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Cell growth- and differentiation-dependent regulation of RNA polymerase III transcription

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RNA polymerase III transcribes small untranslated RNAs that fulfill essential cellular functions in regulating transcription, RNA processing, translation and protein translocation. RNA polymerase III transcription activity is tightly regulated during the cell cycle and coupled to growth control mechanisms. Furthermore, there are reports of changes in RNA polymerase III transcription activity during cellular differentiation, including the discovery of a novel isoform of human RNA polymerase III that has been shown to be specifically expressed in undifferentiated human H1 embryonic stem cells. Here, we review major regulatory mechanisms of RNA polymerase III transcription during the cell cycle, cell growth and cell differentiation.
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Cell Cycle 9:18, 3687-3699; September 15, 2010; © 2010 Landes Bioscience
REVIEW REVIEW
Eukaryotes contain three distinct DNA-dependent RNA poly-
merases (Pol I, Pol II and Pol III)1 that share the task of transcrib-
ing the information contained in genes into mobile RNA entities.
In humans, Pol I transcribes the precursor of the large ribosomal
45S RNA, Pol II transcribes all messenger RNAs, most snRNAs,
snoRNAs and micro RNAs and Pol III transcribes a diverse
group of small untranslated RNAs that participate in the regu-
lation of transcription, splicing and translation. After transcrip-
tion, Pol III transcripts are either directly degraded or modified
for participation in the regulation and execution of processes in
the nucleus and cytoplasm (transcription regulation; RNA pro-
cessing; ribosome assembly; translation) that ultimately lead to
protein synthesis. In the past few years, in part due to the dis-
covery of novel classes of regulatory RNAs such as micro (mi)
RNAs and small interfering (si)RNAs, it has become clear that
the three classical eukaryotic RNA polymerases have acquired
additional layers of complexity during evolution from unicellu-
lar to multicellular eukaryotes. For instance, derivatives of Pol II
that fulfill specific functions in transcription of siRNAs (Pol IV)
or of noncoding RNAs at target loci (Pol V) have been found in
Arabidopsis.2,3 More recently, an isoform of human Pol III has
*Correspondence to: Martin Teichmann; Email: Martin.Teichmann@inserm.fr
Submitted: 06/16/10; Revised: 07/28/10; Accepted: 08/01/10
Previously published online:
www.landesbioscience.com/journals/cc/article/13203
DO I: 10. 4161/cc.9.18.13203
RNA polymerase III transcribes small untranslated RNAs that
fulll essential cellular functions in regulating transcription,
RNA processing, translation and protein translocation. RNA
polymerase III transcription activity is tightly regulated during
the cell cycle and coupled to growth control mechanisms.
Furthermore, there are reports of changes in RNA polymerase
III transcription activity during cellular dierentiation,
including the discovery of a novel isoform of human RNA
polymerase III that has b een shown to be specically expresse d
in undierentiated human H1 embryonic stem cells. Here,
we review major regulatory mechanisms of RNA polymerase
III transcription during the cell cycle, cell growth and cell
dierentiation.
Cell growth- and dierentiation-dependent
regulation of RNA polymerase III transcription
Hélène Dumay-Odelot,1 Stéphanie Durrieu-Gaillard,1 Daniel Da Silva,1 Robert G. Roeder2 and Martin Teichmann1,*
1Institut Europ éen de Chimie et Biolog ie (I.E.C.B.); Unive rsité de Bordeaux; Ins titut National de la Santé e t de la Recherche Médic ale (INSERM) U869; Pessac, Fran ce;
2The Rockef eller University; L aboratory of Bioc hemistry and Mole cular Biology; New York , NY USA
Key words: RNA polymerase III, cell growth, cell cycle, differentiation, embryonic carinoma cell, embryonic stem cell,
cell transformation
been described that is specifically expressed in embryonic stem
cells and in certain tumor cell lines. This isoform of Pol III is able
to contribute to cell transformation under tissue culture condi-
tions.4 In this review, we will describe our current understand-
ing of how Pol III transcription is regulated during growth and
differentiation, with emphasis on the mammalian Pol III system
where appropriate.
Introduction to Pol III Transcription
Promoters. Three types of promoters are recognized by RNA
polymerase III in higher eukaryotes (Fig. 1). Type 1 and type 2
promoters are internal to the gene, whereas type 3 promoters are
located entirely upstream of the transcription initiation site.5 Type
1 (5S ribosomal RNA gene) and type 2 promoters [e.g., those in
tRNA, VA1 RNA and VA2 RNA genes and in short interspersed
nuclear elements (SINEs)] are composed of A- and C- or A- and
B-boxes, respectively (Fig. 1). Transcription of type 1 and 2 genes
is initiated about 8 to 50 nucleotides upstream of the 5'-end of
Box A and terminates as soon as Pol III encounters four or more
consecutive thymidines. Type 3 promoters (U6 RNA, 7SK RNA,
RNAse P RNA, RNAse MRP RNA and Y RNA genes) are com-
prised of a distal sequence element (DSE) that is typically located
about 200–250 nt upstream of the transcription initiation site, a
proximal sequence element (PSE) at around -50 and a TATA box
at -30. Type 3 promoters arose during evolution from unicellular
to multicellular eukaryotes and are found in plants and animals,
but not in yeast, which instead utilize a type 2 promoter for tran-
scription of the U6 gene. Pol III-transcribed genes that contain
both gene-internal and gene-external promoter elements have
also been described and include the 7SL RNA, vault RNA, BC1
RNA, BC200 RNA, EBER1 RNA and EBER2 RNA genes.5
Transcription factors. The yeast and human transcription
factors that recognize these promoters have been identified by
genetic or biochemical means and their cognate subunits have
been cloned (Fig. 1). The primary DNA-binding transcription
factors that are required for the recognition of the evolutionary
conserved type 1 and type 2 promoters have likewise been con-
served from yeast to human. Type 2 genes are directly recog-
nized by the six subunit transcription factor TFIIIC.6,7 The type
1 promoter of the 5S gene requires prior binding of TFIIIA, the
prototype gene-specific transcriptional activator in eukaryotes,8
for recruitment of TFIIIC to the gene.6,9 The type 3 promoters of
3688 Cell Cycle Volume 9 Issue 18
promoters or to type 3 promoters (TFIIIB-α). As for the evolu-
tion of PTF/SNAPc/PBP, the appearance of a second isoform
of TFIIIB may again have been necessary for allowing promoter
type- or gene-specific regulation of Pol III transcription in higher
eukaryotes. Human TFIIIB-α and TFIIIB-β both contain
TATA-binding protein (TBP) and BDP1 but differ with respect
to the presence of the TFIIB-related factors BRF1 (TFIIIB-β)
and BRF2 (TFIIIB-α).22, 23 Interestingly, it was demonstrated
that purified S. cerevisiae BDP1 could replace its human ortholog
in transcription of the human U6 gene reconstituted with human
transcription factors, indicating that essential protein-protein-
interactions with other components of the PIC at type 3 promot-
ers could be established by S. cerevisiae BDP1.24 An alternative
model to the stepwise recruitment of the Pol III transcription
machinery to promoters has been suggested by the discovery of
an RNA polymerase III holoenzyme that contains TFIIIC and
TFIIIB-β subunits in addition to Pol III.25 This holoenzyme as
an entity may directly recognize Pol III promoters and direct
multiple rounds of transcription by mechanisms involving facili-
tated reinitiation of transcription.26,27
Accessory factors. A variety of proteins have been described
that, while not necessarily essential, are highly stimulatory for
U6 and 7SK genes appeared during the evolution of multicellu-
lar eukaryotes, along with transcription factors required for their
expression. Type 3 promoters are directly bound by the multi-
subunit PSE-binding transcription factor (PTF; also referred to
as SNAPc or PBP),10-12 for which no ortholog has been identified
in yeast. The interaction of PTF/SNAPc/PBP with the PSE is
reinforced by DSE binding proteins such as Oct-1,13-16 and Staf
(Z N F143 ) .17 For transcription activation of type 3 promoter-con-
taining genes, DSE and PSE sequences may be juxtaposed by a
positioned nucleosome.18,19
In the model of a stepwise assembly of preinitiation complexes
(PIC) on Pol III promoters, the DNA-binding proteins, once
bound to their promoters, recruit the initiation factor TFIIIB
to their respective genes. After its recruitment to the promoter,
TFIIIB alone is able to direct multiple rounds of transcription
in the absence of TFIIIC and TFIIIA,20 demonstrating that
interactions of Pol III with TFIIIC and TFIIIA are not essential
for transcription initiation, at least in vitro, but rather serve for
the recruitment of TFIIIB. Only a single isoform of TFIIIB has
been described in yeast.6 In contrast, human cells contain two
isoforms of TFIIIB (TFIIIB-α and TFIIIB-β)21 that are specifi-
cally recruited either to gene-internal type 1 and 2 (TFIIIB-β)
Figure 1. Mammalian RNA Polymerase III promoters and transcription factors. Promoter types 1–3. IIIA = TFIIIA. TFIIIB subunits are TBP, BDP1, BRF1 and
BRF2; TFIIIC subunits are indicated as C35, C63, C90, C102, C110 and C220. PTFα, β, γ and δ subunits are also denoted according to the molecular weight
of the respective SNAPc subunits (190, 50, 43 and 45). In addition, SNAP19 is shown, which has not been described in PTF. The ternary complexes com-
posed of RPC39, RPC62 and RPC32α or RPC32β are high lightened within the symbolic representation of RNA polymerase III.
