MOLECULAR AND CELLULAR BIOLOGY, Apr. 2006, p. 2728–2735
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 26, No. 7
Uncleaved TFIIA Is a Substrate for Taspase 1 and Active in Transcription
Huiqing Zhou,1Salvatore Spicuglia,1† James J.-D. Hsieh,2‡ Dimitra J. Mitsiou,3Torill Høiby,1
Gert Jan C. Veenstra,1Stanley J. Korsmeyer,2and Hendrik G. Stunnenberg1*
NCMLS, Department of Molecular Biology, 191, Radboud University of Nijmegen, 6500 HB Nijmegen, The Netherlands1;
Howard Hughes Medical Institute, Dana-Farber Cancer Institute, Harvard Medical School, Boston,
Massachusetts 021152; and Molecular Endocrinology Program, Institute of Biological Research and
Biotechnology, The National Hellenic Research Foundation, 11635 Athens, Greece3
Received 3 January 2006/Accepted 3 January 2006
In higher eukaryotes, the large subunit of the general transcription factor TFIIA is encoded by the single
TFIIA?? gene and posttranslationally cleaved into ? and ? subunits. The molecular mechanisms and bio-
logical significance of this proteolytic process have remained obscure. Here, we show that TFIIA is a substrate
of taspase 1 as reported for the trithorax group mixed-lineage leukemia protein. We demonstrate that
recombinant taspase 1 cleaves TFIIA in vitro. Transfected taspase 1 enhances cleavage of TFIIA, and RNA
interference knockdown of endogenous taspase 1 diminishes cleavage of TFIIA in vivo. In taspase 1?/?MEF
cells, only uncleaved TFIIA is detected. In Xenopus laevis embryos, knockdown of TFIIA results in phenotype
and expression defects. Both defects can be rescued by expression of an uncleavable TFIIA mutant. Our study
shows that uncleaved TFIIA is transcriptionally active and that cleavage of TFIIA does not serve to render
TFIIA competent for transcription. We propose that cleavage fine tunes the transcription regulation of a subset
of genes during differentiation and development.
In eukaryotes, initiation of RNA polymerase II transcription
requires the assembly of a preinitiation complex. Specific bind-
ing of TBP to promoters is a key step in the formation of PIC,
which is followed by recruitment of general transcription fac-
tors and polymerase II. The basal transcription factor TFIIA
interacts with TBP and stabilizes its binding to DNA (26, 28).
TFIIA has also been shown to interact with several activators
(11, 12, 18, 27) and is required for transcriptional activation of
certain genes (10, 13, 14, 20, 21).
In higher eukaryotes, purified TFIIA is composed of three
subunits, ?, ?, and ?. TFIIA?? is encoded by a single gene and
cleaved posttranslationally into ? and ? subunits. The ? sub-
unit is conserved among different species, whereas sequence
similarity in TFIIA?? is limited mostly to the N-terminal re-
gion of the ? subunit and the C terminus covering most of the
? subunit (19). Recently, the cleavage recognition site (CRS)
that is essential for TFIIA cleavage has been identified as
QVDG (amino acids [aa] 272 to 275), and the N terminus of
the ? subunit was determined to be at D278, located 3 amino
acids downstream of the CRS (Fig. 1B) (6). The CRS is re-
markably similar in different evolutionarily distinct species and
is embedded in an otherwise nonconserved and probably un-
structured region (1, 4, 24). The germ cell-specific TFIIA-like
factor ALF, a TFIIA variant that contains the CRS, was also
shown to be cleaved (5, 6). TFIIA cleavage was first reported
more than a decade ago (26), and it has been generally as-
sumed that uncleaved TFIIA is the precursor and cleavage
occurs to activate TFIIA for transcription. Both uncleaved ??
and the cleaved ? and ? subunits can be found in association
with the TFIIA? subunit in vivo (15, 16), and both forms
interact with TBP on DNA and support transcription to similar
extents in vitro and in reporter assays (6, 22). TFIIA is mainly
found in the cleaved form (? plus ? plus ?) in differentiated
cells. In embryonal carcinoma P19 cells, a substantial amount
of uncleaved TFIIA (?? plus ?) is detected and stably interacts
with TBP in the TAC complex to mediate transcription (15,
16). Therefore, uncleaved and cleaved forms of TFIIA may
have distinct gene regulatory functions in differentiation. The
observation that cleavage is the prerequisite for proteasome-
mediated degradation of TFIIA (6) indicates that cleavage
regulates TFIIA protein levels and may play a role in tran-
scription. Elucidation of the biological function of TFIIA
cleavage is hampered because the protease(s) that specifically
cleaves TFIIA has not been identified.
