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In the course of evolution of multi-cellular eukaryotes, paralogs of general transcription factors and RNA polymerase subunits emerged. Paralogs of transcription factors and of the RPC32 subunit of RNA polymerase III play important roles in cell type- and promoter-specific transcription. Here we discuss their respective functions.
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Transcription 1:3, 130-135; November/December 2010; © 2010 Landes Bioscience
130 Transcription Volume 1 Issue 3
Key words: RNA polymerase III,
Submitted: 07/15/10
Revised: 07/29/10
Accepted: 07/30/10
Previously published online:
DOI: 10.4161/trns.1.3.13192
*Correspondence to: Martin Teichmann;
In the course of evolution of multi-
cellular eukaryotes, paralogs of gen-
eral transcription factors and RNA poly-
merase subunits emerged. Paralogs of
transcription factors and of the RPC32
subunit of RNA polymerase III play
important roles in cell type- and pro-
moter-specific transcription. Here we
discuss their respective functions.
Three distinct DNA-dependent RNA
polymerases (Pol I, Pol II and Pol III)
have been identified in eukaryotes more
than 40 years ago.1 The presence of several
RNA polymerases in eukaryotes versus
one single RNA polymerase in prokary-
otes2 reflects increased complexity of the
transcription systems that may have been
necessary to accommodate the transcrip-
tion of an increased number of genes. Such
a gain in complexity may have allowed the
evolution of novel genes or groups of genes
that require the presence of more than one
polymerase for individual regulation.
A further level of complexity has been
added to transcription systems in arche-
bacteria and in eukaryotic cells by the
evolution of general transcription fac-
tors, replacing as an entity eubacterial
sigma factors. In addition, in multicellu-
lar eukaryotes, including insects and ver-
tebrates, paralogs of some of the general
transcription factors have evolved that
fulfill cell type or gene-specific functions.
Furthermore, paralogs of RNA poly-
merase subunits arose during evolution. As
a consequence, higher eukaryotes, includ-
ing plants and vertebrates, contain more
than three individual DNA-dependent
RNA polymerases.3,4 Here, we review
General transcription factors and subunits of RNA polymerase III
Paralogs for promoter- and cell type-specific transcription in multicellular eukaryotes
Martin Teichmann,1,* Giorgio Dieci,2 Chiara Pascali1, 2 and Galina Boldina1
1Institut Européen de Chimie et Biologie (I.E.C.B.); Université de Bordeaux; Institut National de la Santé et de la Recherche Médicale (INSERM) U869;
Pessac, France; 2Dipartimento di Biochimica e Biologia Molecolare; Università degli Studi di Parma ; Viale G.P.; Usberti, Parma, Italy
our current knowledge of the existence
and function of paralogous components
of general transcription machineries and
particularly concentrate on components
that have been shown to function in Pol
III transcription or that may contribute
to Pol III transcription under certain cir-
cumstances (Table 1).
Pol III transcribes small untranslated
RNAs that are required for essential cel-
lular processes including the regulation
of transcription (7SK RNA, Alu RNAs),
RNA processing (U6 snRNA, RNase P
RNA, RNase MRP RNA) and translation
(5S rRNA, tRNAs). Distinct types of pro-
moters (Fig. 1A) are recognized by pro-
moter-specific sets of transcription factors
(Fig. 1B). In mammalian cells, the gene-
internal type 1 promoter is recognized
by the gene-specific transcription factor
TFIIIA, which in turn recruits TFIIIC,
TFIIIB-β and finally Pol III itself to the
5S gene. Gene-internal type 2 promot-
ers (e.g., tRNA-, Alu-, adenoviral VA1-
and VA2-genes) are directly recognized
by TFIIIC, allowing the recruitment of
TFIIIB-β and subsequently of Pol III.