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of ScRPC160 is able to suppress a conditional growth pheno-
type of a mutation in the C-terminal part of ScRPC31.44 It was
further shown that ScRPC31, ScRPC34 and ScRPC82 interact
with each other in two hybrid assays.45 The latter and subsequent
experiments also revealed an interaction between ScRPC34
and ScBR F1.45,4 6 Altogether these results indicate that this sub-
complex serves as a bridge between the enzymatic core of Pol
III and its initiation factors. As a consequence, in pre-initiation
complex formation, this subcomplex likely acts in a central posi-
tion close to the transcription initiation site. Such a role was also
supported by cross-linking experiments that projected ScRPC34
and ScRPC31 upstream of, and ScRPC82 also downstream of,
the transcription initiation site.47 By using the short distance
crosslinker 4-S-dTMP, Bartholomew and colleagues47 further
showed that photoaffinity labeling of ScRPC82 and ScRPC31 by
4-S-dTMP, but not by the 9 Å-crosslinker N3RdUMP, occurs in
transcription bubbles of initiation complexes and stalled elonga-
tion complexes, indicating that these two subunits of Pol III may
be in contact with single-stranded DNA.
Complementary data supporting a model in which HsRPC62,
HsRPC39 and HsRPC32 likewise form a ternary complex were
obtained with human Pol III. It was shown that the ternary
complex can be removed from the other 14 subunits of Pol III
by ultracentrifugation or by treatment with mild denaturants
(2 M urea).48 Human Pol III that was stripped of HsRPC32,
HsRPC39 and HsRPC62 was capable of transcription elongation
(following non-specific initiation), but was incapable of specifi-
cally initiating transcription from Pol III promoters, again indi-
cating that this ternary sub-complex may be especially important
for polymerase-transcription factor interactions during initia-
tion. However, the residual elongation activity of the 14-subunit
Pol III transcription in vitro. Complementation assays coupled
with purification of factors from HeLa nuclear extracts showed
that topoisomerase 1 and positive cofactor 4 (PC4) co-frac-
tionate with TFIIIC (holo-TFIIIC) and that they activate Pol
III transcription in a system reconstituted with highly purified
components.28 Similarly, NF1 was found to stimulate Pol III
transcription from type 2 promoters and to extend the TFIIIC
footprint over the transcription termination site.29 A function
similar to that of human PC4 was recently demonstrated for the
S. cerevisiae ortholog Sub1.30,31 Although the mode of action for
PC4 or Sub1 in Pol III transcription has remained a puzzle owing
to the high (50–100 fold) molar excess of PC4 (relative to other
Pol III transcription factors) required for in vitro transcription
activation, it has been speculated that DNA-binding properties
of PC4 may help to structure DNA for facilitating Pol III tran-
scription in vitro. Furthermore, the transcription termination
and recycling factor La32 co-purified with holo-TFIIIC prepara-
tions.28 Although not absolutely required for in vitro transcrip-
tion,33 La was shown to associate with Pol III promoters in yeast34
and human cells.35 Moreover, the HMG-box-containing proteins
NHP6A and NHP6B have been implicated in the activation of
Pol III transcription in S. cerevisiae.36,37 Up to now, there has been
no identification of a mammalian NHP6A or NHP6B ortholog
that might fulfill a similar function Pol III transcription.
RNA polymerase III. Along with the DNA-binding transcrip-
tion factors, the distinct isoforms of human TFIIIB are necessary
and sufficient to recruit Pol III to the transcription initiation site.
Pol III is highly conserved from yeast to humans and is com-
posed of 17 subunits (Tabl e 1). Of these subunits, five (RPB5/
POLR2E, RPB6/POLR2F, RPB8/POLR2H, RPB10/POLR2L
and RPB12/POLR2K) are common to all three polymerases, two
(RPAC40/POLR1C and RPAC19/POLR1D) are shared by Pol I
and Pol III (with paralogous subunits in Pol II) and five (RPC1/
POLR3A, RPC2/POLR3B, RPC8/POLR3H, RPC9 and
RPC11/POLR3K) are paralogous to subunits found in Pol I and
Pol II. The remaining five subunits (RPC5/POLR3E, RPC4/
BN51/POLR3D, RPC3/RPC62/POLR3C, RPC6/RPC39/
POLR3F and RPC7/RPC32/POLR3G) are specific to RNA
polymerase III and no structural or functional counterparts have
been identified in Pol I or Pol II.38,39 However, it has been sug-
gested that some of these subunits may share structural homolo-
gies to basal transcription factors of the Pol II system.40,41
Three of the five Pol III-specific subunits (POLR3C,
POLR3F, POLR3G) form a stable subcomplex (Fig. 1). Thus,
it was shown in the yeast S. cerevisiae (Sc) that the ScRPC31,
ScRPC34 and ScRPC82 subunits (orthologous to human (Hs)
RPC32, HsRPC39 and HsRPC62, respectively) could be sepa-
rated from the other subunits of Pol III by native gel electro-
phoresis.42 In addition, a mutation in the zinc-binding domain
of the largest subunit of Pol III (ScRPC160) led to an increased
dissociation of subunits ScRPC31, ScRPC34 and ScRPC82
from the remainder of the enzyme,43 indicating that ScRPC31,
ScRPC34 and ScRPC82 may form a Pol III sub-complex that
is less stably associated with the residual enzyme. The associa-
tion of this sub-complex with the rest of Pol III may involve a
direct ScRPC160-ScRPC31-interaction, since overexpression
Tab le 1. The subunits of human RNA polymerase III
Gene symbol Protein name Calculated mass
[kDa] Amino acids
POLR3A RPC160 155.641 139 0
POLR3B RPC 128 127. 7 85 1133
POLR3C RPC62 60 .612 534
POLR3D RPC53 44.396 398
POLR3E RPC80 79. 898 708
POLR3F RPC39 35.68 4 316
POLR3G RPC32α25.914 223
POLR3GL RPC32β25.334 218
POLR3H RPC25 22.918 204
-RP C17 16.871 14 8
POLR3K R P C11 12. 336 10 8
POLR1C R PAC40 39. 250 346
POLR1D R PAC19 15. 237 133
POLR2E RPB5 24 .551 210
POLR2F RPB6 14. 478 12 7
POLR2H RPB8 17.14 3 150
POLR2L RPB10 7. 6 45 67
POLR2K R PB12 7.00 4 58
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MAF1 as a downstream effector of PKC. Likewise, MAF1 was
identified as a downstream effector of the repression of Pol III
transcription by TOR,61 suggesting that many, if not all, repres-
sive signals for Pol III transcription in yeast converge on MAF1.
MAF1 was originally identified in a genetic screen that
decreased the nonsense suppressor efficiency of SUP11 tRNA62
and the link to Pol III transcription was established by the iso-
lation of fragments of the largest subunit of Pol III as multi-
suppressor copies of the conditional growth phenotype of the
maf1-1 mutation.63,64 MAF1 was shown to inhibit S. cerevisiae
Pol III transcription as a downstream effector of several nutrition
and stress signaling pathways. Starvation, rapamycin treatment
and oxidative or endoplasmic reticulum stress were shown to be
dependent on MAF1 for the repression of Pol III transcription.61
Under repressive growth conditions, MAF1 is dephosphorylated
by protein phosphatase 2A (PP2A), leading to its translocation
into the nucleus where it inhibits Pol III transcription mainly
through interactions with BRF1 and Pol III itself.65, 66 Favorable
growth conditions lead to phosphorylation of MAF1 by SCH9
and PKA kinases, resulting in its retention in the cytoplasm.60,67-69
Repression of Pol III transcription by MAF1, MAF1 regulation
by phosphorylation and MAF1 interactions with BRF1 and Pol
III have been conserved from yeast to humans.70-72 It was demon-
strated that facilitated recycling of Pol III transcription prevents
repression by MAF1 in HeLa cell extracts, suggesting that the
regulation of Pol III recruitment to preinitiation complexes repre-
sents an important mechanistic component of MAF1 function.27
Interestingly, overexpression of MAF1 in human glioblastoma
cells led to the inhibition of colony formation in soft agar assays,
thus demonstrating functional aspects that are usually attrib-
uted to tumor suppressor proteins.73 Possibly, direct inhibition of
TATA-binding protein expression and its effects on Pol I and Pol
III transcription may contribute to tumor-suppressive activities of
MAF1.73 However, no MAF1 mutation causative for the develop-
ment of tumors has hitherto been described in higher eukaryotes.
