biomolecules

Review
Human Telomerase RNA: Telomerase Component
or More?
Maria Rubtsova 1,2, * and Olga Dontsova 1,2,3, *
1 Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow
State University, 119992 Moscow, Russia
2 Center of Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo, 143026 Moscow, Russia
3 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
117997 Moscow, Russia
* Correspondence: mprubtsova@gmail.com (M.R.); olga.a.dontsova@gmail.com (O.D.)




Received: 18 April 2020; Accepted: 5 June 2020; Published: 6 June 2020


Abstract: Telomerase is a ribonucleoprotein complex that maintains the lengths of telomeres.

Most studies of telomerase function have focused on the involvement of telomerase activation in the
immortalization of cancer cells and cellular rejuvenation. However, some studies demonstrated that
the results do not meet expectations for telomerase action in telomere maintenance. Recent results
give reason to think that major telomerase components—the reverse transcriptase protein subunit and
telomerase RNA—may participate in many cellular processes, including the regulation of apoptosis
and autophagy, cell survival, pro-proliferative effects, regulation of gene expression, and protection
against oxidative stress. However, the difficulties faced by scientist when researching telomerase
component functions often reduce confidence in the minor effects observed in experiments. In this
review, we focus on the analysis of the functions of telomerase components (paying more attention
to the telomerase RNA component), both as a complex and as independent components, providing
effects that are not associated with telomerase activity and telomere length maintenance. Despite the
fact that the data on alternative roles of telomerase components look illusory, it would be wrong to
completely reject the possibility of their involvement in other biological processes excluded from
research/discussion. Investigations to improve the understanding of every aspect of the functioning
of telomerase components will provide the basis for a more precise development of approaches to
regulate cellular homeostasis, which is important for carcinogenesis and aging.

Keywords: telomerase; ribonucleoprotein particles; alternative function; telomere

1. Introduction
Special structures known as telomeres are located at the ends of linear chromosomes and protect
them from shortening and fusion [1]. Telomeres shorten due to the end replication problem [2]
and nucleolytic degradation [3]. Telomerase reverse transcriptase (TERT) uses a telomerase RNA
(TERC—telomerase RNA component) as a component of the telomerase complex and a template
to add telomeric repeats to the 30 -end of chromosomal DNA [4–7]. Telomerase RNA provides the
structural scaffold for telomerase complex assembly [8]. Telomere length defines the cell’s lifespan,
and critically short telomeres promote the activation of DNA damage signaling, resulting in cell
death [9–11]. Elongation of telomeres by telomerase provides unlimited proliferative potential for the
cell [12,13]. Telomerase activity is inherent to stem, germ, and the majority of cancer cells. Traditionally,
telomerase investigations have been associated with cancer and aging. However, the expression of
hTERT has been demonstrated for cells with active telomerase while hTERC gene expression is not
correlated with telomerase activity [14]. hTERC has been detected in the majority of somatic cells where

Biomolecules 2020, 10, 873; doi:10.3390/biom10060873 www.mdpi.com/journal/biomolecules

Biomolecules 2020, 10, 873 2 of 15

it is expressed constitutively. Mutations in hTERT and hTERC genes are involved in the development
of diseases linked to dysfunctional telomeres, such as aplastic anemia, the autosomal dominant form
of dyskeratosis congenita [15–17], and idiopathic pulmonary fibrosis [18]. This review is focused on
different aspects of telomerase ribonucleoprotein (RNP) components functioning in both the complex
and separately, with special attention paid to the RNA component.

2. Structure of Human Telomerase RNP

Catalytically-active RNP purified from cells as a complex contains TERT, TERC, dyskerin (DKC),
NOP10, NHP2, GAR1, and TCAB1 [8]. DKC1, NOP10, NHP2, and GAR1 are shared by telomerase
and the small nucleolar (sno) RNPs, and TCAB1 is a component of small Cajal body (sca) RNPs.
snoRNP and scaRNP catalyze ribosomal and spliceosomal RNA modifications, respectively. However,
telomerase activity may be reconstituted in vitro by the complex of two major components: TERT and
TERC. hTERC contains 451 nucleotides (nts) that form several structural domains (Figure 1A).
However, only two domains are necessary for telomerase activity to occur. Pseudoknot containing
the template region (t/PK) and H/ACA-domain of hTERC expressed separately are able to reconstruct
the minimally catalytically-active telomerase enzyme that is capable of synthesizing the telomere
repeats (Figure 1A). t/PK possesses the template for telomere synthesis and is involved in association
with hTERT. Interestingly, the presence of a phylogenetically-conserved hairpin in equilibrium with
the pseudoknot in the telomerase pseudoknot domain, which is stabilized by a unique uridine helix,
has been demonstrated. A functionally important interconversion between the hairpin and pseudoknot
conformations was proposed [19]. The H/ACA domain of hTERC shared with snoRNAs is important
for hTERC biogenesis as well as for telomerase activity. It contains CR4/5 (conservative regions 4 and
5) which are responsible for the binding of hTERT and CAB-box, providing the signal to localization in
Cajal bodies similar to other known scaRNAs specific to this intranuclear compartment [20,21].
TERT contains four domains (Figure 1B): TEN-domain (telomerase N-end domain),
TRBD (telomerase RNA-binding domain), RT (reverse transcriptase domain), and CTD (C-end
domain). hTERC binding to H/ACA-proteins is the first step of human telomerase RNP assembly.
Two hetero-tetramers of H/ACA proteins interact with the H/ACA-domain of hTERC in different
positions. One molecule of DKC1 attracts the H/ACA-complex to attach to the P4 stem of hTERC.
The other dyskerin molecule strongly interacts with the P7 stem of hTERC, attracting NOP10, NHP2,
and TCAB1 to the P8 stem (Figure 1) [8].
The interaction of TCAB1 is absolutely necessary for the assembly of the catalytically-active
telomerase complex. Binding of TCAB1 with hTERC promotes the formation of the tertiary structure
of the CR4/5-domain, which is preferential for the association with hTERT. The absence of TCAB1 or
mutations of TCAB1 or in the CR4/5-domain of hTERC disturb the interaction of hTERT with hTERC
and lead to a decrease in the telomerase activity and shortening of telomeres in human embryonic
stem cells [22].
The TRBD (telomerase RNA-binding domain)-domain of hTERT interacts with the t/PK and
CR4/5-domains of hTERC (Figure 1A,B) [23,24]. The P6.1 stemloop of hTERC is critical for the formation
of active enzyme. Mutations in P6.1 that disturb its secondary structure impair the interaction between
hTERT and hTERC and decrease telomerase activity, while compensatory mutations that restore the
secondary structure of this element recover the activity of telomerase [20,25]. To form the catalytic
center of the human telomerase holoenzyme, the pseudoknot and CR4/5 domain of hTERC should
wrap around hTERT (Figure 1C). PK adopts an arch-shaped structure where a triplex structure formed
from the P2b and P3 stems brings the TRBD and CTD-domains of hTERT together. The template region
of hTERC occurs nearby to the TEN-domain of hTERT that stabilizes the 30 -part of the RNA-DNA
duplex formed by telomere and hTERC [8].
Stems P5, P6 and P6.1 form the three-way junction (TWJ) element involved in the interaction
with hTERT. P6a stem located along TRBD of hTERT, while P6.1 exudes from the TWJ almost
perpendicularly to P6a located between the TRBD and CTD domains of hTERT [8]. The CTD domain
Biomolecules 2020, 10, 873 3 of 15
Biomolecules 2020, 10, 873 3 of 15