Type 3 promoters (U6-, 7SK-, RNase P-,
RNase MRP-genes) are recognized by the
PSE-binding transcription factor (PTF;
also known as SNAPc and PBP), followed
by the recruitment of TFIIIB-α and of Pol
III (Fig. 1B).5,6
Paralogs of TFIIIC Subunits
TFC7/t55/TFIIIC35-related factors. For
more than 10 years, Saccharomyces cere-
visiae (Sc) TFIIIC has been known to
be composed of six subunits (TFC4, Transcription 131
each of which consists of 5 alpha-helices
that together adopt a cyclin fold.10 In
vertebrate cells, two distinct TFIIIB-
activities have been described that both
comprise TBP and BDP1, but that differ
by either containing TFIIB-related fac-
tor 1 (BRF1; component of TFIIIB-β;
transcription of type 1 and 2 promot-
ers; Fig. 1B) or TFIIB-related factor 2
(BRF2; component of TFIIIB-α).6 ,11,12
TFIIIB-α is active in transcription of Pol
III genes with gene regulatory elements
that are entirely located upstream of the
transcription initiation site (type 3 pro-
moter; Fig. 1B) and the BRF2 component
of TFIIIB-α has also been cross-linked
to genes that contain a combination of
gene-internal and gene-external promoter
elements.13,14 The evolution of a second
TFIIB-related factor at the emergence of
vertebrates may have permitted or may
have been tolerated by the co-evolution
of a novel Pol III promoter type. With
respect to the question of whether the
evolution of BRF2 led to the co-evolution
of a novel promoter type it is notewor-
thy to mention that promoter elements
upstream of the transcription initiation
site have also been identified for the U6
and 7SK genes in Drosophila melanogaster
(Dm; Fig. 1A).15 Despite the gene regula-
tory elements being located upstream of
the transcription initiation site, only one
isoform of a DmTFIIB-related factor has
been described. These results indicate that
the evolution of upstream promoters in
the Pol III transcription system, at least in
insects, may not have been driven by the
evolution of a second isoform of a TFIIB-
related factor, although we cannot exclude
that BRF2 sequences may have been lost
in the course of evolution in these species.
If ever the appearance of type 3 promot-
ers may not be attributable to the evolu-
tion of BRF2 it may be asked whether
BRF1 or BRF2 possess other gene- or cell
type-specific functions. Today, we only
know a single gene in vertebrates (coding
for BC200 RNA in humans or the func-
tionally analogous BC1 RNA in rodents)
that is transcribed by Pol III and that
shows neuronal-specific expression.5 ChIP
sequencing and ChIP-on-CHIP experi-
ments demonstrated that the BC200 gene
is in physical contact with Pol III.13,16
ChIP sequencing further showed the
regions of homology to both a subunit of
TFIIIC and a subunit of PTF raises the
possibility that at least one subunit of
TFIIIC and of PTF may have evolved from
a common ancestor protein. Interestingly,
genomes of Dpp and Dp encode in addi-
tion a smaller protein of 150 amino acids
(XP_002134843 [Dpp]; XP_002021117
[Dp]) that is highly related (82% identical
and 92% homologous) to the N-terminal
144 amino acids of the two larger pro-
teins (537 amino acids [Dp] or 538 amino
acids [Dpp]) and that may represent the
TFC7 subunit of TFIIIC in the respec-
tive Drosophila (Fig. 2). Importantly, the
proteins of Dpp and Dp, containing the
TFIIIC- and the zf-SNAP_C-signature,
were the only proteins related to Drosophila
melanogaster (Dm)PSEA-binding protein
49 kD, suggesting that these proteins par-
ticipate in UsnRNA transcription in these
species. Thus, these proteins may be both
considered as orthologs of the Dm PSEA-
binding protein 49 kD and as paralogs of
TF C 7.
Paralogs of TFIIIB Subunits
ScTFIIIB is composed of three subunits,
the TATA-binding protein (TBP), TFIIB-
related factor 1 (BR F1) and B double prime
1 (BDP1). Together with TFIIIA, TFIIIC
and Pol III, these three components are
required and sufficient for reconstituting
transcription from all Pol III promoters in
Sc.9 ScBRF1 is a paralog of TFIIB and it
possesses a structure that is similar to that
of TFIIB. It contains an N-terminal zinc
ribbon and two direct imperfect repeats,
TFC1, TFC7, TFC8, TFC3 and TFC6),
whereas only five subunits had been
described in humans (TFIIIC102, 63,
90, 220 and 110; Fig. 1B). Recently, we
identified and characterized the sixth sub-
unit of human (Hs) TFIIIC (TFIIIC35;
Fig. 1B), which is orthologous to
Saccharomyces cerevisiae (Sc) TFC7 (τ55).
We identified TFIIIC35 through a Psi-
Blast search by using a 34 amino acid
sequence of a yeast Schizosaccharomyces
pombe (Sp) protein as a seed, which showed
modest sequence conservation with amino
acids 317–350 of ScTFC7.7 This 34 amino
acid sequence also overlaps with the Pfam
10419:TFIIIC_subunit signature.8 In
order to identify novel proteins that may
represent paralogs of TFIIIC subunits,
we now performed a Psi-Blast search
(BLOSUM 62; exclude Saccharomyceta
from search) with amino acids 321–364
of ScTFC7. Upon the second iteration,
we identified two proteins in two dis-
tinct drosophilidae (XP_001360915.2
in Drosophila pseudoobscura pseudoob-
scura [Dpp] and XP_002017815.1 in
Drosophila persimilis [Dp]) that exhibited
34% sequence identity and 62% sequence
homology of amino acids 39 to 72 with
the amino acids 321–354 of Sc TFC7
(Fig. 2). Surprisingly, amino acids 259 to
510 of the two Drosophila proteins con-
tained a second signature that has been
found to be conserved in PTFβ/SNAP50
subunits of PTF/SNAPc/PBP (29% iden-
tity and 46% homology to amino acids
155 to 402 of human PTFβ); pfam12251:
zf-SNAP50_C; Fig. 2). The presence of a
protein in Dp and in Dpp that contains
Tab le 1. Components of the general S accharomyces cerev isiae Pol III transcription machinery and
their paralogs in Drosophila
Transcription factor/
polymerase subunit Orthologous and/or paralogous transcription factors
Saccharomyces cerevisiae Drosophila Vertebrates
TFIIIC:TFC7 (τ55) XP_002134843 and XP_001360915 in Dpp.