Regulation of Pol III Transcription
in Higher Eukaryotes
Gene-specific cellular factor TFIIIA. The prototypical example
of Pol III-mediated transcriptional regulation in vertebrates is the
regulation of 5S RNA transcription by TFIIIA during Xenopus
laevis development. TFIIIA was the first eukaryotic transcrip-
tion factor to be identified and purified to homogeneity,8 allow-
ing molecular cloning of the cognate cDNA encoding TFIIIA74
and deduction of zinc-finger motifs.75 It was shown that TFIIIA
regulates 5S RNA transcription through site-specific binding to
the 5S gene promoter8 and that it also binds to 5S RNA, forming
a 7S ribonucleoprotein storage particle.76 Two types of 5S RNA
genes have been described in X. laevis, an oocyte-specific gene
that is present in about 20,000 copies per haploid genome and a
somatic gene present in about 400 copies per haploid genome.77
The somatic 5S gene is expressed in both oocytes and in adult
animals, whereas expression of the oocyte-specific 5S genes
decreases dramatically after oogenesis. The reduction of oocyte
5S gene transcription has been attributed to multiple mechanisms
core Pol III complex, while significant, was nonetheless reduced,
suggesting that the ternary complex may also function during
transcription elongation.48 Studies with recombinant proteins
further showed that HsTFIIIC63 and HsRPC62, HsTFIIIC90
and HsRPC62 as well as HsTFIIIC90 and HsRPC39 interact in
vitro,49,50 reinforcing the hypothesis that the ternary sub-complex
of RNA polymerase III fulfils important functions for establish-
ing protein-protein-contacts with transcription factors on the one
hand and with Pol III on the other.
Purification of Pol III from mouse myeloma cells (MOPC
315) led to the identification of two chromatographically distinct
enzymes (Pol IIIA and Pol IIIB) that were both active in tran-
scription of class III genes.51 Determination of the subunit com-
positions of these enzymes revealed that they are highly related
and differ only in the presence of either a 32 kDa (IIIA) or a
33 kDa (IIIB) polypeptide.52 Recently, we have been able to iso-
late two distinct isoforms of Pol III from human cells. The major
physical difference in between these two isozymes is the presence
of either HsRPC32α/POLR3G in Pol IIIα or of HsRPC32β/
POLR3GL in Pol IIIβ.4 Taking into account the migration of
these two subunits on SDS-PAGE, it is very likely that the two
isoforms isolated from human cells are orthologous to the previ-
ously reported isoforms of Pol III in mouse myeloma cells.
Regulation of Pol III Transcription—
Lessons Learned from Yeast
Many factors and mechanisms that underlie the regulation of
RNA polymerase III transcription have been identified by study-
ing yeast cells. These unicellular organisms adapt their gene
expression programs by all three RNA polymerases to environ-
mental conditions. Favorable growth conditions lead to higher
rates of transcription, whereas deprivation in nutrients results
in the repression of transcription. Importantly, transcription of
components of the translational apparatus, including rRNAs,
tRNAs and mRNAs encoding ribosomal proteins consume
about 75–80% of cellular nucleotides, mainly through transcrip-
tion by Pols I and III,53,54 and this high energetic cost is restricted
under unfavorable growth conditions.55 Multiple pathways have
been implicated in the regulation of Pol III transcription in
S. cerevisiae. These include the secretory signaling pathway,54 the
TOR pathway,56-58 the DNA damage pathway59 and the PKA
pathway (as a downstream effector of RAS).60 Defects in the
secretory pathway lead to the repression of 5S RNA and tRNA
transcription. Temperature-sensitive mutations in two genes that
function either in endoplasmic reticulum (ER) to Golgi complex
transport (YPT6 ) or in protein and vesicle trafficking between
ER and Golgi (SLY1) lead to repression of Pol III transcription at
the non-permissive temperature. This repression can be partially
rescued by deletion of protein kinase C (PKC1), the central effec-
tor kinase of the cell integrity pathway. Furthermore, deletions of
either WSC1 or WSC2 genes, the farthest upstream components
of the cell integrity pathway, also strongly reduce repression of
Pol III transcription that is induced by secretory mutations.54, 55
Deletion of MAF1 blocks the repression of Pol III transcription
caused by defects in the secretory pathway,59 possibly placing
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hypophosphorylated Rb was able to efficiently repress Pol III
transcription.99 The regulation of Pol III transcription by tumor
suppressor proteins was shown to be exerted either through direct
interactions with TFIIIB102,106 or PTF/SNAPc/PBP107 subunits or
by the activation of signal transduction cascades that ultimately
lead to the phosphorylation and inactivation of TFIIIB sub-
units.104 However, indirect effects of p53 on the stability of BRF1
have also been described. In this case, it was necessary to main-
tain ectopic expression of p53 in p53-/- Li-Fraumeni cells for at
least 4 days in order to reveal an inhibitory effect on Pol III tran-
scription activity; and this effect proved to be mediated by the
disappearance of a hyperphosphorylated form of BRF1, which
was degraded in a proteasome-dependent manner.108 Moreover,
pRb family members p107 and p130 were also reported to
repress Pol III transcription through direct interaction with
BRF1.109,110
It was further reported that Pol III transcription is repressed in
mitosis and that the repression is conferred by a kinase that phos-
phorylates subunits of TFIIIB. In X. laevis it was shown that a
92 kDa protein in fractions with TFIIIB activity becomes pho-
phorylated by p34cdc2 ( CDK1).111,112 In HeLa cells, TBP-associated
subunits of TFIIIB (presumably corresponding to BRF1 or BRF1
and BDP1) that were purified from cycling cells were shown to
reconstitute transcription in mitotic extracts, suggesting that
these components represent the target proteins of repression.113
Subsequently, it was shown that BRF1 is phosphorylated and
inactivated during mitosis by a kinase different from CDK1.114 In
addition, BDP1 within TFIIIB-α was shown to be phosphory-
lated by CK2 during mitosis, leading to the repression of Pol III
transcription.115 Apart from this inhibitory role of CK2, it was
also reported that CK2 is able to stimulate transcription of the
human U6 gene via phosphorylation of Pol III.116 Regulatory roles
for CK2 in Pol III transcription were also reported in the yeast
S. cerevisiae.117,118 The phosphorylation of BRF1 and of BDP1
during mitosis has been confirmed by quantitative analysis of
protein phosphorylation in HeLa cells.119 This report also showed
that subunits of Pol III (POLR3C, POLR3E and POLR3G),
TFIIIB-α (BRF2), TFIIIC (TFIIIC220, TFIIIC110) and PTF
(PTFα/SNAPc190) are likewise phosphorylated during mitosis,
which could possibly contribute to mitotic repression of Pol III
transcription in human cells. Repression of PTFα/SNAPc190
activity was shown to be mediated by CK2,120 but it remains to be
shown whether CK2 or another kinase is responsible for PTFα/
SNAPc190 phosphorylation during mitosis.
Link to growth control. In contrast to the repression exerted
by tumor suppressor proteins, the proto-oncogene c-MYC121 and
components of the MAP kinase signal transduction pathway
(ERK; JNK1)122,123 were found to activate Pol III transcription.
c-Myc, ERK and JNK1, respectively, were shown to function
through recruitment of TFIIIB to Pol III promoters (c-Myc),
through phosphorylation of the BRF1 subunit of TFIIIB (ERK)
or through enhanced expression of BRF1 (JNK1). Subsequently,
it was shown that c-Myc induces the association of GCN5 and
TRR AP with Pol III promoters (tRNALeu; 5S), resulting in acet-
ylation of histone H3.124 The association of c-Myc with TRRAP
and GCN5, as well as Pol III transcription activation is negatively
involving both chromatin modifications78,79 and regulation of the
expression of TFIIIA itself.74,77,80 Regarding the latter mecha-
nism, there are high levels of TFIIIA mRNA and protein per
cell during early oogenesis but dramatically reduced levels per
cell during embryogenesis. Thus, the expression pattern of
X. laevis TFIIIA suggests a regulatory role for TFIIIA in the devel-
opmental changes in 5S gene transcription. Differential expres-
sion of TFIIIA in oocyte and somatic cells has been attributed to
the usage of distinct promoters. Interestingly, it has been reported
that Pol III initiates transcription within the oocyte-specific pro-
moter of TFIIIA, suggesting a Pol III-mediated downregulation
of the oocyte-specific TFIIIA promoter in Xenopus laevis somatic
cells.81
Viral proteins. In the late seventies and the eighties of the
last millennium, after the discovery and partial characteriza-
tion of general transcription initiation factors for Pol III,82 Pol
II,83 and Pol I,84 the focus of transcription research advanced to
the identification of mechanisms that contribute to the regula-
tion of the three transcription systems. The contribution of viral
proteins in the process of transcription regulation was explored
and, in particular, viral proteins with oncogenic functions were
analyzed for their ability to enhance or repress transcription. We
will restrict the description in this review to the regulation of
mammalian Pol III transcription and refer to excellent publica-
tions that review the regulation of RNA polymerases I and II.85 -87
Major breakthrough discoveries with respect to the regulation
of Pol III transcription by viral proteins were obtained with the
adenovirus E1A protein that activates transcription of immediate
early viral genes. Transfection of cDNAs encoding E1A stimu-
lated transcription of co-transfected VA1 and tRNA genes.88 -90
The activation was attributed to indirect mechanisms that acted
on the activity of TFIIIC, since depletion of E1A from HEK cell
extracts did not abolish transcription activation.91 The molecular
cloning of TFIIIC11092 showed that its expression could be regu-
lated by transfection of adenoviral E1A or by serum stimulation
of HeLa cells. However, it was also shown that recombinant E1A
that was produced in baculoviral expression systems could like-
wise stimulate Pol III transcription,93 pointing to the possibility
that E1A may exert its function through alternative mechanisms.