localizes opposite the P6.1 stemloop, stabilizing this structure that is important for human telomerase
activity (Figure
localizes 1) [26].
opposite the P6.1 stemloop, stabilizing this structure that is important for human telomerase
activity (Figure 1) [26].

Figure
Figure1.1.Human
Humantelomerase
telomerasestructure.
structure.(A)
(A)Secondary
Secondary structure hTERC. (B)
structure of hTERC. (B) Domain
Domainarchitecture
architectureof
ofhTERT.
hTERT.(C) (C)Cryo-EM
Cryo-EM structure
structure of of
thethe human
human telomerase
telomerase holoenzyme
holoenzyme in two
in two views
views [26][26] (Adapted
(Adapted with
with permission
permission fromfrom Elsevier).
Elsevier). Subunits
Subunits are colored
are colored as labeled.
as labeled.

3.3.Telomerase
TelomeraseAction
ActionatatTelomeres
Telomeres
Telomerelengthening
Telomere lengtheningdue duetotothe
thereverse
reversetranscriptase
transcriptaseactivity
activityofofTERT
TERTisisrecognized
recognizedasasa amajormajor
intracellular function
intracellular function of ofthe thetelomerase
telomerase complex.
complex.TERT adds adds
TERT telomere repeatsrepeats
telomere using itsusing
own telomerase
its own
RNA as a template and 3 0 -OH group of the telomere as a primer. The TEN domain of hTERT stabilizes
telomerase RNA as a template and 3′-OH group of the telomere as a primer. The TEN domain of
the RNA–DNA
hTERT stabilizesduplex formed byduplex
the RNA–DNA the template
formedregion of telomerase
by the RNA and
template region telomeric DNA
of telomerase RNAnear andto
the catalytic center of the enzyme.
telomeric DNA near to the catalytic center of the enzyme.
Telomerase RNP
Telomerase RNP intracellular
intracellular trafficking between
trafficking Cajal bodies,
between the nucleolar,
Cajal bodies, and the nucleoplasm
the nucleolar, and the
occurs during biogenesis. DKC1, NOP10, NHP2, and NAF1 interact
nucleoplasm occurs during biogenesis. DKC1, NOP10, NHP2, and NAF1 interact with hTERC co- with hTERC co-transcriptionally,
and assembled complexes
transcriptionally, and assembled rapidly transferrapidly
complexes to Cajal bodiestowhere
transfer trimethylation
Cajal bodies of the 50 -capofof
where trimethylation
hTERC 0
the 5′-capoccurs
of hTERC[27].occurs
DHX36 RNA-helicase
[27]. unfolds G-quadruplexes
DHX36 RNA-helicase formed atformed
unfolds G-quadruplexes the 5 -end of 5′-end
at the hTERC.
ofGAR1
hTERC.replaces
GAR1NAF1, andNAF1,
replaces the complex
and theofcomplex
hTERC of with H/ACA
hTERC with proteins
H/ACAinteracts
proteinswith hTERT
interacts and
with
TCAB1 to form a catalytically active enzyme [28]. Approximately 100 molecules
hTERT and TCAB1 to form a catalytically active enzyme [28]. Approximately 100 molecules of active of active telomerase
RNP are formed
telomerase RNP are in every
formed cellincycle [29,30],
every which[29,30],
cell cycle is not enough
which isto not
set up experiments
enough to set upunder conditions
experiments
of an endogenous level of expression. Indeed, the formation of active telomerase
under conditions of an endogenous level of expression. Indeed, the formation of active telomerase RNP in nucleolar
was demonstrated with the exogenous expression of hTERT and a
RNP in nucleolar was demonstrated with the exogenous expression of hTERT and a decreaseddecreased level of H/ACA proteins.
level
ofInH/ACA
absence of TCAB1
proteins. the assembly
In absence of TCAB1 of the
active telomerase
assembly RNP
of active occurs in
telomerase RNPtheoccurs
nucleus without
in the the
nucleus
activation of hTERT expression [31]. Thus, active telomerase complexes may
without the activation of hTERT expression [31]. Thus, active telomerase complexes may be formed be formed in Cajal bodies
inorCajal
nucleoplasm
bodies or(Figure 2).
nucleoplasm (Figure 2).
Biomolecules 2020, 10, 873 4 of 15

Biomolecules 2020, 10, 873 4 of 15

Figure 2.2.Multistage
Figure Multistageprocess
process of assembly
of assembly of theofactive
the active telomerase
telomerase complex. complex.
Scheme Scheme illustrating
illustrating different
different stages of telomerase complex assembly and its binding
stages of telomerase complex assembly and its binding to telomeres. to telomeres.