The recently identified cleavage site in TFIIA??, G277/
D278 (6), did not match known consensus sequences of pro-
teases. The CRS, QVDG, of TFIIA is, however, virtually iden-
tical to the cleavage sites of the MLL (mixed-lineage leukemia)
protein, QVD/G (aa 2664 to 2667) and QLD/G (aa 2716 to
2719) (Fig. 1B) (8, 17). The MLL protein is a 500-kDa nuclear
protein of the trithorax (Trx) group of proteins and is required
for maintenance of proper HOX gene expression. Chromo-
somal translocation results in different MLL fusion proteins
that are involved in various leukemias (3). The MLL protein is
proteolytically cleaved at two adjacent cleavage sites by a single
protease, taspase 1, an endopeptidase with an asparaginase 2
homology domain (7). Moreover, there is an acidic stretch
downstream of the cleavage site in both the MLL protein and
TFIIA. These similarities strongly indicate a molecular and/or
functional link between TFIIA and MLL protein processing.
Here, we show that TFIIA is a genuine substrate of taspase
1. Taspase 1 cleaves TFIIA in vitro and in vivo. RNA interfer-
* Corresponding author. Mailing address: Radboud University De-
partment of Molecular Biology, NCMLS (191), P.O. Box 9101, 6500
HB Nijmegen, The Netherlands. Phone: 31-24-3610524. Fax: 31-24-
3610520. E-mail: email@example.com.
† Present address: Centre d’Immunologie de Marseille-Luminy, Parc
Scientifique et Technologique de Luminy, 13009 Marseille, France.
‡ Present address: Department of Medicine, Molecular Oncology,
Siteman Cancer Center, Washington University, St. Louis, Mo.
ence (RNAi) knockdown of taspase 1 reduces cleavage of
TFIIA, and TFIIA cleavage is undetectable in taspase 1?/?
knockout mouse embryonal fibroblasts (MEFs). In Xenopus
laevis, TFIIA is required in early development and gene ex-
pression, and an uncleavable mutant was able to rescue the
phenotype in development and transcriptional defects in
MATERIALS AND METHODS
Plasmids and antibodies. Mammalian expression vectors, myc-tagged TFIIA??
(pSG5-myc-TFIIA??), its CRS mutants (alanine mutants from L271 to T279),
hemagglutinin-tagged TFIIA? (pSG5-HA-TFIIA?), a green fluorescent protein
(GFP) construct (pEGFP-N1) (6), and pcDNA-taspase 1 and its T234A mutant
form (7) were described previously. TFIIA?? and -? genes were subcloned from
their mammalian expression vectors into a single polycistronic vector, pST39
(23), between the SacI and KpnI sites and between the XbaI and BamHI sites,
respectively, to generate pST-IIA??? for expression in Escherichia coli. The CRS
mutants (alanine mutants from L271 to T279) in the pST vector were generated
by excision of the MscI and NotI fragments containing the mutated sequences
from the mammalian vectors and replacement of the wild-type sequence in
pST-IIA???. By comparative expressed sequence tag analysis, we identified a
cDNA encoding a previously uncharacterized X. laevis TFIIA?? gene (GenBank
accession no. BE575947), referred to as ??3, that is closely related to Xenopus
TFIIA??1 and was previously described (5). Xenopus TFIIA??3 was PCR am-
plified and cloned into the BglII and EcoRV sites of pT7TS (29). A G269A
mutation was introduced into the TFIIA??3 construct by site-directed mutagen-
esis and confirmed by sequencing. ??MO (morpholino)-resistant TFIIA?? con-
structs were obtained by introducing seven silent mutations within the target
sequence. Capped RNAs for injections were transcribed with an in vitro RNA
synthesis kit (Ambion).
Protein C antibody was purchased from Roche Molecular Biochemicals.
Monoclonal myc antibody; polyclonal GFP antibody; polyclonal TFIIA?-,
TFIIA?-, and TFIIA-?-specific (16) antibodies; and taspase 1 (7) antibodies
were previously described. To detect endogenous TFIIA, immunopurified poly-
clonal TFIIA?-, TFIIA?-, and TFIIA?-specific antibodies were used.
Cell culture, transient transfection, RNAi, protein extraction, and immuno-
precipitation. Maintenance and transfection of U2OS cells and extract prepara-
tion were performed as previously described (6). RNAi knockdown of taspase 1
was carried out with duplex RNAi oligonucleotides as described previously (7).
After 48 h of RNAi treatment of U2OS cells, RNAi oligonucleotides were
removed and cells were transfected with TFIIA plasmids. To detect the effect of
taspase 1 RNAi knockdown on endogenous TFIIA, U2OS cells were treated with
RNAi oligonucleotides for 3 and 4 days.