XP_002021117 and XP_002017815 in Dp. TFIIIC35
(NP_788522.1 in Dm)
The Dpp and Dp proteins XP_001360915 and XP_002017815 are 98% identical and 99%
homologous. The proteins XP_002134843 and XP_002021117 are identical. Dp, Drosophila
persimilis; Dpp, Drosophila pseudo obscura pseudoobscura; Dm, Drosophila melanogaster and
132 Transcription Volume 1 Issue 3
Figure 1. For gure legend, see page 133. Transcription 133
TRF3/TBP2. A gene encoding TRF3 was
found in a variety of metazoans, includ-
ing humans, mice, frogs and fish. Specific
functions for TRF3 in differentiation of
mouse myoblasts, in zebrafish hemato-
poiesis and in Xenopus or mouse oocytes
have been suggested.20,22 TRF3/TBP2
knockout studies showed that mice had no
apparent phenotype except females being
sterile due to defective folliculogenesis.23
A possible involvement of TRF3/TBP2
in Pol III transcription has not yet been
reported. However, the high degree of
conservation between the C-terminal part
of human TBP (residues 141–337) and
TRF3 (residues 184–374; 92% identity
and 95% homology) makes it likely that
TRF3 may be functional in Pol III tran-
scription. Moreover, residues in ScTBP
that have been reported to be critical
for the interaction with ScBRF1 (S261;
D263; S282; E284; E286; L287; R299;
V306) or with ScBdp1 (H277) have been
conserved in both, HsTBP and HsTRF3/
TBP2.24 In line with the possibility that
TRF3/TBP2 may be able to replace TBP
transcription of certain genes may not
depend on TBP.
Although first described as a cell type-
specific factor, TRF1 turned later out to
be widely expressed in Dm and to replace
TBP for transcription by Pol III,18 which
was confirmed by ChIP-on-CHIP analy-
ses using affinity-purified anti-TRF1
antibodies and Drosophila genome tiling
arrays.19 In addition to its essential func-
tions in Dm Pol III transcription, TRF1
has also been implicated in the transcrip-
tion of a small subset of Pol II genes.20 No
ortholog of TRF1 could hitherto be iden-
tified in species other than insects.
Several years later, a second para-
log of TBP was identified in mammals,
Drosophila melanogaster, Caenorhabditis
elegans and other metazoans.20 TRF2
was shown to be involved in tran-
scription by Pol II,20 but it was dem-
onstrated that recombinant HsTRF2
was inactive in Pol III transcription in
The third and latest member of the
TBP-family that has been identified is
presence of BDP1 at the BC200 genomic
locus. Interestingly, however, neither
BRF1, nor BRF2 could be detected at the
BC200 gene locus.13,16 This could merely
be a technical problem, but it could also
indicate that transcription initiation of the
BC200 gene may be independent of BRF1
and BRF2. In this case, another, hitherto
unidentified protein may replace BRF1/
BRF2 for transcription of the BC200 gene
in neurons. Taken together, the emergence
of BRF1 and BRF2 during evolution led to
the appearance of two isoforms of TFIIIB
with promoter-specific functions.
TBP-related factors. TBP was once
assumed to be a universal transcrip-
tion factor.17 This point of view was well
understandable at that time, because the
discovery that TBP participates in tran-
scription of all three RNA polymerases
would not have been anticipated some
years earlier and TBP was thought to be
generally indispensable for transcription.
However since then, several paralogs of
TBP have been identified (TRF1; TRF2;
TRF3) and their discovery indicated that
Figure 1 (See opposite page). (A) RNA polymerase III promoter types in humans (Hs), Drosophila melanogaster (Dm) and Saccharo myces cerevisiae (Sc).
Type 1 and 2 promoters are similar, but U6 promoters are dierent in the three species. The complete set of genes that utilize the distinct promoter
types have been determined.5,6 (B) Promoters and transcription factors of the human (vertebrate) Pol III transcription machiner y. Subunits of the PSE-
binding transcription factor (PTF) are indicated by their PTF nomenclature and the molecular masses of the corresponding SNAPc subunits. SNAPc19
has not been reported in PTF.