In addition to the regulation of Pol III transcription by E1A, it
was demonstrated that the SV40 small t antigen also stimulates
Pol III transcription.94 The hepatitis B virus X (Hep B X) protein
also was shown to activate transcription by Pol III.95 It later was
shown that the Hep B X protein acts, at least in part, through
enhanced expression of the TATA-binding protein.96
Link to cell cycle control. Important mechanistic aspects of
Pol III transcription regulation by viral proteins were revealed by
the identification of cellular proteins that are regulated by these
viral proteins. In particular, it was demonstrated that adenoviral
E1A and E1B proteins, papillomaviral E6 and E7 proteins and
SV40 large T antigen interacted with and inactivated p53 and
pRb, thereby severely impacting on the regulation of the host
cell’s cell cycle.97, 98 These findings led subsequently to the dis-
coveries that major tumor suppressor proteins, such as pRb,99,100
p53,101,102 A RF,103 PTEN104 and BRCA1,105 negatively regulate
Pol III transcription. For Rb it was demonstrated that only
3692 Cell Cycle Volume 9 Issue 18
Figure 2. (A) Schematic representation of several regulatory pathways of mammalian RNA polymerase III transcription: Ras-dependent pathways. (B)
Schematic representation of several regulatory pathways of mammalian RNA polymerase III transcription: Regulation of Pol III transcription by tumor
suppressor and oncogene proteins.
regulated upon nucleolar stress (induced by actinomycin D or
serum starvation) by the ribosomal protein L11.125 Myc func-
tions in regulating Pol III transcription were demonstrated to be
independent of its heterodimerization partner Max in Drosophila
melanogaster.126 Moreover, in an independent link of Myc to the
Pol III transcription apparatus, it was shown that c-Myc activates
transcription of the RPC53 (BN51) gene,127 which encodes a
Pol III subunit that has been reported to suppress the
temperature-sensitive G1 cell cycle arrest in the hamster BHK-
21 cell line.128,129 Interestingly, mutation of the gene encoding
RPC53 in yeast also induced cell cycle arrest predominantly in
G1, although no Myc ortholog has been described in this organ-
ism.130 Collectively, these results strongly indicate that the regu-
lation of growth and cell cycle control are intimately connected
to the activity of Pol III transcription. Some of the regulatory
mechanisms have been summarized in Figures 2A and 2B.
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phenylephrine stimulation. Induction of hypertrophy is accom-
panied by enhanced BRF1 and c-Myc expression and by elevated
rates of phosphorylated ERK, as well as phosphorylated Rb.137 As
a result, Pol III transcription is enhanced in cardiomyocytes by
these stimuli. Except for this particular case, there is no detailed
information on how mammalian Pol III transcription is regu-
lated during differentiation and in terminally differentiated cells
and tissues.
Little is known about the regulation of Pol III transcription
during differentiation of mammalian cells. In particular, analy-
ses of Pol III transcription at the level of protein expression and/
or modification during early steps of differentiation (e.g., in
undifferentiated embryonic stem cells and in the course of their
differentiation) are limited by the amounts of cells and extracts
that can be derived from these cells. To overcome these limits,
embryonic carcinoma (EC) cells have been employed for ana-
lyzing the regulation of Pol III transcription during early steps
of differentiation. Mouse F9 or human NTERA2 EC cells are
tumor cell lines with certain stem cell-like features.138 Suspension
cultures of F9 EC cells can be differentiated into embryoid bod-
ies by the addition of retinoic acid and monolayer cultures of F9
cells differentiate into parietal entoderm (PE) upon the addition
of retinoic acid and cAMP (Fig. 3). This cell system has been
employed for analyzing the activity of the Pol III transcription
system during differentiation. It was shown that Pol III activity
decreased for transcription of gene-internal promoters as well as
of type 3 promoters. The reduced transcriptional activity at gene-
internal promoters was attributed to altered TFIIIB activity139
or to a decrease in TFIIIC1 activity.140 The decrease in TFIIIB
activity was shown to result from the combined reduction of the
abundance of TFIIIB subunits (TBP, BRF1 and BDP1).141,142
Furthermore, the protein levels of c-MYC were also dramatically
downregulated during differentiation of EC into PE cells, which
may account in parts for the reduction of Pol III transcription.142
Impaired transcription of type 3 genes was correlated with a
decrease in binding of PTF/SNAPc/PBP to the PSE143 (Fig. 3).
These results, indicating that TFIIIB-, TFIIIC1- and PTF/
SNAPs/PBP-activities are regulated during differentiation of
F9 EC cells could be functionally linked to each other by the
finding that the TFIIIB subunit BDP1 is essential for TFIIIC1-
activity.144 Originally, TFIIIC was separated by B-Box-based
affinity purification or by chromatography over Mono Q into the
TFIIIC1 and DNA-binding TFIIIC2 components, which were
both required for Pol III transcription.145,14 6 TFIIIC1 was shown
to stimulate binding of the six-subunit TFIIIC2 complex to gene-
internal type 2 promoters,145 as well as PTF/SNAPc/PBP binding
to the PSE.147 Extensive purification of TFIIIC1 in conjunction
with analyses of recombinant TFIIIB150 (corresponding to
amino acids 1–846 of BDP1) revealed that BDP1 and TFIIIC1
were functionally replaceable in complementation assays of tran-
scription with extracts that were derived from differentiated F9
cells (and thus contained limiting amounts of TFIIIC1) or in
transcription systems that were reconstituted with partially puri-
fied transcription factors.144 Together, these data indicate that the
activity of BDP1 is negatively regulated during differentiation of
F9 teratocarcinoma cells.
Link to tumor development. The above described discover-
ies that linked Pol III transcription to cellular processes such as
cell cycle or growth suggested that Pol III transcription may be
deregulated during cell transformation. In addition it was shown
that the expression of five TFIIIC subunits (the sixth was not yet
identified) is increased in ovarian carcinomas.131 Importantly, the
samples analyzed in this study were obtained from patients in
clinics and not from cell lines grown under tissue culture con-
ditions, demonstrating that the expression of Pol III transcrip-
tion components is deregulated in tumors that have not been
produced by experimental manipulations. It was nevertheless a
surprise that the inducible overexpression of the BRF1 subunit
of TFIIIB or of the tRNAi
Met gene in mouse 3T3 fibroblasts was
sufficient for transformation of these cells.132 These data sug-
gested that enhanced transcription from type 3 promoters was
not required for tumor formation, but that only transcription by
BRF1-containing TFIIIB-β, and in particular that of the tRNAi-
Met gene, was crucial in this process. However, overexpression of
BRF1 or tRNAi
Met in rat1a fibroblasts proved insufficient for cel-
lular transformation, indicating that cell type-specific differences
may exist between these two model systems.133 The analysis in
rat1a cells has further shown that enhanced Pol III transcription
is required for oncogenic transformation by c-Myc.133 In sum-
mary, and even if the model of BRF1- or tRNAi
Met-mediated cell
transformation may not be applicable to all types of cells, these
two studies clearly demonstrate the importance of Pol III tran-
scription for cell transformation.
Regulation of Pol III Transcription
during Cellular Differentiation
In addition to the mechanisms that coordinate the regulation of
growth and Pol III transcription activity, multicellular organ-
isms must have evolved the means to regulate transcription dur-
ing differentiation in order to ensure that cells with high protein
synthesis rates are capable of producing the components neces-
sary for translation (i.e., ribosomal RNAs and proteins, as well
as tRNAs)—even if they are resting and thus in the G0 phase of
the cell cycle. Some tissues such as the exocrine134 or endocrine135
pancreas probably circumvent this problem in part by balanc-
ing cell growth and apoptosis, resulting in stable cell numbers
whilst allowing cell growth and division. In these tissues, the
required protein synthesis and thus active Pol III transcription,
may presumably occur predominantly in dividing cells, although
the presence of high amounts of endoplasmic reticulum suggests
high protein synthesis rates in both dividing and resting cells.