Protein expression and purification and Edman sequencing. Polycistronic
expression plasmid pST-IIA??? (and its CRS mutants) carrying both the
TFIIA?? (and its mutants) and TFIIA? genes was transformed into
BL21(DE3)plysS cells, and induced with 0.2 mM isopropyl-?-D-thiogalacto-
pyranoside (IPTG). Overexpressed wild-type TFIIA?? and TFIIA? proteins
were purified as a complex through Ni-nitrilotriacetic acid (NTA), Mono Q, and
Mono S columns to nearly homogeneity. The purified TFIIA complex was func-
tionally assayed in an electrophoretic mobility shift assay after each purification
step. TFIIA??? CRS mutants expressed from pST constructs were semipurified
with Ni-NTA resin and eluted with 250 mM imidazole before being subjected to
in vitro protease assays. To purify the protease activity for TFIIA cleavage, HeLa
nuclear extracts were prepared in high-salt buffer containing 20 mM HEPES (pH
7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 10%
glycerol, 0.1 mM, phenylmethylsulfonyl fluoride, and 1? complete protease
inhibitors (Roche Molecular Biochemicals) and fractionated on a P11 column,
followed by step elutions with 100, 300, 500, and 1,000 mM KCl. The PC-C
fraction (500 mM fraction) containing the protease activity was further fraction-
ated on a Mono S column. For the Mono S column, a gradient of 10 to 1,000 mM
KCl was applied and the activity eluted at 300 mM KCl. Protease activity was
monitored with the in vitro protease assay. To perform Edman N-terminal
sequencing analysis, recombinant TFIIA (rTFIIA) was digested with recombi-
nant taspase 1, followed by sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis and staining with Coomassie. The band corresponding to the ? subunit
was excised and subjected to Edman N-terminal sequencing analysis as described
before (8). Around 3 to 4 million taspase 1?/?MEF cells were extracted in 400
?l of low-salt buffer as described previously (7).
FIG. 1. Taspase 1 activity in HeLa nuclear extracts. (A) A protease
assay was performed to detect TFIIA cleavage activity in HeLa nuclear
extracts. Highly purified recombinant TFIIA (rec. TFIIA) was used as
the substrate as indicated, and Western blotting analysis was used to
detect the cleavage products. The protease activity was further frac-
tionated on a P11 column (lanes 4 to 11), followed by a Mono S
column (lanes 12 to 14). The specificity of the protease activity was
tested by incubation of purified wild-type (wt) TFIIA or the cleavage
site G275A mutant with Mono S fraction 18 (lanes 15 and 16). Non-
specific bands are indicated by asterisks. (B) Alignment of the CRS
and the cleavage site of TFIIA and the MLL protein from different
organisms, i.e., humans (h), mice (m), Xenopus (x), pufferfish (p), and
Drosophila (d). The conserved CRS is boxed. Cleavage of TFIIA and
the MLL protein by taspase 1 is at D/G (red arrow). D278 (?) is the
identified N-terminal end of the ? subunit of TFIIA purified from
mammalian cells. The acidic stretch (residues in blue and purple) is
relatively conserved in TFIIA and the MLL protein. (C) Endogenous
taspase 1, full-length taspase 1 (Taspase1-FL), and the autocleaved N-
terminal part (Taspase1-N28) were detected by a taspase 1-specific anti-
body in all fractions containing the protease activity. NE, nuclear extracts.
VOL. 26, 2006 TFIIA CLEAVAGE BY TASPASE 12729
In vitro protease assay. The purified rTFIIA complex and semipurified TFIIA
(and its mutant forms) were incubated at 37°C with 10 ng of recombinant taspase
1 for 1 h or with HeLa nuclear fractions for 12 h. Protease reaction buffer P
contained 20 mM Tris (pH 8.0), 100 mM KCl, 0.2 mM EDTA, 2 mM dithio-
threitol, and 10% glycerol. The reaction mixture was subsequently analyzed by
Western blotting and probed for TFIIA?? ?, ?, and ? subunits with the respec-
tive antibodies. One microliter of extract of taspase 1?/?MEF cells was incu-
bated with 10 ng of recombinant taspase 1 in protease reaction buffer P, and
cleavage was detected by Western blotting with immunopurified TFIIA?-, ?-,
and ?-specific antibodies.
Modified oligonucleotides for Xenopus injection. Twenty nanograms of TFIIA??
antisense MO-modified oligonucleotide (??MO; 5?-GCGGCCTCGGCTAACG
CAAACCCCG; Gene Tools) was generally injected per embryo. The same
amount of standard control MO (cMO) was injected in parallel.
Western blotting for Xenopus extracts. Xenopus embryo extracts were prepared
as previously described (25). Usually, 2 egg equivalents was used for Western
TFIIA-TBP binding assay. Band shift assays were performed as described
previously (5), with 1 ng of recombinant TBP (rTBP), 0.5 to 1 embryo equivalent
of Xenopus embryo extracts, and the adenovirus major late promoter TATA box
RNA isolation and reverse transcription-quantitative PCR (RT-qPCR). Xe-
nopus embryo RNA was isolated by Trizol (Invitrogen) extraction and LiCl
precipitation. RT was performed with 1 to 2 embryo equivalents of total mRNA
with Superscript II (Amersham) and a combination of random hexanucleotides
and pT21V. Levels of cDNA were quantified by qPCR. Designed primer sets for
qPCR were as follows: for Hmg1, 5?-TCGAAAGGAAAGCTGCCAAG-3? (for-
ward) and 5?-GCAGGTTCTGGCTTTCCCTTA-3? (reverse); for 1A11, 5?-CGA
AGATGCTTTTCCTGCCA-3? (forward) and 5?-CACAATCACAGGCTGCATG
TG-3? (reverse); for Hoxb4, 5?-CAAGAGATCCCGCACAGCTTA-3? (forward)
and 5?-CAAAGTGTGCGCAATTTCCA-3? (reverse). Primer sets for Gsc, Gs17,
Xbra, MyoDb, and Efl? are described at http://www.hhmi.ucla.edu/derobertis/index
.html, and that for retinoblastoma (Rb) was described previously (9).