TCAB1 interacts with active and and inactive

inactive telomerase
telomerase complexes and promotes promotes their their trafficking
trafficking
from the nucleolar to the nucleoplasm and Cajal bodies in the G1 phase of the cell cycle [29,32,33].
Coilin dysfunction resulting in the absence of Cajal bodies does not influence the assembly assembly of of active
active
telomerase RNP, RNP, and and the
the telomere
telomere length
length in in cancer
cancer andand embryonic
embryonic stem stem cells
cells suggest the effective
telomerase with
interaction of telomerase with telomeres
telomeres in in the
the nucleoplasm
nucleoplasm [31,34].
[31,34]. Co-localization
Co-localization of of telomerase
telomerase
with telomeres
with telomeresand and Cajal
Cajal bodies
bodies clearlyclearly demonstrates
demonstrates that the that the interaction
interaction of telomerase
of telomerase with telomeres with
telomeres
occurs occurs
in the in the
S-phase of S-phase of theincell
the cell cycle thecycle in the nucleoplasm,
nucleoplasm, but not in Cajalbut not in Cajal
bodies [35].bodies [35].
The interaction of telomerase with telomeres is promoted by the TPP1 protein in vertebrates.
The structural
The structuralOB-fold
OB-folddomain
domainofofTPP1 TPP1binds
bindstotothetheTEN
TENdomain
domain ofof telomerase
telomerase reverse
reverse transcriptase
transcriptase to
to position
position RNP RNP at single
at the the single strand
strand region region of telomeres,
of telomeres, facilitating
facilitating the interaction
the interaction of the template
of the template region
region
of of telomerase
telomerase RNA with RNA thewith
endthe endchromosome
of the of the chromosome[36]. [36].
interaction of telomerase
The interaction telomerase with telomeres facilitates the addition of telomeric repeats to the the
0 -end of the telomere overhang. Telomerase catalyzes the addition of deoxynucleotide triphosphate
33′-end
(dNTPs) to
(dNTPs) to the 0 -OH group of
the 33′-OH 0 -deoxyribose of the last telomeric
of 22′-deoxyribose telomeric nucleotide by the formation formation of
phosphodiester bonds according to the template region of TERC. The efficiency
phosphodiester efficiency of of telomerase
telomerase action
may be characterized
characterizedby bytwotwotypes
typesofof processivity:
processivity: types I and
types II (Figure
I and II (Figure 3). Processivity
3). Processivity of the of type
theI
provides information concerning the number of nucleotides added
type I provides information concerning the number of nucleotides added to the end of telomerase to the end of telomerase without
telomerase–telomere
without complex complex
telomerase–telomere dissociation, which may
dissociation, occurmay
which afteroccur
the addition
after theofaddition
a single of nucleotide
a single
[37]. The [37].
nucleotide number The of telomeric
number repeatsrepeats
of telomeric addedadded to individual
to individual telomeres
telomeres without
withoutdissociation
dissociation of
telomerase characterizes
telomerase characterizes the the type
type IIII processivity
processivity of of the
the enzyme
enzyme (Figure
(Figure 3). 3). The addition of each repeat
followedby
followed bymelting
meltingofofthetheDNA–RNA
DNA–RNAduplex duplex and
and translocation
translocation of telomerase
of telomerase in ainway
a way thatthat transfers
transfers the
the template region of telomerase RNA to stimulate hybridization 0
at
template region of telomerase RNA to stimulate hybridization at the 3 -end of the telomere and correctthe 3′-end of the telomere and
correct arrangement
arrangement of the heteroduplex
of the heteroduplex in thecenter
in the active activeof center of telomerase.
telomerase. The maximal The maximal RNA–DNA-
RNA–DNA-duplex
duplex
may may be by
be formed formed by the complementary
the complementary interaction interaction of 11 nucleotides
of 11 nucleotides of telomerase of telomerase
RNA and RNA and
telomeric
telomeric DNA. The optimal DNA–RNA duplex length is
DNA. The optimal DNA–RNA duplex length is 5–6 nucleotides [38]. The additional nucleotides5–6 nucleotides [38]. The additional
nucleotides
stabilize thestabilize
structure theandstructure
prevent and theprevent themelting
effective effectiveand melting and translocation
translocation of telomerase.
of telomerase. Recent
Recent structural
structural data indicates
data indicates that the that
TENthe TEN of
domain domain
telomeraseof telomerase reverse transcriptase
reverse transcriptase limits the limits
lengththeof
length
the of the product-template
product-template heteroduplex heteroduplex
[8,39]. The [8,39].
TENThe TEN domain
domain binds tobinds to the DNA/RNA
the DNA/RNA duplexduplex at the
at the bifurcation
bifurcation point and point and regions
to some to someofregions of TERC
TERC outside the outside
DNA/RNA the duplex,
DNA/RNA as hasduplex, as has been
been demonstrated
demonstrated for Hansenula polymorpha [39] and confirmed later for human telomerase [8]. The
telomeric protein TPP1 stimulates translocation and reduces the dissociation efficiency of the
Biomolecules 2020, 10, 873 5 of 15

for Hansenula polymorpha [39] and confirmed later for human telomerase [8]. The telomeric protein
Biomolecules 2020, 10, 873 5 of 15
TPP1 stimulates translocation and reduces the dissociation efficiency of the complex of telomerase
and telomere,
complex increasing
of telomerase thetelomere,
and number of telomericthe
increasing repeats added
number during one
of telomeric act ofadded
repeats telomerase action
during one
without dissociation from the telomere [40].
act of telomerase action without dissociation from the telomere [40].

