Uncleaved TFIIA Is a Substrate for Taspase 1 and Active in Transcription

Abstract

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 biological 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 binding of TBP to promoters is a key step in the formation of PIC, which is followed by recruitment of general transcription factors 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 ␥ subunit is conserved among different species, whereas sequence similarity in TFIIA␣␤ is limited mostly to the N-terminal region 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 remarkably similar in different evolutionarily distinct species and is embedded in an otherwise nonconserved and probably unstructured 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 assumed 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 ␤ plu...

Taspase1 processing alters TFIIA cofactor properties in the regulation of TFIID

“…6,15,16,45 How might Taspase1 cleavage of the TFIIAa/b precursor protein affect TFIIA interactions with TFIID? The Taspase1 cleavage site (269-LVLQVDjGTGDT) 23 is located in a functionally poorly defined non-conserved linker region in the TFIIAa/b precursor protein, that connects evolutionary conserved N-and C-termini, which are absolutely required for TFIIA function 46 1. X-ray structure analysis of ternary TBP/ TFIIA/TATA complexes, containing a minimal TFIIA complex composed only of the conserved TFIIAa/b N-and C-termini, revealed a boot-shaped TFIIA structure composed of 2 distinct domains orientated approximately 120 C from each other, in which all 3 TFIIA subunits are in intimate contact.…”

“…To investigate this possibility we digested our rTFIIA (p55/p12) preparation with highly purified recombinant Taspase1 23,35 and re-purified processed rTFIIA (p35/p19/p12; Fig. 3A) by ion exchange chromatography on RESOURCE Q and phosphocellulose (P11) resins.…”

“…3; 15 ) was cleaved with 50 mg recombinant 6His:epitopetagged Taspase1 enzyme 23,35 for 1 h at 37 C in 1.4 ml BC buffer containing 100 mM KCl, 1.5 mM MgCl 2 , 0.1 mg/ml insulin and 2 mM dithiothreitol (DTT). Following protease cleavage, Taspase1-processed rTFIIA (35/19/12 kDa) was separated from Taspase1 by ion exchange chromatography on Resource Q (GE Healthcare Life Sciences) and phosphocellulose (P11; Whatman) resins.…”

“…1,22 TFIIAa and TFIIAb originate from a single gene and are produced by sitespecific cleavage of a 55 kDa TFIIAa/b precursor protein by the protease Taspase1. 23 The relative abundance of unprocessed TFIIA and Taspase1-processed TFIIA in mammalian cells varies significantly between cell types, 22 suggesting that Taspase1 processing of TFIIA is regulated in a cell-type specific manner. Unprocessed TFIIA has been shown to associate with free TBP in the absence of DNA to form a transcriptionally active TBP-TFIIA-containing complex (TAC) lacking TAFs.…”

A transcription factor IIA-binding site differentially regulates RNA polymerase II-mediated transcription in a promoter context-dependent manner

“…The precursor of TFIIAα/β can be digested by taspase1, but uncleaved TFIIAα/β remains active in transcriptional regulation (20). Recent studies showed that the cleavage of TFIIA by taspase 1 is involved in a number of molecular and biological processes (21)(22)(23)(24).…”