Specific protease activity for TFIIA cleavage in HeLa cell
nuclear extracts. To identify the protease for TFIIA, we set up
an in vitro cleavage assay with purified rTFIIA composed of
uncleaved ?? and ? subunits as the substrate to test for a
cleavage activity for TFIIA in HeLa cell extracts. In crude
nuclear extracts, cleavage could readily be monitored by the
appearance of the His-tagged, exogenous ? subunit (Fig. 1A,
compare lanes 2 and 3); the cleaved, exogenous ? subunit
cannot be discriminated from endogenous TFIIA. Fraction-
ation of nuclear extracts on a P11 column showed that the
cleavage activity was eluted at 500 mM KCl (PC-C fraction)
(Fig. 1A, lanes 8 and 9). The PC-C fraction was further frac-
tionated on a Mono S column, and the cleavage activity was
recovered at approximately 300 mM KCl in fractions 17, 18,
and 19 (Fig. 1A, lanes 12 to 14). To assess whether the ob-
served cleavage activity is specific and displays the same amino
acid sequence requirements as in vivo, we tested a G275A
cleavage site mutant of TFIIA (6) in our in vitro assay. The
protease activity in Mono S fraction 18 cleaved wild-type
TFIIA but not the G275A mutant (Fig. 1A, lanes 15 and 16),
showing that the cleavage activity for TFIIA in HeLa nuclear
extracts is specific.
Having identified the CRS for TFIIA (6), we noticed that
the CRS in TFIIA, QVDG (aa 272 to 275), is identical or
similar to the cleavage sites in the MLL protein, QVDG (aa
2664 to 2667) and QLDG (aa 2716 to 2719), which are both
cleaved at D/G by taspase 1 (Fig. 1B). This similarity indicated
that TFIIA and the MLL protein might be cleaved by the same
protease. To test whether HeLa nuclear fractions enriched for
TFIIA cleavage activity contain taspase 1, Western blotting
analysis was performed with an anti-taspase 1 antibody (7).
Figure 1C shows that autocleaved taspase 1 is present in frac-
tions with cleavage activity for TFIIA, including the nuclear
extracts (lane 1), the PC-C fraction (lane 4), and Mono S
fractions 17 to 19 (lanes 7 to 9). Full-length taspase 1 could not
be observed in whole-cell extracts (7) or nuclear extracts (lane
1) but was detectable in the PC-C fraction and was further
enriched in Mono S fractions 17 to 19. These data strongly
suggest that taspase 1 is the protease for TFIIA cleavage.
Cleavage of TFIIA by taspase 1 in vitro. To directly assess
whether TFIIA is a substrate of taspase 1, we first tested
cleavage in vitro with recombinant TFIIA and taspase 1. Re-
combinant wild-type taspase 1 cleaved TFIIA efficiently (Fig.
2A, lanes 1 to 5), while the T234A active-site mutant of taspase
1 did not cleave wild-type TFIIA (Fig. 2A, lanes 6 to 10).
Although the in vitro assays showed that taspase 1 cleaves
TFIIA, the determined cleavage site of the MLL protein is
FIG. 2. Cleavage of TFIIA by taspase 1 in vitro. (A) Coomassie
staining was performed to detect cleavage of the recombinant TFIIA
by recombinant taspase 1. Wild-type (wt) TFIIA was incubated with
different amounts of wild-type taspase 1 (lanes1 to 5) or mutant (mt)
taspase 1 (T234A) (lanes 6 to 10) as indicated. The ? subunit of TFIIA
(arrowhead) was cut out of the gel and subjected to Edman analysis.
Edman analysis showed that G275 is the N-terminal end of the ?
subunit. (B) Western blot analysis was performed to test the cleavage
of mutant TFIIAs covering the CRS. These TFIIA mutants were
expressed in complex with the ? subunit in E. coli, and one-step
Ni-NTA purification was applied to obtain semipurified proteins.
2730 ZHOU ET AL.MOL. CELL. BIOL.
different from that of TFIIA purified from cell extracts and
analyzed by Edman degradation. Edman sequencing showed
that cleavage in the MLL protein occurs at D/G within the
conserved CRS, QVDG or QLDG (8), whereas in TFIIA, the
most N-terminal amino acid of the ? subunit was determined
to be D278, 3 amino acids downstream of the CRS (Fig. 1B)
(6). To resolve this ambiguity, the N terminus of the TFIIA?