Figure 3.
Figure 3. Catalytic
Catalytic cycle
cycle of telomerase. Scheme
of telomerase. Scheme illustrating
illustrating the
the telomerase
telomerase active
active cycle
cycle and its
and its
modulation by TPP1.

Telomerase
Telomerasewas wasfirst recognized
first as a telomere-maintaining
recognized as a telomere-maintainingenzyme due to its reverse
enzyme due totranscriptase
its reverse
activity. However,
transcriptase activity.extensive
However, investigations of telomerase
extensive investigations have provided
of telomerase data detailing
have provided possible
data detailing
additional functionsfunctions
possible additional of components of the telomerase
of components holoenzyme
of the telomerase that are
holoenzyme thatnot
areassociated with
not associated
telomere lengthening.
with telomere Both Both
lengthening. majormajor
components of theoftelomerase
components complex
the telomerase may be
complex mayinvolved not only
be involved not
only
in in oncogenesis
oncogenesis but in different
but in different physiological
physiological intracellular intracellular
mechanisms, mechanisms,
such as tissue such as tissue
homeostasis,
homeostasis,
gene gene
expression, andexpression, and the stress
the stress response. response. functions
The alternative The alternative
of TERTfunctions of TERT
were reviewed were
in detail
reviewed[41,42],
recently in detail recently
so in [41,42],
this review, wesoconcentrate
in this review,
on thewenon-telomeric
concentrate on the non-telomeric
action action of
of telomerase RNA.
telomerase RNA.
4. Effects of the Expression of TERC at the Early Stages of Tumorigenesis Are Not Associated with
Telomerase
4. Effects of Activation
the Expression of TERC at the Early Stages of Tumorigenesis Are Not Associated with
Telomerase
The firstActivation
evidence that telomerase RNA can function independently of telomerase was observed in
investigations of the expression
The first evidence of telomerase
that telomerase RNA can components and the activation
function independently of telomerase
of telomerase wasfollowing
observed
tumorigenesis in mice. The activation of mTerc expression was demonstrated
in investigations of the expression of telomerase components and the activation of telomerase in two different transgenic
mice models
following of multi-stage
tumorigenesis in tumorigenesis: K14-HPV16
mice. The activation of mTercand expression
RIP-TAg2 [43].
was The K14-HPV16
demonstrated in mice
two
expressed the human
different transgenic papillomavirus
mice type 16 region
models of multi-stage in basal keratinocytes.
tumorigenesis: K14-HPV16 and This model allows
RIP-TAg2 the
[43]. The
multistage induction of squamous cell carcinomas of the epidermis to
K14-HPV16 mice expressed the human papillomavirus type 16 region in basal keratinocytes. Thisbe controlled. RIP1-TAg2
mice
modeldevelop isletmultistage
allows the cell carcinomas by a of
induction multistage
squamous process controlledofby
cell carcinomas thethe expression
epidermis of controlled.
to be the SV40 T
antigen (TAg) oncogene in pancreatic β-cells. The analysis of telomerase activity
RIP1-TAg2 mice develop islet cell carcinomas by a multistage process controlled by the expression and mTerc expression of
revealed upregulation of the telomerase RNA component at very early stages of
the SV40 T antigen (TAg) oncogene in pancreatic β-cells. The analysis of telomerase activity and mTerc tumorigenesis in
both used models,
expression revealed whereas telomerase
upregulation of activity was detected
the telomerase RNAincomponent
end-stage tumors
at very[43]. Thestages
early possible
of
alternative
tumorigenesis in both used models, whereas telomerase activity was detected in end-stage explain
function of TERC as a response to the initiation of continuous cell proliferation may tumors
the
[43].upregulation TERC expression
The possibleofalternative functionat the early stages
of TERC of tumorigenesis
as a response independent
to the initiation of telomerase
of continuous cell
activation.
proliferation However,
may explainwe cannot exclude thatofearly
the upregulation TERC overexpression
expression at of thehTERC is necessary
early stages in order to
of tumorigenesis
obtain a sufficient
independent concentration
of telomerase of this molecule
activation. However,ready for effective
we cannot telomerase
exclude that earlycomplex assembly of
overexpression at
later
hTERC stages when theinoverexpression
is necessary order to obtainofahTERT willconcentration
sufficient occur. of this molecule ready for effective
telomerase complex assembly at later stages when the overexpression of hTERT will occur.
Additional evidence of the noncanonical role of the telomerase RNA component in
tumorigenesis was observed in K5-Tert mice overexpressing TERT in basal keratinocytes from
stratified epithelia [44]. K5-Tert mice demonstrated increased susceptibility to skin tumorigenesis and
an increased rate of wound healing than wild-type mice without the telomere length being affected.
Biomolecules 2020, 10, 873 6 of 15

