TFIIA transcriptional activity is controlled by a 'cleave-and-run' Exportin-1/Taspase 1-switch.

Transcription factor TFIIA is controlled by complex regulatory networks including proteolysis by the protease Taspase 1, though the full impact of cleavage remains elusive. Here, we demonstrate that in contrast to the general assumption, de novo produced TFIIA is rapidly confined to the cytoplasm via an evolutionary conserved nuclear export signal (NES, amino acids 21VINDVRDIFL30), interacting with the nuclear export receptor Exportin-1/chromosomal region maintenance 1 (Crm1). Chemical export inhibition or genetic inactivation of the NES not only promotes TFIIA's nuclear localization but also affects its transcriptional activity. Notably, Taspase 1 processing promotes TFIIA's nuclear accumulation by NES masking, and modulates its transcriptional activity. Moreover, TFIIA complex formation with the TATA box binding protein (TBP) is cooperatively enhanced by inhibition of proteolysis and nuclear export, leading to an increase of the cell cycle inhibitor p16INK, which is counteracted by prevention of TBP binding. We here identified a novel mechanism how proteolysis and nuclear transport cooperatively fine-tune transcriptional programs.

Evolutionary divergence of Threonine Aspartase1 leads to species-specific substrate recognition.

Proteases are key regulators of life. Human Threonine Aspartase1 processes substrates, such as the mixed-lineage leukemia (MLL) protein, containing two cleavage sites, CS1 and CS2. Likewise, MLL's Drosophila ortholog trithorax is cleaved by Drosophila Threonine Aspartase1 (dTasp), suggesting a mechanistic coevolution. However, a detailed analysis of dTasp's function was missing so far. Here, active and inactive dTasp mutants allowed to compare substrate recognition and cleavage site selectivity of human and Drosophila enzymes. In contrast to the human protease, our cell-based assay revealed a preferential processing of CS2-like (QLD↓Gx[xD/Dx]) targets for dTasp, whereas cleavage of CS1-like targets (QVD↓Gx[xD/Dx]) was significantly impaired. Systematic mutagenesis of the CS2 sequence defined the motif x[FILMW]D↓Gx[xD/Dx] as the consensus cleavage sequence for dTasp. Substrate species selectivity of the enzymes was uncovered by demonstrating that dTasp cleaves Drosophila TFIIA, but not the human ortholog, suggesting evolutionary divergence of TFIIA downstream networks. Also, Drosophila USF2 was neither predicted nor cleaved by dTasp. Moreover, we found that dTasp cleavage site selectivity is independent of heterocomplex formation, as dTasp exists predominantly as an αß-monomer. Collectively, we provide novel insights into evolutionary similarities and divergence concerning Threonine Aspartase1 function in different species, which may aid to dissect and better target human Threonine Aspartase1 in malignancies.

Fly versus man: evolutionary impairment of nucleolar targeting affects the degradome of Drosophila's Taspase1.

Human Taspase1 is essential for development and cancer by processing critical regulators, such as the mixed-lineage leukemia protein. Likewise, its ortholog, trithorax, is cleaved by Drosophila Taspase1 (dTaspase1), implementing a functional coevolution. To uncover novel mechanism regulating protease function, we performed a functional analysis of dTaspase1 and its comparison to the human ortholog. dTaspase1 contains an essential nucleophile threonine(195), catalyzing cis cleavage into its α- and ß-subunits. A cell-based assay combined with alanine scanning mutagenesis demonstrated that the target cleavage motif for dTaspase1 (Q(3)[F/I/L/M](2)D(1)↓G(1')X(2')X(3')) differs significantly from the human ortholog (Q(3)[F,I,L,V](2)D(1)↓G(1')x(2')D(3')D(4')), predicting an enlarged degradome containing 70 substrates for Drosophila. In contrast to human Taspase1, dTaspase1 shows no discrete localization to the nucleus/nucleolus due to the lack of the importin-α/nucleophosmin1 interaction domain (NoLS) conserved in all vertebrates. Consequently, dTaspase1 interacts with neither the Drosophila nucleoplasmin-like protein nor human nucleophosmin1. The impact of localization on the protease's degradome was confirmed by demonstrating that dTaspase1 did not efficiently process nuclear substrates, such as upstream stimulatory factor 2. However, genetic introduction of the NoLS into dTaspase1 restored its nucleolar localization, nucleophosmin1 interaction, and efficient cleavage of nuclear substrates. We report that evolutionary functional divergence separating vertebrates from invertebrates can be achieved for proteases by a transport/localization-regulated mechanism.