generated in vitro by recombinant taspase 1 (Fig. 2A, the
FIG. 3. Cleavage of TFIIA by taspase 1 in vivo. (A) Wild-type (wt) TFIIA was transfected either alone or together with either wild-type or mutant
(mt) taspase 1 (T234A) in U2OS cells, and cleavage was analyzed by Western blotting. This experiment was performed more than 10 time, and the ratio
of uncleaved to cleaved TFIIA was consistent. (B) TFIIA mutants covering the CRS were tested either alone or together with taspase 1 for their cleavage
in U2OS. GFP was cotransfected as the internal control. Nonspecific bands detected by taspase 1 antibody are indicated by asterisks. (C) Endogenous
(end.) taspase 1 was knocked down by RNAi duplex oligonucleotides (oligos). Control oligonucleotides (C) and taspase 1 oligonucleotides (T) were used
in this experiment. To test the effect on transiently transfected TFIIA, oligonucleotides were transfected for 48 h and removed, followed by transfection
of TFIIA constructs. To test the effect on endogenous TFIIA, U2OS cells were treated with oligonucleotides for 3 and 4 days as indicated. Nonspecific
bands detected by TFIIA?-specific antibody are marked by asterisks. (D) TFIIA cleavage was tested in taspase 1?/?MEF cells. Extracts from wild-type
(lane1) and taspase 1?/?MEF cells incubated without (lane 2) and with recombinant taspase 1 (rTaspase1) (lane 3) were subjected to Western blot
VOL. 26, 2006 TFIIA CLEAVAGE BY TASPASE 12731
marked bands on the left side) was subjected to Edman deg-
radation. The analysis yielded the amino acid sequence GTG
DTSSE, showing that cleavage of TFIIA by taspase 1 occurred
at D274/G275 (Fig. 2A). This cleavage site is within the con-
served CRS that is essential for TFIIA cleavage, and it is
consistent with the sites of MLL protein cleavage by taspase 1
Having shown that TFIIA is cleaved by taspase 1 in vitro and
the cleavage site is identical to that of the MLL protein, we
tested whether cleavage of TFIIA by taspase 1 has the same
amino acid requirement as cleavage of TFIIA in vivo, as shown
previously (6). A panel of mutants covering the CRS which was
tested previously in vivo were expressed in E. coli and purified
with Ni-NTA resin, and subsequently, the Ni-NTA eluates
were analyzed in our in vitro assay with recombinant taspase 1.
In this assay, wild-type TFIIA (Fig. 2B, lanes 1 and 2) and
mutant forms with changes flanking the CRS, L271A (lanes 3
and 4) and T276A (lanes 13 and 14), were readily cleaved by
taspase 1. Cleavage of the CRS mutant forms was either com-
pletely blocked (D274A and G275A) or occurred weakly
(Q272A and V273A) (Fig. 2B, lanes 9 to 12 and 5 to 8, respec-
tively), which matches the cleavage profile observed for the
endogenous protease (Fig. 3B) (6). In conclusion, our in vitro
data show that cleavage by taspase 1 requires the CRS and that
taspase 1 cleaves TFIIA at D274/G275.
Cleavage of TFIIA by taspase 1 in vivo. To corroborate and
extend our in vitro observations, we tested whether TFIIA is
cleaved by taspase 1 in vivo in transient-transfection assays. In
U2OS cells, expression of wild-type taspase 1, followed by
Western blot analysis with taspase 1 antibody against the N-
terminal region of taspase 1 (7), revealed two polypeptides of
approximately 50 kDa and 28 kDa (Fig. 3A, lanes 3 and 5)
corresponding to full-length taspase 1 (taspase 1-FL) and the
autocleaved N terminus (taspase 1-N28) (7). Coexpression of
TFIIA and taspase 1 led to complete cleavage of TFIIA??
(lanes 5), while expression of T234A mutant taspase 1, which
cannot undergo autocleavage, did not change the ratio of un-
cleaved and cleaved TFIIA (compare lanes 6 and 2). These
data show that TFIIA is cleaved specifically by taspase 1 in
vivo. Interestingly, we did not observe a clear increase in the
TFIIA? and TFIIA? subunits upon complete cleavage of
TFIIA?? (compare lanes 5 and 6), suggesting that in vivo the
FIG. 4. An uncleavable G269A mutant of xTFIIA is transcription-
ally active in early Xenopus development. (A) TFIIA is required for
early development. Embryos at the one-cell stage were injected with 20
ng of cMO or ??MO, and pictures were taken at stage 37 (tadpole).
(B) Knockdown of endogenous xTFIIA was assessed by a TBP-TFIIA
band shift assay. Embryos were injected with ??MO alone or together
with ??MO-resistant xIIAwtRmRNA and collected at stage (St.) 11. A
32P-labeled TATA box probe was incubated in the presence of rTBP
(except for lane 3) and either rTFIIA or extracts from stage 11 em-
bryos and analyzed as described in Materials and Methods. The TA
complex is represented by the symbol Š, and nonspecific bands are
marked by asterisks. (C) Embryos were injected with ??MO alone or
together with xIIAwtRor G269ARmRNA and analyzed at stage 11.
??MO-resistant xIIAwtR(lanes 2 and 3) and G269AR(lanes 4 and 5)
were expressed at similar levels. Ctr, control. (D) TFIIA is required for
gene expression during early stages of Xenopus development. Expres-
sion levels of several genes were analyzed at the indicated stages (St.)
by RT-qPCR from extracts of embryos injected with either cMO or
??MO. (E) An uncleavable G269A mutant form is able to rescue the
expression of TFIIA-dependent genes. Embryos were injected with
??MO alone or together with xIIAwtRor G269ARmRNA and ana-
lyzed at stage 11 by RT-qPCR. The expression values of ??MO plus
xIIAwtRversus ??MO or ??MO plus G269ARversus ??MO are
significantly different (P ? 0.05), except for Rb (P ? 0.05). The dif-
ference in expression values between ??MO plus xIIAwtRand ??MO
plus G269ARwas not significant (P ? 0.05).