Additional evidence of the noncanonical role of the telomerase RNA component in tumorigenesis
was observed in K5-Tert mice overexpressing TERT in basal keratinocytes from stratified epithelia [44].
K5-Tert mice demonstrated increased susceptibility to skin tumorigenesis and an increased rate of
wound healing than wild-type mice without the telomere length being affected. Model mice were
generated in a Terc-/- genetic background to reveal the role of the telomerase complex in tumorigenesis.
An impairment of tumorigenesis in K5-Tert/Terc-/- mice in comparison to control Terc-/- mice was
observed. The authors concluded that Tert overexpression inhibits tumorigenesis in the absence of
Terc, and this effect is independent of telomerase activity and telomere length. A delayed rate of
wound healing in K5-Tert/Terc-/- mice in comparison to control Terc-/- mice indicates that Terc is necessary
for a higher wound healing rate, and this effect is independent of telomerase activity and telomere
length [44]. It is interesting that the overexpression of Tert does not promote increased wound healing
and increased tumorigenesis. Expression of Terc might be necessary at the early stages of tumorigenesis
in mammals, which correlates with the amplification of genome locus 3q26 containing Terc in human
tumors [45]. However, the data obtained in this research must be interpreted very carefully, and it is
very difficult to separate effects provoked by the telomerase components or telomere length.
The influence of telomerase RNA on the regulation of the cell growth rate was uncovered in
experiments involving the inhibition of hTERC expression by RNA interference [46]. The distinct effects
of inhibition telomerase activity and telomere shortening were observed after long-term treatment.
The depletion of the telomerase RNA component reduced the cell proliferation rate quickly, without
having a significant influence on the level of telomerase activity. At this stage, hTERC knockdown
does not cause telomere uncapping or a DNA damage response, but it induces changes in global gene
expression [46]. Rapid downregulation of genes involved in cell cycle progression, such Cyclin G2 and
Cdc27, was observed. Interestingly, the knockdown of TERC also rapidly lowered the expression of
specific genes coding for proteins important for tumor growth, angiogenesis, and metastasis, such as
integrin αV and Met oncogene protein [26].
Interestingly, two copies of the telomerase RNA component are encoded in Marek’s disease virus
(MDV) [47]. MDV is a lymphotropic alphaherpesvirus that causes Marek’s disease (MD) in chickens.
This condition is associated with neurological disorder, immune suppression and, primarily, malignant
T cell lymphomas [48]. Viral telomerase RNA (vTERC), which shares 88% sequence identity with
chicken telomerase RNA (chTERC), is expressed during both lytic and latent MDV infection. vTERC can
reconstitute telomerase activity with chicken vTERT in vitro [29]. However, it was demonstrated that
the tumorigenesis effect in virus-induced lymphomas of vTERC occurs independently of telomerase
activity. The mutant form of vTERC that does not interact with chTERT and is incapable of activating
telomerase activity demonstrated the same tumorigenesis effect as wild type vTERC [49]. Expression
of vTERC in chicken fibroblasts (DF-1) led to a two-times increase in integrin αV expression [48].
RNA pull-down assay can identify RPL22, a ribosomal protein that is involved in T-cell development
and virus-induced transformation, as a protein interacting with vTERC and chTERC [49]. However,
the interaction of RPL22 with vTERC was found to be two times stronger in comparison with chTERC.
The expression of vTERC led to the re-localization of RPL22 from the nucleolus to the nucleoplasm,
which is similar to the effect of EBER-1, a small type of RNA that contributes to tumor formation
upon Epstein–Barr virus infection. The re-localization of RPL22 due to its interaction with EBER-1 is
associated with an enhanced proliferative potential of cells [50,51]. Notably, the interaction of hTERC
with RPL22 has been demonstrated in vivo [52].
These observations, therefore, suggest that telomerase activation promotes tumorigenesis and
immortalization at different levels of regulation, and telomerase components may have additional
contributions, independent of their essential functions in telomere elongation. However, the fact
that the expression levels of telomerase components and the rate of telomerase activity influence
the telomere length, which is strongly associated with cell cycle progression and cell proliferation,
make it impossible to separate effects initiated by the telomere length or noncanonical functions of
the telomerase component. This question should be investigated carefully to allow to confidently
Biomolecules 2020, 10, 873 7 of 15

conclude that telomerase RNA has some functions in the regulation of the proliferation rate as an
individual molecule.

5. TERC in the Regulation of Gene Expression

The potential
Biomolecules mechanism
2020, 10, 873 of TERC’s participation in the regulation of global genome expression 7 of 15
may be derived from experimental genome mapping of long noncoding RNA occupancy. The improved
The potential
method, termed CHIRPmechanism
(Chromatin of TERC’s
Isolationparticipation in the regulation
by RNA Purification), allowsof global genome expression
the discovery of RNA-bound
may be derived from experimental genome mapping of long noncoding RNA occupancy. The
DNA and proteins. Significant enrichment of telomerase RNA at telomeric regions has been
improved method, termed CHIRP (Chromatin Isolation by RNA Purification), allows the discovery
demonstrated, as was expected. Surprisingly, numerous specific binding sites for hTERC have been
of RNA-bound DNA and proteins. Significant enrichment of telomerase RNA at telomeric regions
detected throughout the genome. A total of 2198 hTERC binding sites have been identified in the genome
has been demonstrated, as was expected. Surprisingly, numerous specific binding sites for hTERC
with have
a covering pattern
been detected very similar
throughout to that of
the genome. the transcription
A total of 2198 hTERC factors. It was
binding sites haveobserved that TERC
been identified
preferably binds towith theacytosine-rich motif 0 0
in the genome covering pattern very5similar
-GGCCACCACCCC-3 , whichfactors.
to that of the transcription is complementary
It was observedto the
sequence 0 -GGGGUGGUGGCC-30 at nucleotides 25–36 of hTERC. It was shown that hTERC binding
that 5TERC preferably binds to the cytosine-rich motif 5′-GGCCACCACCCC-3′, which is
complementary
sites are located at the to multiple
the sequence
Wnt5′-GGGGUGGUGGCC-3′
genes and a series of Myc at nucleotides 25–36 of4),
genes [53] (Figure hTERC.
whichItcorrelates
was
with the previously documented binding sites of TERT. The activation of Wnt reporters by [53]
shown that hTERC binding sites are located at the multiple Wnt genes and a series of Myc genes TERT in
TERC(Figure
-/- mouse 4), which correlates
embryonic with thewas
fibroblasts previously documented
demonstrated binding [54].
previously sites of TERT.
Thus, The
one activation
can speculateof that
Wnt reporters by TERT in TERC-/- mouse embryonic fibroblasts was demonstrated previously [54].
the binding of TERC to specific sites on DNA may have a direct effect on transcription or an indirect
Thus, one can speculate that the binding of TERC to specific sites on DNA may have a direct effect
effect by attracting TERT via the telomerase complex. Independent TERT binding to promoter regions
on transcription or an indirect effect by attracting TERT via the telomerase complex. Independent
cannot be excluded.
TERT binding to promoter regions cannot be excluded.