Figure 2. Schematic representation of the amino acid homology of a Drosophila pseudo obscura pseudoobscura protein (Genbank accession #
XP_001360915) with ScTFC7, with HsPTFβ and with a Drosophila pseudo obscura pseudoobscura protein (Genbank accession # XP_002134843). The
percentage of identical amino acids is indicated by “id” and of homologous amino acids by “ho.” The numbers of amino acids of the individual
proteins, as well as the regions showing homology in between individual proteins are appropriately indicated. The Pfam signature 12251:zf-SNAP50_C
is found in HsPTFβ (amino acids 201–407) and in Dpp XP_001360915 (amino acids 309–515). The Pfam signature 10419:TFIIIC_subunit is contained in
ScTFC7 (amino acids 317–351) and the Dpp proteins XP_001360915 and XP_002134843 (amino acids 35– 69 in both proteins).
134 Transcription Volume 1 Issue 3
to determine how RPC32α/Pol IIIα is
involved in cellular differentiation and
de-differentiation processes and whether
the existence of two Pol III isoforms is
somehow related to cell type- or stage-
specific expression of both classical house-
keeping29 and non-canonical regulatory30
Pol III genes.
This work has been supported by grants
from the Conseil Régional d’Aquitaine
and the European Regional Development
Fund (to M.T.); by grants from the
Agence Nationale de la Recherche (ANR)
“REGPOLSTRESS” (to M.T.), the
Ligue Contre le Cancer-Comités Gironde
and Dordogne (to M.T.), the Italian
Ministry of Education, University and
Research (PRIN 2007 grant to G.D.), the
AICCRE-Regione Emilia Romagna (to
G.D.). C.P. was supported by a doctoral
fellowship from the “Università Italo-
Francese/Université Franco-Italienne.
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RT-qPCR or western blot confirmed low
RPC32α mRNA or protein expression
levels in human embryonic IMR90 fibro-
blasts as well as increased expression levels
during tumoral transformation, whereas
the RPC32β mRNA expression pattern
remained unchanged.4 Functional impor-
tance for RPC32α expression was demon-
strated by RNAi-mediated knockdown of
RPC32α expression. The suppression of
RPC32α led to reduced colony formation
of HeLa cells in soft-agar assays. Thus, the
expression of RPC32α changes cellular
characteristics and it will be important
to determine the underlying molecu-
lar mechanisms of how RPC32α affects
soft-agar growth. It is likely that Pol IIIα-
mediated transcription of genes, which
cannot be expressed by Pol IIIβ represents
the molecular key to understanding the
cellular functions of RPC32α (Pol IIIα).
Overexpression of RPC32α in partially
transformed human IMR90 fibroblasts
strongly enhances the expression of the
7SK gene. However, it can currently not
be assessed to which extent transcription
of the 7SK gene by RPC32α (Pol IIIα)
contributes to the changes in growth
behavior that have been observed.4 7SK
RNA participates in the regulation of
Pol II transcription through alteration
of P-TEFb function.27 However, since
Pol IIIα-induced effects are likely to be
restricted to a limited number of Pol II
genes, rather than being due to a general
deregulation of Pol II transcription,4 it
seems plausible that Pol IIIα-dependent
transcription of other RNAs may also
contribute to RPC32α-induced induction
or maintenance of cell transformation.
The existence of such a gene or group of
genes is likely, because the acquisition
of a novel function for a duplicated gene
product enhances its probability of being
preserved.28 If ever such genes exist, does
their transcription depend on known or
novel promoter structures (types 1–3) and
transcription factors (TFIIIA, TFIIIC,
TFIIIB, PTF)? Today, these questions
cannot be answered but will be of interest
for future research.
Furthermore, from the expression
pattern of RPC32α,4 it may be inferred
that Pol IIIα has a role in maintaining
embryonic stem cells in an undifferenti-
ated state. Therefore, it will be of interest
in Pol III transcription, it has been sug-
gested that TRF3/TBP2 may be a TBP
replacement factor in cells that contain
low levels of TBP.22
Paralogs of RNA
Polymerase III Subunits
Pol III is composed of 17 subunits. Five
of these subunits are shared with Pol I
and II, two with Pol I only (with paralo-
gous subunits in Pol II) and another five
subunits are paralogous to subunits of
Pol I and Pol II. However, five subunits
are Pol III-specific and no paralogous
subunits have been identified in Pols I
and II. Recently, it has been proposed
that four of the five Pol III-specific sub-
units (POLR3E/HsRPC80, POLR3D/
POLR3F/HsRPC39) exhibit sequence/
structure homology to the heterodimeric
general Pol II transcription factors TFIIE
and TFIIF and that they may be consid-
ered as permanently recruited forms of
these transcription factors.25, 26 POLR3G/
HsRPC32 is the only Pol III subunit for
which no homologous polypeptide has
been identified within the Pol I and Pol II
transcription machineries.
