Uncleaved TFIIA is a substrate for taspase 1 and active in transcription - PubMed (original) (raw)
Uncleaved TFIIA is a substrate for taspase 1 and active in transcription
Huiqing Zhou et al. Mol Cell Biol. 2006 Apr.
Abstract
In higher eukaryotes, the large subunit of the general transcription factor TFIIA is encoded by the single TFIIAalphabeta gene and posttranslationally cleaved into alpha and beta 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.
Figures
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 fractionated 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). Nonspecific 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 β 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 antibody in all fractions containing the protease activity. NE, nuclear extracts.
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 β 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 β 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 γ subunit in E. coli, and one-step Ni-NTA purification was applied to obtain semipurified proteins.
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 TFIIAα-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 analysis. The nonspecific signal masking the TFIIAγ subunit in lane 3 is from cross-reaction of the TFIIAγ antibody with the recombinant taspase 1 preparation.
FIG. 4.
An uncleavable G269A mutant of xTFIIA is transcriptionally 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 αβMO, 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 αβMO alone or together with αβMO-resistant xIIAwtR mRNA 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 embryos and analyzed as described in Materials and Methods. The TA complex is represented by the symbol ◂, and nonspecific bands are marked by asterisks. (C) Embryos were injected with αβMO alone or together with xIIAwtR or G269AR mRNA and analyzed at stage 11. αβMO-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. Expression levels of several genes were analyzed at the indicated stages (St.) by RT-qPCR from extracts of embryos injected with either cMO or αβMO. (E) An uncleavable G269A mutant form is able to rescue the expression of TFIIA-dependent genes. Embryos were injected with αβMO alone or together with xIIAwtR or G269AR mRNA and analyzed at stage 11 by RT-qPCR. The expression values of αβMO plus xIIAwtR versus αβMO or αβMO plus G269AR versus αβMO are significantly different (P < 0.05), except for Rb (_P_ > 0.05). The difference in expression values between αβMO plus xIIAwtR and αβMO plus G269AR was not significant (P > 0.05).
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