FigureFigure 4. Model
4. Model of hTERC
of hTERC biogenesis,depicting
biogenesis, depicting the
the competition
competitionbetween
between processing andand
processing degradation,
degradation,
and trafficking through cellular organelles with the distribution of functions. Telomerase RNA is
and trafficking through cellular organelles with the distribution of functions. Telomerase RNA is
synthesized as a long precursor that may be degraded, processed to the mature form following
synthesized as a long precursor that may be degraded, processed to the mature form following
association with hTERT or it may work outside of the telomerase complex. TERC is involved in gene
association with hTERT or it may work outside of the telomerase complex. TERC is involved in gene
expression regulation due to its interaction with promoter regions of genes involved in the regulation
expression regulation
of proliferation anddue
celltocycle
its interaction
progression.with
TERCpromoter regions
is transported of genes
from involved
the nucleus in cytoplasm
to the the regulation
of proliferation and cell cycle progression. TERC is transported from the nucleus to the
where it may associate with TERT and be reimported to the nucleus to elongate the telomeric repeats. cytoplasm
whereOn it the
may associate with TERT and be reimported to the nucleus to elongate the telomeric
other hand, TERC is imported to the mitochondria where it is processed and reexported to the repeats.
On thecytoplasm. The precursor of TERC is translated and the obtained TERP protein protects cells underto the
other hand, TERC is imported to the mitochondria where it is processed and reexported
stressfulThe
cytoplasm. conditions.
precursor of TERC is translated and the obtained TERP protein protects cells under
stressful conditions.
Evidence shows that the binding of TERC to DNA can directly influence the transcription
pattern was obtained in recent work on the identification of the upregulation of genes related to the
immune system in telomerase-negative U2OS cells that ectopically express hTERC [55]. The
Biomolecules 2020, 10, 873 8 of 15

Evidence shows that the binding of TERC to DNA can directly influence the transcription pattern
was obtained in recent work on the identification of the upregulation of genes related to the immune
system in telomerase-negative U2OS cells that ectopically express hTERC [55]. The stimulation of
TERC-U2OS with TNF-α led to the upregulation of cytokine expression and increased its secretion in
culture medium independently of telomerase activity. The depletion of hTERC by RNA interference
resulted in decreases in both the expression and secretion levels of cytokines. The activation of the
NF-κB pathway in TERC-expressing cells in comparison with TERC-negative cells was shown. Analysis
of binding sites of TERC on chromosomes demonstrated that the majority of them were located within
±1000 bp of transcriptional start sites. For several genes related to the NF-κB pathway—LIN37, TPRG1L,
TYROBP, and USP16—the direct interactions with hTERC were demonstrated in vitro. Further in vivo
experiments revealed upregulation of these genes as a response to TERC expression. Interestingly,
the increased expression level of hTERC was determined in CD14+ cells from type II diabetes and
multiple sclerosis patients displaying increased chronic inflammation [55]. The expression levels of
TPGRL1 and TYROBP and IL-8 and TNF-α increased for diabetic patients, whereas TYROBP and USP16
and IL-6, IL-8, CSF2, and TNF-α increased for multiple sclerosis patients. The fact that U2OS cells are
telomerase-negative allows us to affirm that the regulation of NF-κB-pathway occurs due to direct
action of TERC and is independent of the telomerase complex and telomere length.
TERC occupied sites near CSF1 and CSF2 genes encode macrophage colony-stimulating factor
(MCSF) and granulocyte-macrophage colony-stimulating factor (GMCSF), as was revealed by CHIRP
approach [53]. Decreased expression was demonstrated for the CSF1 and CSF3 genes in TERC-deficient
larvae of zebrafish [56], while the overexpression of TERC increased the levels of both transcripts.
The expression of the transcriptional factors spi1 and gata1a, which are essential for the differentiation
of hematopoietic stem cells (HSCs), was affected in zebrafish embryos when TERC was depleted.
GCSF overexpression has been shown to restore the expression level of the major myeloid transcription
factor spi1 and does not affect TERC levels. This indicates that TERC participates in the regulation of
the expression of gcsf and mcsf genes, which maintain a critical balance between the major myeloid
(spi1) and erythroid (gata1) transcriptional factors [56]. Finally, the depletion of TERC in zebrafish
embryos results in strong neutropenia and monocytopenia that is characterized by a decreased number
of fully functional neutrophils and macrophages. This effect is fully rescued by the overexpression of
TERC. The effect of TERC in developmental myelopoiesis is independent of telomerase activity and
telomere length.
Thus, the interaction of TERC with the promoter areas of specific genes (Figure 4) provides a
basis for the participation of TERC in transcription regulation, at least in genes of the immune system
and myelopoiesis.

