Mouse Dfa Is a Repressor of TATA-box Promoters and Interacts with the Abt1 Activator of Basal Transcription

Christopher S Brower,Lucia Veiga,Richard H Jones,Alexander Varshavsky

doi:10.1074/jbc.m110.118638

Christopher S Brower, Lucia Veiga + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.118638

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Abstract

Our study of the mouse Ate1 arginyltransferase, a component of the N-end rule pathway, has shown that Ate1 pre-mRNA is produced from a bidirectional promoter that also expresses, in the opposite direction, a previously uncharacterized gene (Hu, R. G., Brower, C. S., Wang, H., Davydov, I. V., Sheng, J., Zhou, J., Kwon, Y. T., and Varshavsky, A. (2006) J. Biol. Chem. 281, 32559-32573). In this work, we began analyzing this gene, termed Dfa (divergent from Ate1). Mouse Dfa was found to be transcribed from both the bidirectional P(Ate1/Dfa) promoter and other nearby promoters. The resulting transcripts are alternatively spliced, yielding a complex set of Dfa mRNAs that are present largely, although not exclusively, in the testis. A specific Dfa mRNA encodes, via its 3'-terminal exon, a 217-residue protein termed Dfa(A). Other Dfa mRNAs also contain this exon. Dfa(A) is sequelogous (similar in sequence) to a region of the human/mouse HTEX4 protein, whose physiological function is unknown. We produced an affinity-purified antibody to recombinant mouse Dfa(A) that detected a 35-kDa protein in the mouse testis and in several cell lines. Experiments in which RNA interference was used to down-regulate Dfa indicated that the 35-kDa protein was indeed Dfa(A). Furthermore, Dfa(A) was present in the interchromatin granule clusters and was also found to bind to the Ggnbp1 gametogenetin-binding protein-1 and to the Abt1 activator of basal transcription that interacts with the TATA-binding protein. Given these results, RNA interference was used to probe the influence of Dfa levels in luciferase reporter assays. We found that Dfa(A) acts as a repressor of TATA-box transcriptional promoters.

Highlights

Gron is a destabilizing N-terminal residue of a substrate protein
We show that Dfa is transcribed from both the bidirectional PAte1/Dfa promoter and other nearby promoters
We found that DfaA acts as a repressor of this promoter but does not influence the bidirectional PAte1/Dfa promoter, which contains a CpG island and lacks the TATA-box

Summary

Introduction

Gron is a destabilizing N-terminal residue of a substrate protein. Among such residues, some are primary destabilizing N-terminal residues, which are recognized directly by ubiquitin ligases of the N-end rule pathway, called N-recognins. This analysis (Fig. 1B) showed that, to the Ate1 exons 1A, 1B, and 2 and the bidirectional PAte1/Dfa promoter, the genomic sequences of specific mouse Dfa exons that had been identified by RT-PCR contained more gap-free DNA segments with greater than 50% identity to the corresponding human genomic DNA than nearby less sequelogous (less similar in sequence) [18] DNA regions.

Results

Conclusion