RPC32-related factors. As pointed
out above, the emergence of three
DNA-dependent RNA polymerases in
eukaryotes may have allowed a more
sophisticated regulation of individual sets
of genes. Recently, two distinct isoforms
of human Pol III have been reported,
which may allow even further specializa-
tion for transcription of individual genes.
The two isoforms of human Pol III differ
at least in containing either the RPC32α
(Pol IIIα) or the RPC32β (Pol IIIβ) sub-
unit, and they exhibit isoform-specific
expression patterns in distinct cell types.
Northern blot analyses of multiple human
tissues and cell lines demonstrated that
Pol IIIβ is widely expressed, whereas Pol
IIIα expression is only detectable in sev-
eral lymphoma and leukemia cell lines.
As a consequence, RPC32β-containing
Pol IIIβ may be considered as the gen-
eral form of Pol III, whereas RPC32α-
containing Pol IIIα may only be required
in a subset of cells and it may be impor-
tant for sustaining cell type-specific func-
tions. Analyses of RPC32α expression by Transcription 135
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... Chez l'homme, on trouve deux isoformes de l'ARN Pol III selon qu'elle contienne la sous-unité RPC32α (ARN Pol IIIα) ou RPC32β (ARN Pol IIIβ). La forme β est considérée comme la forme générale de l'ARN Pol III, la forme α étant exprimée dans des types cellulaires spécifiques et de manière intéressante dans les cellules indifférenciées (Haurie et al., 2010 ;Teichmann et al., 2010). ...
... On notera l'existence chez les mammifères de trois paralogues de TBP : les TBP-related factors (TRF). Parmi eux, TRF1 et TRF3 sont vraisemblablement les seules formes impliquées dans la transcription par l'ARN Pol III (pour revue, Teichmann et al., 2010). ...
RNA polymerase III synthetizes many small untranslated RNA, including tRNA and 5S rRNA which are essential to cell growth. In this work, we took an interest in RNA polymerase III transcription regulation in the baker’s yeast, Saccharomyces cerevisiae. We have detected Sub1 on all class III genes in vivo. We also observed that Sub1 is able to stimulate RNA polymerase III transcription which has been reconstituted in vitro with TFIIIB et TFIIIC recombinants factors and purified RNA polymerase III. Sub1 stimulates two steps of RNA polymerase III transcription : initiation and facilitated reinitiation. Supplementary experiments established that Sub1 directly interacts with TFIIIB and TFIIIC transcription factors. Finally, we showed that Sub1 deletion in yeast leads to a decrease in RNA polymerase III transcription during exponential phase. Then, we tried to determine which link could exist between Sub1, the activator, and Maf1, the repressor of RNA polymerase III transcription. Furthermore, we attempted to identify other elements which could interact with Sub1 during transcription regulation.
... Type III are distinct from Type I and II promoters as they do not require TFIIIC for Pol III mediated transcription but employ SNAPc, a TF also associated with Pol II transcription (Schramm and Hernandez, 2002). This general set of promoter types and TFs is essentially conserved across wide evolutionary distances but with some phyla-specific differences (Schramm and Hernandez, 2002;Teichmann et al., 2010). Additional TFs regulating Pol III activity include Myc, a transcriptional activator that can act on all three Pols (Gomez-Roman et al., 2003;Campbell and White, 2014), as well as the protein Maf1, a highly conserved repressor of Pol III activity (Upadhya et al., 2002;Vorländer et al., 2020). ...
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Transcription in eukaryotic cells is performed by three RNA polymerases. RNA polymerase I synthesises most rRNAs, whilst RNA polymerase II transcribes all mRNAs and many non-coding RNAs. The largest of the three polymerases is RNA polymerase III (Pol III) which transcribes a variety of short non-coding RNAs including tRNAs and the 5S rRNA, in addition to other small RNAs such as snRNAs, snoRNAs, SINEs, 7SL RNA, Y RNA, and U6 spilceosomal RNA. Pol III-mediated transcription is highly dynamic and regulated in response to changes in cell growth, cell proliferation and stress. Pol III-generated transcripts are involved in a wide variety of cellular processes, including translation, genome and transcriptome regulation and RNA processing, with Pol III dys-regulation implicated in diseases including leukodystrophy, Alzheimer’s, Fragile X-syndrome and various cancers. More recently, Pol III was identified as an evolutionarily conserved determinant of organismal lifespan acting downstream of mTORC1. Pol III inhibition extends lifespan in yeast, worms and flies, and in worms and flies acts from the intestine and intestinal stem cells respectively to achieve this. Intriguingly, Pol III activation achieved through impairment of its master repressor, Maf1, has also been shown to promote longevity in model organisms, including mice. In this review we introduce the Pol III transcription apparatus and review the current understanding of RNA Pol III’s role in ageing and lifespan in different model organisms. We then discuss the potential of Pol III as a therapeutic target to improve age-related health in humans.
