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 cleavage 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 endogenous, 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 experiments with X. laevis. An uncleavable G269A mutant of TFIIA (corresponding to human G275A) was able to rescue phenotypic and transcriptional defects caused by TFIIA knockdown, showing that uncleaved TFIIA is sufficient for bulk transcription.
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 β 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 β 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. ) (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 TFIIAαβ, TFIIA-like factor (ALF), showed that the C terminus of the α subunit of endogenous mouse ALF is D341 (2
), indicating that the cleavage site of ALF in vivo is at D341/G342 (corresponding to D274/G275 in human TFIIA). Mass spectrometric analysis to identify the C terminus of the human TFIIAα subunit is complicated due to the lack of arginine and lysine residues in the region around the cleavage site (unpublished data).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 TFIIAβ subunit reported previously (6
) is probably generated by a secondary protease. This could be either an endo- or an exopeptidase activity that removes three more amino acids and yields D278 as the N terminus. 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 obtained from our transient-transfection experiments, in which a clear increase in the levels of the cleaved α and β subunits could not be observed upon complete cleavage of TFIIAαβ. Moreover, we only observed a slight decrease in the levels of the cleaved subunits in the RNAi experiments while a clear increase in uncleaved TFIIAαβ was detected (Fig. ). These in vivo observations suggest that the level of cleaved TFIIA is measured and maintained in cells. One possibility is that excessive amounts of the cleaved subunits are degraded through the proteasome-dependent pathway. However, upon proteasome inhibitor treatment, we could not observe an increase in the level of the cleaved α and β subunits, even when the uncleaved αβ form was completely processed by overexpressed taspase 1 (data not shown), which suggests that, apart from the proteasome-dependent pathway, there may be other mechanisms 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 different species (Fig. ), 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. ). 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 probably 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. Searching for the CRS sequence QV/LDG in the SwissProt database revealed about 150 proteins that contain the QV/LDG sequence, and about 1/10 of these proteins contain acidic 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 uncleaved, the cleaved, or both forms of TFIIA are transcriptionally competent. Since cleaved TFIIA is the major form detected 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
), suggesting that uncleaved TFIIA is transcriptionally active. Taspase 1−/−
knockout mice in which only the uncleaved TFIIA form is present (Fig. ) 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. TFIIAαβ knockdown in Xenopus
resulted 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. ). Importantly, an uncleavable TFIIA mutant (G269A in Xenopus
) was able to rescue phenotypic and transcriptional defects in TFIIA knockdown 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 reinforces our hypothesis that the biological role of TFIIA cleavage 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 TFIIA.