Other cells, including Schwann cells that induce myelin gene
transcription after cell cycle exit,136 require transcription of ribo-
somal components after entering the G0 phase of the cell cycle.
Only one study addresses the regulation of Pol III transcrip-
tion in cells that have entered the G0 phase of the cell cycle.
The model system for this study is the hypertrophic growth
of cardiomyocytes that is induced by serum, endothelin-1 or
phenylephrine. Even if these cells are in the G0 phase of the
cell cycle, they are still able to increase cell mass (hypertrophy)
upon serum stimulation and to a lesser extent by endothelin-1 or
3694 Cell Cycle Volume 9 Issue 18
demonstrated that RPC32α is important for anchorage-indepen-
dent growth of a HeLa S3 carcinoma cell line. Most importantly,
overexpression of RPC32α in IMR90 human embryonic lung
fibroblasts with altered functions of p53 and pRb (through the
expression of papillomaviral E6 and E7 proteins) led to full trans-
formation and immortalization of these cells. Altogether, these
data indicated that the changes in expression of RPC32α were
not only coincident with differentiation or cell transformation,
but that RPC32α may play an active role in these processes.4
In view of these results it is relevant to ask why studies that
examined Pol III transcription during the differentiation of F9
teratocarcinoma cells did not identify the stem cell-specific iso-
form of RNA polymerase III. A possible explanation is that the
genes analyzed in these studies were the well-known Pol III genes
(transfer-, 5S-, U6- or VA1-RNA genes), that can be transcribed
in vitro either by Pol IIIα or by Pol IIIβ.4 As a consequence,
the expression of these genes presumably does not change dur-
ing the process of stem cell differentiation. If this supposition
holds true, the downregulation of RPC32α during differentia-
tion may affect the transcription of genes other than the 5S and
tRNA genes—and these hypothetical yet-to-be-identified genes
may turn out to be important for maintaining cells in an undif-
ferentiated state. The possible existence of such Pol IIIα-specific
genes also implies that the phenotypes observed in the zebraf-
ish (which according to BLAST analyses contains orthologs of
RPC32α and RPC32β) expressing the slim jim mutant may, at
least in part, be attributable to a dysfunction of Pol IIIα. Thus,
the identification of genes that are specifically transcribed by Pol
The importance of Pol III transcription for proliferation and
differentiation and thus for organ development, was further dem-
onstrated in Danio rerio. Expression of a splice mutant of the sec-
ond largest subunit of Pol III (POLR3B Δ239-279 ; slim jim) in
zebrafish led to disruption of the development of digestive organs
and to size reductions in the exocrine pancreas, liver, retina and
terminal branchial arches. Although the strongest expression of
POLR3B was reported in the nervous system 24 hours post fer-
tilization, no neuronal phenotype other than that of the retina
was reported for the slim jim mutant.148 These data suggest that
normal Pol III transcription rates may be crucial for the develop-
ment of rapidly growing cells. In support of this notion, it was
proposed that tissue-specific expression of tRNAs may be coordi-
nated with the codon usage of individual cells, which may in turn
be crucial for tissue or cell type-specific translation regulation.149
Recent data indicate that expression of a subunit of Pol III
itself is also subject to regulation during the process of differenti-
ation. It was shown that RPC32α mRNA expression is negatively
regulated during differentiation of human H1 embryonic stem
cells and that RPC32α mRNA could not be detected in RNA
blots from differentiated tissues (Fig. 4). In addition, RPC32α
mRNA and protein expression were found to increase during
transformation of human embryonic lung IMR90 fibroblasts
by defined genetic elements.4 These data collectively suggested
that RPC32α mRNA and protein expression is negatively regu-
lated during differentiation and upregulated during the malig-
nant transformation of human cells. Furthermore, by employing
siRNAs that specifically suppress RPC32α-expression, it was
Figure 3. Regulation of RNA polymerase III transcription during dierentiation of mouse F9 embryonic carcinoma cells.
www.landesbioscience.com Cell Cycle 3695
(HeLa cells)15 3 analyses, but was not identified by the other stud-
ies. The differential detection of the BC200 gene in individual
studies may be attributable to distinct cut-off values that were
used to distinguish positive and negative results and/or a cell
type-specific expression pattern of the BC200 gene.155 A greater
discrepancy between the different studies and distinct cell types
was observed with respect to the occupancy of tRNA genes by Pol
III and/or subunits of TFIIIC and TFIIIB. This variation may be
attributable to the copy number of individual tRNA genes and
cell type-specific nuclear organization of the genome. In general,
the ChIP-sequencing results do not support earlier reports156 ,157
indicating that micro RNAs may be transcribed by Pol III. In
fact, the only miRNA loci that were found to be associated with
Pol III are hsa-mir565 (a tRNA fragment),150 hsa-m ir-1975 (over-
laps with the hy5 gene)151,15 2 and hsa-mir-886 (overlaps with a
vault gene).151-15 4 Thus, all these micro RNA genes overlap genes
that possess classical Pol III promoters. One single microRNA
gene (has-mir-498) has been described to associate with Pol III
in CD4+ cells but not in HeLa cells.150 The number of novel Pol
III genes is difficult to assess, although tens to hundreds of can-
didate genes, depending on the study, have been identified. These
genes include SINEs, snaR loci, sn/snoRNA loci and unanno-
tated genes.
Interestingly, two of the five studies employed antibod-
ies directed against RPC32α for ChIPs.153 ,15 4 Some of the
genes identified in these studies may represent Pol IIIα-specific
genes, although the specificity of the antibodies for Pol IIIα
versus Pol IIIβ in immunoprecipitation assays remains to be
proven. At present, it cannot be excluded that the genomic loci
IIIα and that cannot be transcribed by Pol IIIβ will be important
for understanding the roles of Pol IIIα and Pol IIIβ in growth
and development. A first step towards the identification of such
genes that may fulfil important functions in growth and differ-
entiation has been made by establishing a genome-wide map of
RNA polymerase III DNA interactions.
The Human Pol III Transcriptome
Recently, the results from five independent genome-wide chroma-
tin immunoprecipitation (ChIP) sequencing projects in human
cells have been published.150 -15 4 Antibodies against several dis-
tinct subunits of Pol III (RPC155/POLR3A; RPC53/POLR3D;
RPC32/POLR3G) and its transcription factors (BRF1; BRF2;
BDP1; TFIIIC220; TFIIIC110; TFIIIC63; PTFδ/SNAPc2/
SNAP45) were employed for determining the genes that are in
contact with these proteins and presumably transcribed in a vari-
ety of untransformed, immortalized or transformed cell lines
(IMR90 fibroblasts/TERT; human foreskin fibroblasts/TERT;
Jurkat; HEK; HeLa; K562; CD4+ cells). As expected, at least
one copy of each multicopy gene (individual tRNA, 5S, 7SL and
U6 genes) is associated with Pol III and BRF1 (TFIIIB-β) or
BRF2 (TFIIIB-α).151-15 4 Single-copy Pol III-transcribed genes
include the 7SK, RNAse MRP, RNAse P, BC200, U6atac and
vault-1, -2 and -3 genes). Of these, the 7SK, U6atac, RNAse
MRP, RNAse P and vault genes were reported to be associated
with Pol III in each of the studies. BC200 was crosslinked to Pol
III and identified in the ChIP-seq (K562 cells)152 and microarray
Figure 4. Regulation of RNA polymerase III transcription during dierentiation of human H1 embryonic stem cells.
3696 Cell Cycle Volume 9 Issue 18
facilitated by a direct TFIIIC-mediated recruitment of the his-
tone acetyl transferase p300,159 which could contribute to the his-
tone acetylation161 observed at these promoters in cells.
In addition to local chromatin modifications that are impor-
tant for Pol III transcription, tRNA-induced and condensin-
mediated organization of chromatin domains results in the
recruitment of tRNA genes to the nucleolus in S. cerevisiae.162,163
This could result in the co-recruitment of Pol II genes to these
sites and placement of the tRNA genes into a chromatin con-
text that may be favorable for their expression or repression.
Moreover, perinucleolar domains (PNC) have been shown to
form in tumor cells in a Pol III-dependent manner164 and may
represent sites that are favorable for gene expression. With respect
to PNCs, it remains to be established whether Pol IIIα contrib-
utes to the formation of these subcellular structures. If so, the
ability of Pol IIIα to contribute to cell transformation may be
attributable to repositioning Pol II genes involved in cell trans-
formation or tumor suppression into actively transcribed or
repressed domains, respectively. In summary, the genome-wide
determination of Pol III-DNA associations provides important
information about the genes that are in contact with Pol III in
several distinct cell lines and a closer analysis of these genes may
reveal the ones that contribute to essential functions of Pol III in
cell growth and differentiation.