... To verify these results, a second approach was used to repress RNA pol III-dependent transcription. Downregulation of BRF1, an RNA pol III-specific TFIIIB transcription factor subunit (45), also produced a decrease in cell viability and an increase in cleaved caspase-3 expression in the presence of doxorubicin ( Fig. 6C-D). We also confirmed our results using MAF1-deficient (Maf1 −/− ) mouse embryonic fibroblast cells (MEFs). ...
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MAF1 homolog, negative regulator of RNA polymerase III (MAF1) is a key repressor of RNA polymerase (pol) III-dependent transcription and functions as a tumor suppressor. Its expression is frequently down-regulated in primary human hepatocellular carcinomas (HCCs). However, this reduction in MAF1 protein levels does not correlate with its transcript levels, indicating that MAF1 is regulated posttranscriptionally. Here, we demonstrate that MAF1 is a labile protein whose levels are regulated through the ubiquitin-dependent proteasome pathway. We found that MAF1 ubiquitination is enhanced upon mTOR complex 1 (TORC1)-mediated phosphorylation at Ser-75. Moreover, we observed that the E3 ubiquitin ligase cullin-2 (CUL2) critically regulates MAF1 ubiquitination and controls its stability and subsequent RNA pol III-dependent transcription. Analysis of the phenotypic consequences of modulating either CUL2 or MAF1 protein expression revealed changes in actin cytoskeleton reorganization and altered sensitivity to doxorubicin-induced apoptosis. Repression of RNA pol III-dependent transcription by chemical inhibition or knockdown of BRF1 RNA pol III transcription initiation factor subunit (BRF1) enhanced HCC cell sensitivity to doxorubicin, suggesting that MAF1 regulates doxorubicin resistance in HCC by controlling RNA pol III-dependent transcription. Together, our results identify the ubiquitin proteasome pathway and CUL2 as important regulators of MAF1 levels. They suggest that decreases in MAF1 protein underlie chemoresistance in HCC and perhaps other cancers and point to an important role for MAF1 and RNA pol III-mediated transcription in chemosensitivity and apoptosis.
... The high level of crystal protein synthesis in Bt and its co-ordination with the stationary phase are controlled by a variety of mechanisms occurring at the transcriptional, post transcriptional and post translational levels. In Bt, most of the cry genes are expressed only during sporulation; few genes are expressed during the vegetative phase (Moran 1993 ). ...
Bacillus thuringiensis (Bt) is used to control agriculturally-important pests. It is a Gram positive spore-forming bacterium which produces parasporal proteinaceous inclusions during the sporulation phase. These crystalline parasporal inclusions are toxic to a wide spectrum of insects including the orders Lepidoptera, Coleopteran, Diptera, etc. The Bt insecticide proteins are toxic only after ingestion by the susceptible insects. The main steps involved when the Cry protein is ingested by the insect is comprised of solubilization of the protoxin, its enzymatic activation by terminal cleavage, receptor binding in brush border membrane of the midgut, pore formation, consequent disruption of ionic potential and destruction of the epithelial membrane leading to cell death. The first discovery of Bt was in 1901 when Ishiwata discovered a bacterium in Japan and in 1915, Berliner in Germany renamed it as Bacillus thuringiensis. Following a brief introduction, this chapter addresses the classification, the general structure of Cry toxin, its mode of action, strategies to improve the insecticidal activity of Cry proteins, transgenic plants developed using Bt genes, resistance to Bt toxins and resistance management, and an overall brief account of Bt and its insecticidal proteins, from 1901 to the present.
... Earlier studies have shown that Tfc7 is required for RNA polymerase (Pol) III in transcribing 5S rRNA, which is a part of ribosomes (Teichmann et al., 2010;Acker et al., 2013). In S. cerevisiae, Tfc7 is essential. ...
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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.
LTR-retrotransposons are widespread transposable elements in eukaryotes. Like retroviruses, they replicate by reverse transcription of their RNA into cDNA, which is integrated into the host genome by their own integrase (IN). High-throughput sequencing studies clearly established that integration does not occur randomly throughout the host-cell genome. Deep insights on retroviral biology have been gained by their study in yeast using the Ty1 LTR-retrotransposon as a working model. The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae integrates upstream of class III genes, the genes transcribed by RNA polymerase III (Pol III). Recent data revealed the importance of AC40, a Pol III subunit in this targeting. An interaction between the Ty1 IN and AC40 is necessary for integration site choice at the Pol III genes. Nevertheless, the molecular mechanism remains largely unknown. To obtain a global view of the entire phenomenon that occurs on the integration site we would like to exhaustively determine the proteins that interact with Ty1 IN and analyze their role in both Ty1 integration and RNA Pol III transcription. To achieve this goal, we have developed proteomic approaches to identify new Ty1 integrase cellular partners. We have identified several novel Ty1 IN partners that seem interesting and their molecular role in Ty1 retrotransposition will be studied. However, in the tenure of my PhD, I have particularly worked to decipher the molecular role of the casein kinase II protein in Ty1 retrotransposition.