6. TERC and Cell Protective Mechanisms

TERC can affect cellular processes, not only by the modulation of gene expression but by its
influence on cellular signaling systems.
Multiple studies have provided evidence concerning the implications of TERC in cell protective
mechanisms. Protein kinase ATR (ATM (ataxia telangiectasia mutated) and Rad3-related) participates
in the modulation of cellular responses to DNA damage. ATR is activated when single-stranded DNA
ends emerge upon the formation of DNA adducts, during the processing of double-strand breaks
(DSBs), or during termination of the replicative fork [57,58]. Suppression of ATR kinase activity is
observed under conditions of increased expression of hTERC [59]. Reduction of the hTERC level
stimulates ATR activity. These processes are independent from the level of telomerase activity and the
telomere length. A reduction of hTERC expression in cells results in an increase in the amount of p53,
the tumor suppressor and major contributor to the signaling pathway under conditions of oncogenic
stress. Meanwhile, the cell content of the protein CHK1, the cell cycle regulator, increases. p53 and
CHK1 are the major substrates of ATR kinase. hTERC inhibits ATR kinase and disrupts the regulation
of cell cycle checkpoints following DNA damage in vivo [59].
Biomolecules 2020, 10, 873 9 of 15

The activation of DNA-dependent protein kinase (DNA-PK), related to the same PIKK
(phosphatidylinositol 3-kinase-related kinases) family as ATR, by hTERC [60] was described several
years later. It was demonstrated that DNA-PK phosphorylates the hnRNP A1 protein in the presence
of hTERC in vitro and in vivo. hnRNP A1 is involved in the regulation of alternative splicing and
was identified as a telomere-associated protein and suggested to be a regulator of the recruitment of
telomerase to chromosome ends. Unfortunately, the activation of DNA-PK by hTERC has only been
studied in terms of its relationship with hnRNP A1. However, it was shown that the activation of
DNA-PK occurs in a hTERC-dependent manner in vitro and in vivo. The exogenous expression of
hTERC in VA13 cells lacking telomerase RNA was shown to increase the level of phosphorylated hnRNP
A1 [60], which was reduced by treatment with the inhibitor of DNA-PK. Data obtained in this study
allowed the proposal that hTERC is involved in the regulation of the DNA damage response by the
activation of DNA-PK, which is required for the repair of double-strand breaks via a nonhomologous
end-joining pathway [61,62]. Although evidence that telomerase activity and the telomere length do
not influence the effect of the hTERC level on the regulation of the DNA damage response has been
provided, it is very difficult to exclude the idea that telomerase RNA may be involved in the structural
organization of telomeres. Indeed, DNA-PKs are known to associate with telomeres and regulate the
cellular response to telomere dysfunction through the p53 pathway. Ectopically-expressed telomerase
RNA in telomerase-negative cells may interact with telomeres and disturb their structure, which will
influence the DNA damage response.
Surprisingly, mitochondrial trafficking of TERC was recently demonstrated (Figure 4) [63].
The mitochondria import signal—the first 52 nucleotides—was identified in hTERC. The processing of
hTERC to the shorter form hTERC-53 corresponding to the 52–248 nt of hTERC occurs in mitochondria
followed by export back to the cytoplasm [63]. hTERC-53 accumulates in the cytoplasm when the
membrane potential of mitochondria is impaired (Figure 4). More severe damage to mitochondrial
functions could lead to dramatic changes in the cytosolic hTERC-53 level because of mitophagy or
apoptosis [63]. The cellular and physiological functions of hTERC-53 and the mitochondrial trafficking
and processing of hTERC should be investigated more carefully.
The presence of an additional alternative TERC gene in mice was revealed by searching the
mouse genome by BLAT [64]. Alternative TERC (alTERC) showed an 87.9% similarity to mTERC.
The differences were caused by the deletion of 18 nt in the CR4 region of alTERC. The expression
of alTERC in mouse brain was determined by reverse transcription followed by PCR. AlTERC is
associated with TERT and supports telomerase activity. Overexpression of mTERC and alTERC protects
motor neuron cells from oxidative stress [64]. The survival of cells expressing alTERC increased in
comparison with the survival rate of cells with an increased level of canonical mTERC. The observed
protective effect occurred independently of TERT expression or telomerase activity.
The increased expression of telomerase components and telomerase activity are associated with
the stimulation of T-cell proliferation [65–68]. Interestingly, an increased level of hTERC expression
is important for short-term CD4 T-cell survival, while telomerase activity is needed for long-term
survival [69]. The cell protective effect of telomerase RNA overexpression independent of telomerase
activity was demonstrated under dexamethasone treatment. Overexpression of wild type hTERC
results in the stimulation of telomerase activity and does not have an antiapoptotic effect. In addition,
the expression of chimeric RNA hTERC-U64, which contains the 50 -half of hTERC (pseudoknot and
template region), occurs, but the 30 half (CR4/CR5 and box H/ACA regions) is replaced by the H/ACA
domain of the similarly sized U64 small nucleolar (sno)RNA [69]. Chimeric RNA cannot associate
with hTERT and activate telomerase. Overexpression of mutant forms of telomerase RNA ∆96–97 and
G305A, but not wild type or chimeric TERC, protects cells against dexamethasone-induced apoptosis.
The hTERC mutation ∆96–97 shifts the structural equilibrium from a pseudoknot structure to a hairpin,
which abolishes telomerase activity [19]. The point mutation G305A in hTERC reduces the binding
to hTERT by up to 80%. Moreover, wild-type hTERC, as well as both mutant forms of hTERC,
protect CD4 T-cells against apoptosis when the hTERT level is reduced. As expected, the reduction of
Biomolecules 2020, 10, 873 10 of 15

