with the human disease
(Bessler et al. 2004). The mouse model was created by
integration of a targeting construct 9 kb downstream
from Dkc1 in the neighboring Mpp1 gene, which reduced
dyskerin mRNA by fourfold (males) or twofold (hetero-
zygous females) in cultured embryo fibroblasts. B-lym-
phocytes from the mouse model have reduced levels of
TER and telomerase activity but unaltered telomere
lengths. Instead of critically short telomeres, defects in
ribosome biogenesis were detected. A substantial de-
crease in the pseudouridine content of mature 18S and
28S rRNAs was evident in six of eight independent lym-
phocyte pools, along with a dramatic kinetic delay in
rRNA precursor processing (Ruggero et al. 2003). These
biogenesis defects are proposed to underlie the impair-
ment of IRES-mediated translation that has been re-
ported for cells from the mouse model as well as human
patients (Yoon et al. 2006). However, the premature telo-
mere shortening and heterogeneous senescence charac-
teristic of all commercially available X-linked DC pa-
tient primary cell cultures is a caveat to conclusions
about ribosome biogenesis, because the altered growth
properties can impact ribosomes independently of the
DC genetic lesion. This caveat also applies to previous
ribosome biogenesis studies that reported normal pseu-
douridine modification at specific residues of rRNA in
X-linked DC patient cells (Mitchell et al. 1999).
Here, for the first time, we test whether X-linked DC
patient cells and family-matched control cells differ in
their capacity to support telomerase function at telo-
meres. We show that telomerase activation in X-linked
DC patient cells does not allow telomere elongation or
stable telomere length maintenance, in contrast to re-
sults from control cells cultured in parallel. Strikingly,
we find that this X-linked DC telomere maintenance
defect can be rescued by an increase in TER. Also, for the
first time, we used long-term nonsenescent cultures of
X-linked DC patient cells to investigate putative defects
in ribosome biogenesis. We find that X-linked DC pa-
tient cells from two different families have normal levels
of rRNA pseudouridine modification and no dramatic
kinetic delay in rRNA precursor processing. These re-
sults support the significance of telomerase deficiency as
a mechanism of X-linked DC, and suggest future evalu-
ation of telomerase activation as a disease therapy.
TERT expression in DC patient cells produces
catalytically active telomerase
Primary dermal fibroblast cultures from an X-linked DC
patient and his asymptomatic maternal grandmother
were obtained with minimal prior passage in culture (see
Materials and Methods). The DC patient cells express a
dyskerin variant lacking leucine at position 37, desig-
nated ?L37 dyskerin, and the family-matched control
cells express only wild-type dyskerin despite carrying a
mutant allele (Mitchell et al. 1999). The DC patient pri-
Telomerase RNA limits telomere length
GENES & DEVELOPMENT2849
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