In eukaryotes, RNA polymerase (RNAP) III transcribes the tRNAs, the 5S ribosomal RNA and a half a dozen known untranslated RNA. Mammalian genome contains several thousand of repeated elements, the Short interspersed repetitive elements (SINE). In vitro, they are transcribed by RNAP III. RNAP III transcription levels determine cell growth and proliferation and, importantly, its deregulation is associated with cancer. Looking at the genome-wide distribution of RNAP III and its transcription factors, TFIIIB and TFIIIC, we develop a highly specific tandem ChIP-sequencing method. We have determined the set of genes that are transcribed by RNAP III in mouse embryonic stem cells. We discovered that not all known class III genes were transcribed in ES cells. We also observed that RNAP III and its transcription factors were present at thirty unannotated sites on the mouse genome, only one of which was conserved in human. Only a couple of hundreds of SINEs out of more than half a million are associated with RNAP III in mouse ES cells. Our study reveals numerous 'TFIIIC-only' sites, called ETC for extra-TFIIIC loci in yeast. These sites are correlated with association of CTCF and the cohesin. Cohesin has been shown to occupy sites bound by CTCF and to contribute to DNA loop formation associated with gene repression or activation. This observation suggests that TFIIIC may play a role in chromosome organization in mouse. We also demonstrated that TCEA1, the ubiquitous isoform of TFIIS RNAP II elongation factor, is associated with active class III genes suggesting that TFIIS is a RNAP III transcription factor in mammals. Finally, the distribution of TFIIS on RNAP II-transcribed genes indicated that its recruitment does not control the transition of RNAP II paused at genes 5' end into elongation.
SIRT1, member of the sirtuins family, is an NAD-dependent deacetylase, playing an essential role in controlling gene expression. In addition to modifying histones, SIRT1 can affect the activity of several transcription factors and their target genes. A fundamental question is to understand the molecular mechanisms by which SIRT1 controls the expression of genesinvolved in cell proliferation and energy metabolism. To identify protein partners of SIRT1, we used the method of TAP-TAG purification from a soluble nuclear fraction and a chromatin anchored fraction of Mef cells stably expressing ectopic copy of SIRT1 (SIRT1-e). We were able to identify a SIRT1 complex associated with both cell proliferation factor Ki67, and TFIIIC,subunit required for assembly of the RNA polymerase III pre-initiation complex. By deleting Sirt1, and by specifically inhibiting Ki67 expression, we showed that the RNA Polymerase III transcription machinery and cell proliferation were strongly affected. All of my results clearly shows that SIRT1, Ki67, and TFIIIC are within a same protein complex, SIRT1 and Ki67, acting in coordination to regulate the expression level of SINES and LINES, transcribed from RNA polymerase III transcription machinery.
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Neuroblastoma (NB) is a pediatric cancer characterized by remarkable cell heterogeneity within the tumor nodules. Here, we demonstrate that the synthesis of a pol III-transcribed noncoding (nc) RNA (NDM29) strongly restricts NB development by promoting cell differentiation, a drop of malignancy processes, and a dramatic reduction of the tumor initiating cell (TIC) fraction in the NB cell population. Notably, the overexpression of NDM29 also confers to malignant NB cells an unpredicted susceptibility to the effects of antiblastic drugs used in NB therapy. Altogether, these results suggest the induction of NDM29 expression as possible treatment to increase cancer cells vulnerability to therapeutics and the measure of its synthesis in NB explants as prognostic factor of this cancer type.
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Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III-associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.
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RNA polymerase (Pol) III transcribes many noncoding RNAs (for example, transfer RNAs) important for translational capacity and other functions. We localized Pol III, alternative TFIIIB complexes (BRF1 or BRF2) and TFIIIC in HeLa cells to determine the Pol III transcriptome, define gene classes and reveal 'TFIIIC-only' sites. Pol III localization in other transformed and primary cell lines reveals previously uncharacterized and cell type-specific Pol III loci as well as one microRNA. Notably, only a fraction of the in silico-predicted Pol III loci are occupied. Many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer-binding proteins such as ETS1 and STAT1. Moreover, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. These results suggest that active chromatin gates Pol III accessibility to the genome.