hTERC level was shown to induce apoptosis in CD4 T-cells. The genome-wide microarray analysis
did not reveal any transcriptional changes in genes involved in apoptosis in cells with a reduced
level of hTERC. The overexpression of wild type hTERT but not of the catalytically inactive isoform
led to apoptosis induction and the simultaneous expression of catalytically inactive hTERC mutants
restored the protective potential [69]. Altogether, these data provide evidence that the telomerase RNA
component may protect cells against apoptosis through its alternative function.
The data described above represent a set of evidence of the involvement of hTERC in various
cellular processes in the nucleus, cytoplasm, and even the mitochondria, independently of telomerase
and transcription activation, but do not explain the molecular mechanisms of the telomerase RNA
functions outside the telomerase complex (Figure 4).
The set of hTERC transcripts with heterogenous 30 -ends appears in the process of transcription of
the hTERC gene [70]. The position of the point of transcription termination is undetermined and the
length of primary transcript of hTERC is unknown at this moment. However, the application of the
deep-sequencing has revealed the major fraction of hTERC transcripts corresponding to the mature
form (451 nt). Additionally, a fraction of transcripts lengthened for 5–6 nts are encoded in the genome,
followed by the oligo(A)-sequence [71]. The stable intermediates with diverse lengths should appear
during the rapid processing of the elongated (up to 1000 nts) primary transcript of hTERC [70].
Multisubunit’s complex Integrator regulates the termination of hTERC transcription in a
promoter-dependent manner. Depletion of the Integrator results in the accumulation of the precursor
elongated by up to 571 nucleotides [72]. Different extended forms of the hTERC primary transcript
have been detected: some of them contain only few additional nucleotides, whereas some of them
exceed 1500 nt in length [70].
The main function of telomerase is in the nucleus, and the majority of hTERC is located in the
nucleus. However, under certain conditions, hTERC can be found in the cytoplasm. It was shown
that the depletion of DKC1 leads to the appearance of hTERC in the cytoplasm [73]. The loss and
mutations of TGS1 promote the accumulation of hTERC in the cytoplasmic fraction [74]. Moreover,
accurate analysis of wild type HEK293T cells revealed that around 20% of hTERC is localized in the
cytoplasm [75].
The transcript of hTERC contains an open reading frame (ORF) that starts at position 176 nt
and codes for the protein named hTERP. hTERP contains 121 amino acid residues and is encoded
in a premature transcript. It should be mentioned that hTERC was shown to be associated with the
ribosomes according to ribosome profiling data [76,77] and it is present in the polysome fraction [78].
The localization of the AUG codon in the pseudoknot domain should impede its translation; however,
the appearance of an alternative hairpin structure [19] allows start codon recognition and translation
of the protein hTERP.
The existence of hTERP has been confirmed by several experimental approaches, including mass
spectrometry and immunodetection [75]. It was revealed that overexpression of wild-type hTERC,
but not of the mutant which is incapable of directing hTERP synthesis (mutation in start-codon),
protects HEK293T cells from doxorubicin-induced apoptosis. Moreover, mutations at the N-terminus
of hTERP and a reduced level of hTERP affect autophagosome formation [75]. hTERP may be involved
in the protection of cells from stress and helps cells to survive and adapt to unfavorable conditions
(Figure 4).
The coding potential of hTERC explains the constitutive expression of hTERC independently from
telomerase activity. Upregulation of the expression of TERC in the early stages of tumorigenesis may
be necessary to increase the level of TERP protein that protects cells from apoptosis and promotes
survival in the period when the cell should adapt to the new metabolism. Mutations in TERC promote
severe phenotypes associated with telomeropathies, such as dyskeratosis congenita [17]. Mutant forms
of TERC, which are not able to associate with TERT but save open reading frames protect cells from
apoptosis [69]. In this case, the accumulation of unbound hTERC may promote translation, and an
increased level of hTERP protects cells from apoptosis (Figure 4).
Biomolecules 2020, 10, 873 11 of 15

Recent work revealed that hTERC produces small RNA named terc-sRNA at positions 425–447 nt.
The interaction of terc-sRNA with AGO2 affects telomerase complex formation and telomere
lengthening. The expression level of AGO2 correlates with telomerase activity. Surprisingly, authors
demonstrated that the overexpression of terc-sRNA in cells knocked out by AGO2 stimulates telomerase
activity. Telomerase RNA itself has been identified as target RNA for terc-sRNA, so it may be involved
not only in the regulation of telomerase assembly but it may also influence translation and the level of
hTERP protein expression in cells [79].
Thus, the majority of cell protective functions of telomerase RNA, excluding transcription
regulation, may be explained by the appearance of the protein hTERP, which is encoded in this RNA.
Indeed, the protective effect of the hTERP protein encoded in hTERC was demonstrated in cells
under apoptosis-inducing conditions. The hTERP protein is involved in autophagosome formation.
The coding capacity of hTERC may explain the protective effect under apoptosis-induction treatment
of immune cells of mutant hTERC, which is incapable of forming a complex with hTERT.

7. Conclusions and Perspectives

The major function of telomerase complex components is related to the maintenance of the
telomere length. The majority of investigations on telomerase have been concentrated in the field of
aging and cancer biology for a long period. Investigations aiming to understand the roles of TERT or
TERC in telomerase activation and disease development have provided data showing the possible
functional role of telomerase components outside of telomerase. The overexpression of TERC and TERT,
which is independent of telomerase activation, may promote the increased survival of transformed
cells due to the protective function of TERC itself or may activate the expression of components of
regulatory cascades involved in proliferation and cell protection.
The data concerning the alternative function of telomerase components in cell protective
mechanisms may be considered to show a new level of regulation related to tumorigenesis, telomere
syndromes, and cell survival. However, at this moment, we have fragmented data concerning the
action of telomerase components outside of the telomerase complex, and it is very difficult to separate
their functions as individual components or as compounds of the telomerase machine. More precise
investigations of telomerase components’ functions and the mechanisms of expression regulation
under different states of cellular homeostasis will enhance the development of approaches for the
modulation of carcinogenesis and aging.

Author Contributions: Conceptualization, writing: M.R.; conceptualization, editing: O.D. All authors have read
and agreed to the published version of the manuscript.
Funding: This work was supported by Russian Foundation for Basic Research (18-29-07031 mk) (section about
hTERC alternative functions); Russian Science Foundation (19–14–00065) (section about hTERC biogenesis and
action in telomerase complex); and the Lomonosov Moscow State University Development Program (PNR 5.13).
Conflicts of Interest: The authors declare no conflict of interest.

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