2732ZHOU ET AL.MOL. CELL. BIOL.
levels of cleaved TFIIA are measured and maintained in cells.
To assess whether the CRS is essential for taspase 1 cleavage
in vivo, we again utilized the alanine scanning mutants cover-
ing the CRS. Without overexpression of taspase 1, mutations in
the CRS either completely abolished cleavage of TFIIA
(Q272A, D274A, G275A) (Fig. 3B, top, lanes 4, 6, and 7) or
yielded only small amounts of the cleaved products (V273A)
(lane 5), as observed previously (6). Coexpression of taspase 1
with wild-type TFIIA and mutants with amino acid changes
outside the CRS resulted in significant reduction of the un-
cleaved ?? subunits (Fig. 3B, bottom, lanes 2 and 3 and 8 to
11). Q272A and V273A mutant forms showed elevated cleav-
age in the presence of overexpressed taspase 1, but the cleaved
products remained at low levels (lanes 4 and 5). Importantly,
mutant forms with changes at the cleavage site, D274A and
G275A, cannot be cleaved, even upon overexpression of ta-
spase 1 (lanes 6 and 7), demonstrating that D274 and G275 are
absolutely essential for cleavage by taspase 1, which is consis-
tent with the requirement of TFIIA cleavage by the endoge-
nous protease (6).
The role of endogenous taspase 1 in TFIIA cleavage was
further tested by an RNAi approach. In an experiment with
transfected TFIIA, treatment of U2OS cells with RNAi oligo-
nucleotides for 2 days led to a clear accumulation of the un-
cleaved TFIIA?? subunit and a small decrease in the cleaved
products (Fig. 3C, compare lanes 2 and 4). To investigate the
effect on endogenous TFIIA, U2OS cells were treated with
RNAi oligonucleotides for 3 and 4 days, respectively. This
treatment gave rise to a clear decrease in the endogenous
taspase 1 level and, concomitantly, accumulation of the un-
cleaved form of endogenous TFIIA?? and a slight decrease in
the cleaved ? and ? subunits (Fig. 3C, compare lanes 5 and 6
and lanes 7 and 8). To gain insights into the role of taspase 1 in
TFIIA cleavage, we used MEF cells established from taspase
1?/?mice (J. J. Hsieh, unpublished data). In extracts of these
MEFs, only uncleaved TFIIA?? could be detected (Fig. 3D,
compare lanes 1 and 2) and cleavage could be recovered in
vitro by adding recombinant taspase 1 to taspase 1?/?MEF
cell extracts (lane 3). In summary, our in vivo results show
unambiguously that TFIIA is a genuine substrate for taspase 1.
Uncleaved TFIIA is transcriptionally active during early
stages of Xenopus development. It has long been assumed that
the uncleaved form is a nonfunctional precursor and that
cleavage renders TFIIA functional for transcription because
cleaved TFIIA is the major form present in most cells. The fact
that taspase 1?/?MEF cells could be established and main-
tained in culture indicates that uncleaved TFIIA is most prob-
ably functional and sufficient for bulk transcription. To provide
further evidence, we turned to X. laevis as a model organism
where knockdown and rescue experiments can be performed
more easily. As Xenopus TFIIA?? mRNA is maternally con-
tributed and TFIIA is detected after maturation (5), we used a
morpholino antisense oligonucleotide directed against the
three TFIIA?? isoforms (??MO) identified in X. laevis to
knock down the endogenous TFIIA. Injection of ??MO, but
not cMO, into one-cell-stage embryos gave rise to a variety of
phenotypes, ranging from complete developmental arrest dur-
ing late gastrulation to severe axial defects resulting in short-
ened and twisted tadpoles (Fig. 4A and data not shown). We
took advantage of the TATA box binding property of the
TBP-TFIIA complex (TA complex) to assess the efficiency of
TFIIA knockdown. Extracts from stage 11 embryos supple-
mented with rTBP yielded a TA complex migrating at the same
position as a TA complex from rTFIIA and rTBP (Fig. 4B,
compare lane 4 and lane 2). Extracts from embryos injected
with the ??MO antisense oligonucleotide did not yield a TA
band shift, showing that endogenous TFIIA is efficiently
knocked down (Fig. 4B, lane 6). To rescue TFIIA expression,
we utilized an antisense-resistant TFIIA?? synthetic mRNA
(hereafter xIIAwtR) in which silent mutations blocked knock-
down by the antisense oligonucleotide. Coinjection of xIIAwtR
mRNA together with ??MO restored the TA complex (Fig.