Conclusion
Research in recent years has contributed to an improved under-
standing of how RNA polymerase III-mediated transcription
is regulated in yeast and mammals. There has been consider-
able progress with respect to an understanding of the molecular
mechanisms that link cell growth and cell cycle control to Pol
III transcription in lower and higher eukaryotes. This under-
standing has also allowed an appreciation of the importance of
Pol III transcription for cell transformation and for sustaining
tumor growth. Recent data have linked Pol III-mediated tran-
scription to the differentiation of EC and of ES cells, indicat-
ing that the transcription of small untranslated RNAs by Pol III
is essential not only for the regulation of growth, but also for
the regulation of differentiation. Future research will provide a
more detailed understanding of how Pol III contributes to these
processes, including the specific functions of the two Pol III
isoforms. Acknowledgements
We thank Giorgio Dieci for critically reading the manuscript.
This work has been supported by grants from the Conseil
Régional d’Aquitaine and the European Regional Development
Fund (to M.T.), from the Agence Nationale de la Recherche
(ANR) “REGPOLSTRESS” (to M.T.) and from the Ligue
Contre le Cancer-Comités Gironde and Dordogne (to M.T).
R.G.R. was supported by grants (CA113872, DK071900 and
CA113872) from the National Institutes of Health.
identified in these studies also contain Pol IIIβ-transcribed genes,
such that Pol IIIα-specific genes remain unequivocally to be
determined.
Pol III, Chromatin and Higher Order Genome
Organization
Chromatin modifications found near Pol III-bound genes
share features with the histone modifications found at actively
transcribed Pol II genes, including histone H3K4me3 or
H3K9Ac.150 ,152 ,15 3 However, other positively acting histone modi-
fications described at Pol II transcribed genes, namely the RPB1
CTD-dependent, Set2-mediated H3K36me3 or H3K79me2
were absent at transcribed Pol III genes. Furthermore, H3K27me3
was rarely found at actively transcribed Pol III promoters.150
In addition to the discovery of novel genes that may be tran-
scribed by Pol III, two of the publications also described sites
within the genome that are occupied by TFIIIC, but not by Pol
III (extra TFIIIC [ETC] loci).152 ,153 These sites are often found
in close vicinity to genes transcribed by Pol II and may represent
boundary or insulator elements, similar to what was reported in
yeast.158 TFIIIC may bind to ETCs via a novel DNA recogni-
tion sequence152 and might also directly recruit histone modify-
ing factors.159 CCCTC-binding factor (CTFC) also was detected
at these ETC sites152 and at a subset of tDNAs with the highest
enrichment of Pol III.153 The association of the cohesin-interact-
ing protein CTCF with TFIIIC-bound ETCs or tDNA genes
is reminiscent of a situation described in yeast wherein tDNAs
and Pol III-associated proteins have been reported to contribute
to the establishment of sister chromatid cohesion at the
HMR locus.160
c-Myc was crosslinked close to Pol III promoters, in sup-
port of a role for c-Myc in activating Pol III transcription.154
Surprisingly, in addition to associations with Pol III transcription
factors, c-Myc and Pol III itself, many of the transcribed Pol III
genes were also in close proximity to Pol II transcription activa-
tors such as Fos and Jun15 4 or Ets1 and STAT1.153 Moreover, basal
Pol II transcription factors and the cyclin T1 subunit of elonga-
tion factor PTEFb could be crosslinked close to sites occupied
by Pol III.150 Most importantly, Pol II was similarly enriched at
Pol III-transcribed genes,151-15 4 often upstream of the transcrip-
tion initiation site and sometimes attributable to the presence of
Pol II promoters within a distance of 2 kb.153 Notably, in about
two-thirds of the cases these Pol II promoters directed transcrip-
tion by Pol II in the opposite direction of that of Pol III. The co-
occupation of these sites by Pol II and Pol III has been interpreted
as being advantageous for Pol III-mediated transcription as a
result of Pol II-dependent chromatin modifications. However,
this assumption has not been experimentally validated and alter-
native scenarios can be envisaged. In favor of direct interactions
of Pol III components with chromatin-modifying enzymes, it has
been shown that Pol III transcription of a chromatin template is
www.landesbioscience.com Cell Cycle 3697
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... Expression of genes regulated by intragenic promoters requires the six subunit transcription factor TFIIIC (type 1 and 2 promoters) and the transcription factor TFIIIA (type 1 promoter only) to recruit the transcription initiation factor TFIIIB-β (Figures 2A,B). The regulatory elements upstream of the TSS in type 3 and the promoter of the selenocysteine tRNA (tRNA Sec ) gene are recognized by STAF/ZNF143 and OCT1 (DSE), as well as by SNAPc/PTF (PSE), which stimulate the recruitment of TFIIIB-α to the TSS, whereupon Pol III is recruited (reviewed in Schramm and Hernandez (2002), Dumay-Odelot et al. (2010); Figure 2C). TFIIIB-α is composed of the TATA-binding protein (TBP), the B double prime 1 (BDP1) component and the TFIIB-related factor 2 (BRF2), whereas TFIIIB-β contains the TFIIB-related factor 1 (BRF1) instead of BRF2 ( Figure 2) (Teichmann and Seifart, 1995;Teichmann et al., 2000;Schramm et al., 2000;reviewed in Schramm and Hernandez (2002), Dumay-Odelot et al. (2010)). ...
... The regulatory elements upstream of the TSS in type 3 and the promoter of the selenocysteine tRNA (tRNA Sec ) gene are recognized by STAF/ZNF143 and OCT1 (DSE), as well as by SNAPc/PTF (PSE), which stimulate the recruitment of TFIIIB-α to the TSS, whereupon Pol III is recruited (reviewed in Schramm and Hernandez (2002), Dumay-Odelot et al. (2010); Figure 2C). TFIIIB-α is composed of the TATA-binding protein (TBP), the B double prime 1 (BDP1) component and the TFIIB-related factor 2 (BRF2), whereas TFIIIB-β contains the TFIIB-related factor 1 (BRF1) instead of BRF2 ( Figure 2) (Teichmann and Seifart, 1995;Teichmann et al., 2000;Schramm et al., 2000;reviewed in Schramm and Hernandez (2002), Dumay-Odelot et al. (2010)). Hybrid promoters display gene-specific transcription factor requirements. ...
... Frontiers in Molecular Biosciences | www.frontiersin.org July 2021 | Volume 8 | Article 696438 3 promoters in multicellular organisms (Carbon and Krol, 1991;Meissner et al., 1994;reviewed in Dieci et al. (2007), Dumay-Odelot et al. (2010)). Furthermore, only the promoters that depend on a PSE and SNAPc/PTF transcription factors recruit the BRF2-containing TFIIIB-α transcription initiation factor, whereas other enhancer-activator combinations with geneinternal A-and B-Boxes result in the recruitment of the BRF1-containing TFIIIB-β. ...
Article
Full-text available
RNA polymerase (Pol) III transcribes small untranslated RNAs such as 5S ribosomal RNA, transfer RNAs, and U6 small nuclear RNA. Because of the functions of these RNAs, Pol III transcription is best known for its essential contribution to RNA maturation and translation. Surprisingly, it was discovered in the last decade that various inherited mutations in genes encoding nine distinct subunits of Pol III cause tissue-specific diseases rather than a general failure of all vital functions. Mutations in the POLR3A, POLR3C, POLR3E and POLR3F subunits are associated with susceptibility to varicella zoster virus-induced encephalitis and pneumonitis. In addition, an ever-increasing number of distinct mutations in the POLR3A, POLR3B, POLR1C and POLR3K subunits cause a spectrum of neurodegenerative diseases, which includes most notably hypomyelinating leukodystrophy. Furthermore, other rare diseases are also associated with mutations in genes encoding subunits of Pol III (POLR3H, POLR3GL) and the BRF1 component of the TFIIIB transcription initiation factor. Although the causal relationship between these mutations and disease development is widely accepted, the exact molecular mechanisms underlying disease pathogenesis remain enigmatic. Here, we review the current knowledge on the functional impact of specific mutations, possible Pol III-related disease-causing mechanisms, and animal models that may help to better understand the links between Pol III mutations and disease.
... POLR3B is the second largest subunit of Pol III and, together with POLR3A, forms the catalytic center of the enzyme. 6 In 2011, biallelic mutations in POLR3B were reported to cause a rare hypomyelinating leukodystrophy, 7 whereas in 2021, de novo missense mutations in POLR3B were associated with afferent ataxia, spasticity, variable intellectual disability, and epilepsy, and predominantly demyelinating sensorimotor peripheral neuropathy. 8 Heterozygous mutations in POLR3B could result in a demyelinating CMT phenotype without any additional neurological or extraneurological involvement. ...