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Transcription in eukaryotic nuclei is carried out by DNA-dependent RNA polymerases I, II, and III. Human RNA polymerase III (Pol III) transcribes small untranslated RNAs that include tRNAs, 5S RNA, U6 RNA, and some microRNAs. Increased Pol III transcription has been reported to accompany or cause cell transformation. Here we describe a Pol III subunit (RPC32beta) that led to the demonstration of two human Pol III isoforms (Pol IIIalpha and Pol IIIbeta). RPC32beta-containing Pol IIIbeta is ubiquitously expressed and essential for growth of human cells. RPC32alpha-containing Pol IIIalpha is dispensable for cell survival, with expression being restricted to undifferentiated ES cells and to tumor cells. In this regard, and most importantly, suppression of RPC32alpha expression impedes anchorage-independent growth of HeLa cells, whereas ectopic expression of RPC32alpha in IMR90 fibroblasts enhances cell transformation and dramatically changes the expression of several tumor-related mRNAs and that of a subset of Pol III RNAs. These results identify a human Pol III isoform and isoform-specific functions in the regulation of cell growth and transformation.
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The C53 and C37 subunits of RNA polymerase III (pol III) form a subassembly that is required for efficient termination; pol III lacking this subcomplex displays increased processivity of RNA chain elongation. We show that the C53/C37 subcomplex additionally plays a role in formation of the initiation-ready open promoter complex similar to that of the Brf1 N-terminal zinc ribbon domain. In the absence of C53 and C37, the transcription bubble fails to stably propagate to and beyond the transcriptional start site even when the DNA template is supercoiled. The C53/C37 subcomplex also stimulates the formation of an artificially assembled elongation complex from its component DNA and RNA strands. Protein-RNA and protein-DNA photochemical cross-linking analysis places a segment of C53 close to the RNA 3′ end and transcribed DNA strand at the catalytic center of the pol III elongation complex. We discuss the implications of these findings for the mechanism of transcriptional termination by pol III and propose a structural as well as functional correspondence between the C53/C37 subcomplex and the RNA polymerase II initiation factor TFIIF.
Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Pol III transcription machinery and transcriptome. We have localized, genome-wide, Pol III and some of its transcription factors. Our results reveal broad usage of the known Pol III transcription machinery and define a minimal Pol III transcriptome in dividing IMR90hTert fibroblasts. This transcriptome consists of some 500 actively transcribed genes including a few dozen candidate novel genes, of which we confirmed nine as Pol III transcription units by additional methods. It does not contain any of the microRNA genes previously described as transcribed by Pol III, but reveals two other microRNA genes, MIR886 (hsa-mir-886) and MIR1975 (RNY5, hY5, hsa-mir-1975), which are genuine Pol III transcription units.
Mammalian short interspersed elements (SINEs) are abundant retrotransposons that have long been considered junk DNA; however, RNAs transcribed from mouse B2 and human Alu SINEs have recently been found to control mRNA production at multiple levels. Upon cell stress B2 and Alu RNAs bind RNA polymerase II (Pol II) and repress transcription of some protein-encoding genes. Bi-directional transcription of a B2 SINE establishes a boundary that places the growth hormone locus in a permissive chromatin state during mouse development. Alu RNAs embedded in Pol II transcripts can promote evolution and proteome diversity through exonization via alternative splicing. Given the diverse means by which SINE encoded RNAs impact production of mRNAs, this genomic junk is proving to contain hidden gems.
Gene duplications and their subsequent divergence play an important part in the evolution of novel gene functions. Several models for the emergence, maintenance and evolution of gene copies have been proposed. However, a clear consensus on how gene duplications are fixed and maintained in genomes is lacking. Here, we present a comprehensive classification of the models that are relevant to all stages of the evolution of gene duplications. Each model predicts a unique combination of evolutionary dynamics and functional properties. Setting out these predictions is an important step towards identifying the main mechanisms that are involved in the evolution of gene duplications.
The number of subunits of RNA polymerases (RNAPs) increases during evolution from 5 in eubacteria to 12 in archaea. In eukaryotes, which have at least three RNAPs, the number of subunits has expanded from 12 in RNA polymerase II (RNAPII) to 14 in RNA polymerase I (RNAPI) and to 17 in RNA polymerase III (RNAPIII). It was recently demonstrated that the two additional subunits found in RNAPI relative to RNAPII are homologous to TFIIF, a dimeric general transcription factor of RNAPII. Here, we extend this finding by demonstrating that four of the five RNAPIII-specific subunits are also homologous to transcription factors of RNAPII. We use the available evidence to propose an evolutionary history of the eukaryotic RNAPs and argue that the increases in the number of subunits that occurred in RNAPs I and III are due to the permanent recruitment of preexisting transcription factors.