4B, lane 7), showing that ??MO specifically knocked down
endogenous xTFIIA but not morpholino-resistant xIIAwtr. To
investigate whether uncleaved TFIIA is functional, silent mu-
tations were also introduced into an uncleavable mutant
form of xTFIIA (G269A; corresponding to human G275A)
mRNA (G269AR), and the xIIAwtRand G269ARmRNAs
were tested in two different amounts (150 ng and 300 ng) in
rescue experiments. In two independent experiments, pheno-
types of TFIIA knockdown by ??MO were largely rescued by
injection of xIIAwtrmRNA, 66.1% and 78.8% at amounts of
150 ng and 300 ng, respectively (Table 1, Total), indicating that
the observed phenotypic defects are specific for TFIIA knock-
down. Importantly, injection of G269ARmRNAs resulted in a
similar rescue of the ???? phenotype, 66.1% and 69.5% at
TABLE 1. Rescue of TFIIA function by the G269A mutant in X. laevis embryosa
??MO ? xIIAwtR
??MO ? xIIAwtR
??MO ? G269AR
??MO ? G269AR
No. of injected embryos in expt 1
No. of injected embryos in expt 2
Total no. of injected embryos
aPhenotypes were scored at stage 37.
bThe abnormal phenotypes observed in MO-injected embryos ranged from arrested gastrulation to severe axial defects.
VOL. 26, 2006 TFIIA CLEAVAGE BY TASPASE 12733
amounts of 150 ng and 300 ng, respectively (Table 1, Total),
showing that G269A is able to replace endogenous TFIIA in
embryonal development. Note that wild-type TFIIA and the
G269A mutant were expressed at similar levels (Fig. 4C).
Therefore, the uncleavable G269A mutant form of TFIIA is
functional during the early stages of Xenopus development.
To study the transcriptional role of TFIIA, we set out to
identify TFIIA-dependent genes. We screened a low-density X.
laevis cDNA microarray and identified several candidate genes
that were consistently down-regulated upon ??MO injection
(unpublished data). To verify that these genes are TFIIA de-
pendent, we analyzed the expression of these genes at different
stages of development after the onset of embryonic transcrip-
tion at the mid-blastula transition (stage 8.5) by RT-qPCR
(Fig. 4D). Several maternally contributed mRNAs, such as
those encoding the high-mobility group 1 (Hmg1) and Rb
proteins, were not affected by TFIIA knockdown. In contrast,
a number of genes transcribed de novo during early embryo-
genesis (GS17, Xbra, 1A11, MyoDb, Ef1?, and Hoxb4) were
down-regulated at different stages of development. Induction
of the homeobox transcription factor Goosecoid (Gsc), how-
ever, was not affected upon TFIIA knockdown, which excludes
the possibility that the observed effects on gene expression
resulted from a general developmental delay. Therefore, the
defects in gene-specific expression caused by TFIIA knock-
down showed that TFIIA is essential for gene expression in
early Xenopus development. Rescue of gene expression of the
TFIIA-dependent genes was tested by injection of xIIAwtror
uncleavable G269ARmRNA together with ??MO and subse-
quent RT-qPCR analysis of these genes (GS17, Xbra, 1A11,
MyoDb, Ef1?, and Hoxb4) from extracts of embryos at stage
11. As shown in Fig. 4E, wild-type and G269A mutant TFIIA
could rescue expression defects in these genes caused by
??MO to significant levels (P ? 0.05 in both cases), while the
levels of rescue by xIIAwt and G269A were not significantly
different (P ? 0.05), showing that the uncleavable G269A
mutant is fully functional in transcription during early stages of
In this study, we have provided several lines of evidence that
taspase 1 is the protease for TFIIA. First, taspase 1 cleaves
TFIIA efficiently in vitro and in vivo, whereas the TFIIA cleav-
age site mutants D274A and G275A cannot be cleaved by
taspase 1. Second, knockdown of endogenous taspase 1 by
RNAi reduces cleavage of overexpressed, as well as endog-
enous, TFIIA, and most conclusively, uncleaved TFIIA is
the only form detected in taspase 1?/?MEFs. The fact that
taspase 1?/?MEF cells could be established and maintained in
culture indicates that the uncleaved TFIIA is transcriptionally
active rather than a nonfunctional precursor. This conclusion
was corroborated and extended by MO knockdown experi-
ments with X. laevis. An uncleavable G269A mutant of TFIIA
(corresponding to human G275A) was able to rescue pheno-
typic and transcriptional defects caused by TFIIA knockdown,
showing that uncleaved TFIIA is sufficient for bulk transcrip-
We showed that taspase 1 cleaves TFIIA at D274/G275
within the highly conserved CRS and that the N terminus G275
generated by taspase 1 is different from the N terminus of the
? subunit identified by us purified from mammalian extracts
(6). Our new finding that G275 rather than D278 is the primary
N-terminal residue of the ? subunit of TFIIA is supported by
the observation that mutations of D274 and G275 prevented
cleavage completely, even upon overexpression of taspase 1,
whereas mutation of D278 diminished but did not abolish
cleavage (Fig. 3B) (6). Furthermore, TFIIA cleavage could not
be detected in taspase 1?/?MEF cells, which unequivocally
demonstrates that taspase 1 is the primary protease for TFIIA.