Article
Full-text available
Background: Biallelic POLR3B mutations cause a rare hypomyelinating leukodystrophy. De novo POLR3B heterozygous mutations were recently associated with afferent ataxia, spasticity, variable intellectual disability, and epilepsy, and predominantly demyelinating sensorimotor peripheral neuropathy. Methods: We performed whole-exome sequencing (WES) of DNA samples from 804 Charcot-Marie-Tooth (CMT) cases that could not be genetically diagnosed by DNA-targeted resequencing microarray using next-generation sequencers. Using WES data, we analyzed the POLR3B mutations and confirmed their clinical features. Results: We identified de novo POLR3B heterozygous missense mutations in two patients. These patients presented with early-onset demyelinating sensorimotor neuropathy without ataxia, spasticity, or cognitive impairment. Patient 1 showed mild cerebellar atrophy and spinal cord atrophy on magnetic resonance imaging and eventually died of respiratory failure in her 50s. We classified these mutations as pathogenic based on segregation studies, comparison with control database, and in silico analysis. Conclusion: Our study is the third report on patients with demyelinating CMT harboring heterozygous POLR3B mutations and verifies the pathogenicity of POLR3B mutations in CMT. Although extremely rare in our large Japanese case series, POLR3B mutations should be added to the CMT-related gene panel for comprehensive genetic screening, particularly for patients with early-onset demyelinating CMT.
... POLR3A (#607694) and POLR3B (#614381): POLR3A, the largest of the 17 subunits that make up RNA polymerase III (Pol III), and POLR3B, the second-largest subunit, together form the catalytic center of the enzyme, which transcribes small untranslated RNAs such as tRNA [86]. Recessive mutations in these genes have been reported in association with the 4H syndrome, which is characterized by hypomyelination, hypodontia, and hypogonadotropic hypogonadism [87,88]. ...
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Idiopathic hypogonadotropic hypogonadism (IHH) is a group of rare developmental disorders characterized by low gonadotropin levels in the face of low sex steroid hormone concentrations. IHH is practically divided into two major groups according to the olfactory function: normal sense of smell (normosmia) nIHH, and reduced sense of smell (hyposmia/anosmia) Kallmann syndrome (KS). Although mutations in more than 50 genes have been associated with IHH so far, only half of those cases were explained by gene mutations. Various combinations of deleterious variants in different genes as causes of IHH have been increasingly recognized (Oligogenic etiology). In addition to the complexity of inheritance patterns, the spontaneous or sex steroid-induced clinical recovery from IHH, which is seen in approximately 10–20% of cases, blurs further the phenotype/genotype relationship in IHH, and poses challenging steps in new IHH gene discovery. Beyond helping for clinical diagnostics, identification of the genetic mutations in the pathophysiology of IHH is hoped to shed light on the central governance of the hypothalamo-pituitary-gonadal axis through life stages. This review aims to summarize the genetic etiology of IHH and discuss the clinical and physiological ramifications of the gene mutations.
... In vitro experiments also support a direct role of RNF216 in GN11 cell migration [117]. No murine studies are available for OTUD4, but its biological role has been validated through morpholino-mediated knockdown in zebrafish [114] Polymerase III is an enzyme involved in the transcription of small untranslated RNAs and is required for the regulation of essential cellular processes [118]. This enzyme is composed of many subunits including POLR3A (chr 10q22.3) ...
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Gonadotropin releasing hormone (GnRH) neurons are hypothalamic neuroendocrine cells that control sexual reproduction. During embryonic development, GnRH neurons migrate from the nose to the hypothalamus, where they receive inputs from several afferent neurons, following the axonal scaffold patterned by nasal nerves. Each step of GnRH neuron development depends on the orchestrated action of several molecules exerting specific biological functions. Mutations in genes encoding for these essential molecules may cause Congenital Hypogonadotropic Hypogonadism (CHH), a rare disorder characterized by GnRH deficiency, delayed puberty and infertility. Depending on their action in the GnRH neuronal system, CHH causative genes can be divided into neurodevelopmental and neuroendocrine genes. The CHH genetic complexity, combined with multiple inheritance patterns, results in an extreme phenotypic variability of CHH patients. In this review, we aim at providing a comprehensive and updated description of the genes thus far associated with CHH, by dissecting their biological relevance in the GnRH system and their functional relevance underlying CHH pathogenesis.
... Pol III transcription is up-regulated with cell growth and cell proliferation (Goodfellow and White 2007;Dumay-Odelot et al. 2010), and TFIIIB is a target of both tumor suppressors and oncoproteins. This raises the possibility of cancer-associated deregulation of TFIIIB subunits and subunits of other Pol III transcription factors. ...
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RNA polymerase (Pol) III is responsible for transcription of different noncoding genes in eukaryotic cells, whose RNA products have well-defined functions in translation and other biological processes for some, and functions that remain to be defined for others. For all of them, however, new functions are being described. For example, Pol III products have been reported to regulate certain proteins such as protein kinase R (PKR) by direct association, to constitute the source of very short RNAs with regulatory roles in gene expression, or to control microRNA levels by sequestration. Consistent with these many functions, deregulation of Pol III transcribed genes is associated with a large variety of human disorders. Here we review different human diseases that have been linked to defects in the Pol III transcription apparatus or to Pol III products imbalance and discuss the possible underlying mechanisms.
... Pol III is the largest RNA polymerase with 17 subunits (1), 5 of which are specific to Pol III (10). Three of the five Pol IIIspecific subunits (POLR3C/RPC62, POLR3F/RPC39, and POLR3G/ RPC32) form a stable subcomplex with a selective and critical function in transcription initiation (11). ...
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Significance Mammalian cells contain two RNA polymerase III isoforms that differ only in ubiquitous POLR3GL and developmentally regulated POLR3G subunits. Here, in contradiction to previous conclusions from POLR3G knockdown analyses, we show that POLR3G and POLR3GL are functionally redundant and, in the context of embryonic stem cell differentiation, can largely compensate for each other when expressed at appropriate levels. Moreover, whereas Polr3g knockout mice die at an early embryonic stage, Polr3gl knockout mice complete embryonic development but die at weaning with signs of both general growth defects and potential cerebellum-related neuronal defects. As interests grow in reported Pol III-related disorders, Polr3gl knockout mice provide a convenient model to study the physiological effect of variations in Pol III functions in more detail.
... Pol III is an enzyme involved in the transcription of small noncoding RNAs (such as tRNAs, 5S RNA, 7SK RNA, and U6 RNA) that play a role in processes such as transcription regulation, RNA processing, ribosome assembly, and translation, which ultimately lead to protein synthesis. The transcription of small noncoding RNAs by Pol III plays an essential role in cell growth and differentiation (Dumay-Odelot, Durrieu-Gaillard, Da Silva, Roeder, & Teichmann, 2010). Recently, it was discovered that 4H leukodystrophy can also be caused by biallelic pathogenic variants in POLR1C, another subunit of Pol III (Thiffault et al., 2015). ...
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4H leukodystrophy, also known as Pol III‐related leukodystrophy, is a rare autosomal recessive neurodegenerative disorder characterized by hypomyelination, hypodontia, and hypogonadotropic hypogonadism. It is caused by biallelic mutations in POLR3A, POL3RB, or POLR1C. So far, only two patients have been described with homozygosity for the common c.1568T>A (p.Val523Glu) POLR3B mutation, both of them showing a remarkably mild clinical course. Here, we report another patient with homozygosity for the same mutation, but with a more severe phenotype including ataxia, developmental delay, and intellectual disability. This information is of importance for clinicians to provide comprehensive counseling to patients with 4H leukodystrophy and their families.
... 1,2 The largest subunit of human RNA polymerase III is encoded by POLR3A; the RNA polymerase III transcribes untranslated RNA that are necessary for translation, transcription, and protein translocation; and RNA polymerase III is involved in the regulation of growth and the differentiation of cells. 3,4 Herein we describe a case of a POLR3A gene mutation presenting with a phenotype characterized by adult-onset spastic paraplegia and dystonia and minor changes in brain magnetic resonance imaging (MRI). ...
... POLR2C encodes the largest subunit of RNA polymerase II, which synthesizes messenger RNA in eukaryotes (125). A heterozygous nonsense mutation in POLR2C (c.454A>T:p.Lys152*) was identified in a woman with familial POI who was also diagnosed with immune thrombocytopenia, pernicious anemia, and hypothyroidism. ...
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Hypomyelinating leukodystrophies are a group of genetic disorders characterized by insufficient myelin deposition during development. A subset of hypomyelinating leukodystrophies, named RNA polymerase III (Pol III or POLR3)-related leukodystrophy or 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) leukodystrophy, was found to be caused by biallelic variants in genes encoding subunits of the enzyme Pol III, including POLR3A, POLR3B, POLR3K, and POLR1C. Pol III is one of the three nuclear RNA polymerases that synthesizes small non-coding RNAs, such as tRNAs, 5S RNA, and others, that are involved in the regulation of essential cellular processes, including transcription, translation and RNA maturation. Affinity purification coupled with mass spectrometry (AP-MS) revealed that a number of mutations causing POLR3-related leukodystrophy impair normal assembly or biogenesis of Pol III, often causing a retention of the unassembled subunits in the cytoplasm. Even though these proteomic studies have helped to understand the molecular defects associated with leukodystrophy, how these mutations cause hypomyelination has yet to be defined. In this review we propose two main hypotheses to explain how mutations affecting Pol III subunits can cause hypomyelination.
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The mitotic state is associated with a generalized repression of transcription. We show that mitotic repression of RNA polymerase III transcription can be reproduced by using extracts of synchronized HeLa cells. We have used this system to investigate the molecular basis of transcriptional repression during mitosis. We find a specific decrease in the activity of the TATA-binding-protein (TBP)-containing complex TFIIIB. TBP itself is hyperphosphorylated at mitosis, but this does not appear to account for the loss of TFIIIB activity. Instead, one or more TBP-associated components appear to be regulated. The data suggest that changes in the activity of TBP-associated components contribute to the coordinate repression of gene expression that occurs at mitosis.
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RNA polymerase III (Pol III) transcription is subject to repression by the retinoblastoma protein RB, both in vitro and in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88–90, 1996). This is achieved through a direct interaction between RB and TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061–2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755–14761, 1997). p107 and p130 are two closely related proteins that display 30 to 35% identity with the RB polypeptide and share some of its functions. We show that p107 and p130 can both repress Pol III transcription in transient transfection assays or when added to cell extracts. Pull-down assays and immunoprecipitations using recombinant components demonstrate that a subunit of TFIIIB interacts physically with p107 and p130. In addition, endogenous TFIIIB is shown by cofractionation and coimmunoprecipitation to associate stably with both p107 and p130. Disruption of this interaction in vivo by using the E7 oncoprotein of human papillomavirus results in a marked increase in Pol III transcription. Pol III activity is also deregulated in fibroblasts derived from p107 p130 double knockout mice. We conclude that TFIIIB is targeted for repression not only by RB but also by its relatives p107 and p130.
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One of the major challenges for developmental biologists and investigators in the field of diabetes over the last few decades has been to dissect the origin of pancreatic endocrine cells and to accurately understand the mechanisms that regulate islet cell regeneration. While significant advances have been made recently, there continues to be a paucity of knowledge regarding the growth factor signalling pathways that directly regulate the proteins involved in islet cell cycle control. We will discuss recent work in these areas and provide insights from our studies into age-dependent alterations in the expression of growth factor signalling proteins and cell cycle proteins in islet cells.
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Transcriptional coactivators that regulate the activity of human RNA polymerase III (Pol III) in the context of chromatin have not been reported. Here, we describe a completely defined in vitro system for transcription of a human tRNA gene assembled into a chromatin template. Transcriptional activation and histone acetylation in this system depend on recruitment of p300 by general initiation factor TFIIIC, thus providing a new paradigm for recruitment of histone-modifying coactivators. Beyond its role as a chromatin-modifying factor, p300 displays an acetyltransferase-independent function at the level of preinitiation complex assembly. Thus, direct interaction of p300 with TFIIIC stabilizes binding of TFIIIC to core promoter elements and results in enhanced transcriptional activity on histone-free templates. Additional studies show that p300 is recruited to the promoters of actively transcribed tRNA and U6 snRNA genes in vivo. These studies identify TFIIIC as a recruitment factor for p300 and thus may have important implications for the emerging concept that tRNA genes or TFIIIC binding sites act as chromatin barriers to prohibit spreading of silenced heterochromatin domains.
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The c-Myc oncoprotein and its dimerization partner Max bind the DNA core consensus sequence CACGTG (E-box) and activate gene transcription. However, the low levels of induction have hindered the identification of novel Myc target genes by differential screening techniques. Here, we describe a computer-based pre-selection of candidate Myc/Max target genes, based on two restrictive criteria: an extended E-box consensus sequence for Myc/Max binding and the occurrence of this sequence within a potential genomic CpG island. Candidate genes selected by these criteria were evaluated experimentally for their response to Myc. Two Myc target genes are characterized here in detail. These encode nucleolin, an abundant nucleolar protein, and BN51, a co-factor of RNA polymerase III. Myc activates transcription of both genes via E-boxes located in their first introns, as seen for several well-characterized Myc targets. For both genes, mutation of the E-boxes abolishes transcriptional activation by Myc as well as repression by Mad1. In addition, the BN51 promoter is selectively activated by Myc and not by USF, another E-box-binding factor. Both nucleolin and BN51 are implicated in the maturation of ribosomal RNAs, albeit in different ways. We propose that Myc, via regulation of these and probably many other transcriptional targets, may be an important regulator of ribosome biogenesis.
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The hepatitis B virus X gene product transactivates a variety of cellular and viral genes. The mechanism for X induction of RNA polymerase (pol) III genes was investigated. By using Drosophila S-2 cells stably transformed with the X gene, the transient expression of a tRNA gene is enhanced. Comparing the transcriptional activities of extracts derived from these cells, all three types of RNA pol III promoters are stimulated by X. Interestingly, both S-2 and rat 1A cells stably transformed with the X gene produce increased cellular levels of the TATA-binding protein (TBP). By using various kinase inhibitors, it was found that the X-mediated increases in both transcription and TBP are dependent upon protein kinase C activation. Since TBP is a subunit of TFIIIB, the activity of this component fractionated from extracts derived from control and X-transformed cells was analyzed. These studies reveal that TFIIIB activity is substantially more limiting in control cells and that TFIIIB isolated from X-transformed cells has increased activity in reconstitution assays compared with TFIIIB isolated from control cells. Conversely, comparison of TFIIIC from control and X-transformed cell extracts revealed that there is relatively little change in its ability either to reconstitute transcription or to bind to DNA and that there is no change in the catalytic activity of RNA pol III. Studies were performed to determine whether directly increasing cellular TBP alone could enhance RNA pol III gene transcription. Transient expression of a TBP cDNA in rat 1A cells was capable of stimulating transcription activity from the resultant extracts in vitro. Together, these results demonstrate that one mechanism by which X mediates transactivation of RNA poll III genes is by increasing limiting TBP via the activation of cellular signaling pathways. The discovery that X increases cellular TBP, the universal transcription factor, provides a novel mechanism for the function of a viral transactivator protein and may explain the ability of X to produce such large and diverse effects on cellular gene expression.
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The 7S particle of Xenopus laevis oocytes contains 5S RNA and a 40‐K protein which is required for 5S RNA transcription in vitro. Proteolytic digestion of the protein in the particle yields periodic intermediates spaced at 3‐K intervals and a limit digest containing 3‐K fragments. The native particle is shown to contain 7‐11 zinc atoms. These data suggest that the protein contains repetitive zinc‐binding domains. Analysis of the amino acid sequence reveals nine tandem similar units, each consisting of approximately 30 residues and containing two invariant pairs of cysteines and histidines, the most common ligands for zinc. The linear arrangement of these repeated, independently folding domains, each centred on a zinc ion, comprises the major part of the protein. Such a structure explains how this small protein can bind to the long internal control region of the 5S RNA gene, and stay bound during the passage of an RNA polymerase molecule.
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Transcription initiation at RNA polymerase III promoters requires transcription factor IIIB (TFIIIB), an activity that binds to RNA polymerase III promoters, generally through protein–protein contacts with DNA binding factors, and directly recruits RNA polymerase III.Saccharomyces cerevisiae TFIIIB is a complex of three subunits, TBP, the TFIIB-related factor BRF, and the more loosely associated polypeptide β″. Although human homologs for two of the TFIIIB subunits, the TATA box–binding protein TBP and the TFIIB-related factor BRF, have been characterized, a human homolog of yeast B″ has not been described. Moreover, human BRF, unlike yeast BRF, is not universally required for RNA polymerase III transcription. In particular, it is not involved in transcription from the small nuclear RNA (snRNA)–type, TATA-containing, RNA polymerase III promoters. Here, we characterize two novel activities, a human homolog of yeast B″, which is required for transcription of both TATA-less and snRNA-type RNA polymerase III promoters, and a factor equally related to human BRF and TFIIB, designated BRFU, which is specifically required for transcription of snRNA-type RNA polymerase III promoters. Together, these results contribute to the definition of the basal RNA polymerase III transcription machinery and show that two types of TFIIIB activities, with specificities for different classes of RNA polymerase III promoters, have evolved in human cells. Keywords • human TFIII • human B″ • BRFU • U6 snRNA gene • RNA polymerase III
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Transcription of class III genes is conducted by multi-protein complexes consisting of polymerase III itself and several transcription factors. We established a reconstituted in vitro transcription system from which the autoantigen La was removed by immunodepletion, This system showed no RNP formation, but was still fully active in transcription. Supplementing such La-free transcription reactions with recombinant La restored the formation of La complexes with the newly synthesised RNA, but did not lead to enhanced transcription efficiency. Furthermore, we developed a technique for the generation and isolation of transcription complexes, assembled from purified transcription factors and isolated by glycerol centrifugation. These complexes were fully competent to re-initiate RNA synthesis but they were not associated with La and their transcription rate could not be stimulated by addition of recombinant La. Therefore, we conclude that La does not act as a human polymerase III transcription factor.