Studies on the germ cell-specific paralogue of TFIIA??,
TFIIA-like factor (ALF), showed that the C terminus of the ?
subunit of endogenous mouse ALF is D341 (2), indicating that
the cleavage site of ALF in vivo is at D341/G342 (correspond-
ing to D274/G275 in human TFIIA). Mass spectrometric anal-
ysis to identify the C terminus of the human TFIIA? subunit is
complicated due to the lack of arginine and lysine residues in
the region around the cleavage site (unpublished data). N-
terminal residue D278 in the TFIIA? subunit reported previ-
ously (6) is probably generated by a secondary protease. This
could be either an endo- or an exopeptidase activity that re-
moves three more amino acids and yields D278 as the N ter-
minus. The secondary cleavage generates a destabilizing N
terminus for the destruction pathway and might be part of an
intricate regulatory circuitry to fine tune the level of TFIIA (6).
Support for tight regulation of the levels of TFIIA was ob-
tained from our transient-transfection experiments, in which a
clear increase in the levels of the cleaved ? and ? subunits
could not be observed upon complete cleavage of TFIIA??.
Moreover, we only observed a slight decrease in the levels of
the cleaved subunits in the RNAi experiments while a clear
increase in uncleaved TFIIA?? was detected (Fig. 3). These in
vivo observations suggest that the level of cleaved TFIIA is
measured and maintained in cells. One possibility is that ex-
cessive amounts of the cleaved subunits are degraded through
the proteasome-dependent pathway. However, upon protea-
some inhibitor treatment, we could not observe an increase in
the level of the cleaved ? and ? subunits, even when the
uncleaved ?? form was completely processed by overexpressed
taspase 1 (data not shown), which suggests that, apart from the
proteasome-dependent pathway, there may be other mecha-
nisms involved in maintaining the cleaved-protein levels.
TFIIA is the second substrate for taspase 1 identified so far.
The CRS of TFIIA is evolutionarily conserved between differ-
ent species (Fig. 2B), with the exception of the large subunit of
yeast TFIIA, TOA1, which does not contain a CRS and is not
cleaved (19). In addition to the CRS, a downstream acidic
stretch is also conserved in TFIIA in different species, as well
as in Trx group proteins (Fig. 2B). Apart from the CRS and the
acidic stretch, there is little homology in surrounding regions in
different TFIIA proteins and no overall homology between
TFIIA and the MLL protein. These findings suggest that the
CRS, together with the acidic stretch, is necessary and proba-
bly sufficient for cleavage by taspase 1. The acidic stretch may
play a role in cleavage recognition or facilitate docking or
positioning of the active site of taspase 1 on the CRS. Search-
ing for the CRS sequence QV/LDG in the SwissProt database
revealed about 150 proteins that contain the QV/LDG se-
quence, and about 1/10 of these proteins contain acidic
2734ZHOU ET AL.MOL. CELL. BIOL.
stretches (data not shown). It will be of interest to test whether
they are also substrates of taspase 1.
It has remained elusive for a long time whether the un-
cleaved, the cleaved, or both forms of TFIIA are transcription-
ally competent. Since cleaved TFIIA is the major form de-
tected in most cell lines, it has been assumed that the cleaved
form is the active form in transcription. We have previously
shown that uncleaved TFIIA interacts with TBP to form a
distinct TAC complex in embryonal carcinoma P19 cells (15,
16), suggesting that uncleaved TFIIA is transcriptionally ac-
tive. Taspase 1?/?knockout mice in which only the uncleaved
TFIIA form is present (Fig. 3D) survived until birth and
showed minor overall defects (Hsieh, unpublished), indicating
that uncleaved TFIIA is transcriptionally competent and that
cleavage of TFIIA does not serve to render TFIIA competent
for transcription. Taking advantage of an uncleavable mutant
form of TFIIA and the identification of the TFIIA protease,
we provide evidence that uncleaved TFIIA is functional during
Xenopus development. TFIIA?? knockdown in Xenopus re-
sulted in reduced expression of a number of genes induced
during embryogenesis, such as those for GS17 and Xbra,
whereas the expression pattern of Gsc, which is also regulated
during embryogenesis, was not altered (Fig. 4D). Importantly,
an uncleavable TFIIA mutant (G269A in Xenopus) was able to
rescue phenotypic and transcriptional defects in TFIIA knock-
down embryos, showing that the uncleaved form of TFIIA is
functional in early embryogenesis. Our study shows that
cleaved TFIIA is dispensable for bulk transcription and rein-
forces our hypothesis that the biological role of TFIIA cleav-
age is to regulate the levels of TFIIA by degradation through
the proteasome-dependent pathway (6) and cleavage of TFIIA
might be important for the expression of a subset of genes.
Resolving these important issues will require the generation of
conditional knock-in mice carrying an uncleavable mutant of
Salvatore Spicuglia was supported by a Marie Curie Fellowship of
the European Community program Training and Mobility of Re-
searchers under contract HPMF-CT-2002-01646. This work was sup-
ported by grants 812.08.006 NWO (ALW) and 700.53.311 WWO-CW-
TOP in The Netherlands.
We thank Xavier Le Guezennec and Michiel Vermeulen for pro-
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