TAF7: A Check-point Regulator in Transcription Initiation and Translation

Division Of Clinical Sciences - Nci

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Abstract

As noted above, TAF7 serves as a critical checkpoint regulator that ensures each step of transcription is completed before proceeding to the next step. Although most studies have focused on the transcriptional functions of TAF7, there is some evidence to suggest that the role of TAF7 extends beyond transcription regulation. For example, TAF7 has been shown to associate with the 160 kD subunit of cleavage-polyadenylation specificity factor (CPSF), which is required for 3' polyadenylation of mRNA. In S. pombe, mutations in Ptr6p, a homologue of human TAF7, results in the accumulation of RNA in the nucleus, suggesting it plays a role in mRNA -transport to the cytoplasm. Considering our finding that TAF7 travels with Pol II and the nascent transcript during elongation, we hypothesized that TAF7 associates with RNA and executes functions beyond transcriptional regulation in the nucleus. Indeed, we have discovered a novel cytoplasmic, TFIID-independent and non-transcriptional, function for TAF7. We have discovered that in addition to the regulatory role it plays in transcription, TAF7 is an RNA binding protein that binds a large spectrum of RNAs. Using both PAR-CLIP and RNA-immunoprecipitation approaches, we find that TAF7 preferentially binds to 3' UTR regions of transcripts to the consensus motif CUG within an RNA bulge. Intriguingly, we found that TAF7 binds to the HIV TAR element with affinity equal to HIV Tat. Surprisingly, TAF7, a transcription factor that regulates transcription, is present in both the nucleus and the cytoplasm, where it is bound to RNA. This finding leads to the hypothesis that TAF7 has a TFIID- and transcription-independent function in the cytoplasm. Consistent with TAF7 playing a functional role in the cytoplasm, we have mapped both NLS and NES elements within the protein. Mutation of the NLS prevents TAF7 from entering the nucleus; conversely, mutation of the NES sequence retains TAF7 in the nucleus. Export of TAF7 from the nucleus to the cytoplasm is actively regulated since leptomycin B, which inhibits crm1-mediated export, results in nuclear retention of TAF7. We have also mapped an RNA binding domain (RBD); interestingly, the RBD overlaps the NLS. In further support of a cytoplasmic function for TAF7, cytoplasmic TA7 is found in a large 450kD complex that also contains many ribosomal proteins. Both immunoprecipitation and proximity ligation studies have validated the binding of TAF7 to ribosomal proteins and demonstrated that this binding is RNA dependent. Furthermore, TAF7 is recovered from purified polysomes. Of particular interest, the TAF7 NES mutation, which prevents TAF7 export from the nucleus results in reduced translation, without affecting transcription. Similarly, a TAF7 RBD mutant is unable to transport its target RNAs to the cytoplasm, similarly resulting in reduced protein synthesis. Taken together, these findings demonstrate that TAF7 is not only a checkpoint regulator of transcription but also functions to promote translation. To further examine the role of TAF7 in global gene regulation, we have examined the spectrum of cellular proteins to which TAF7 binds in both the nucleus and cytoplasm by bio-ID. These studies have provided new insights into the full functional repertoire of TAF7. They identified nuclear TAF7as largely associated with TFIID. In the cytoplasm, TAF7 was found to be associated with two major complexes involved in proteostasis: the chaperonin CCT complex, dysregulation of which is associated with various cancers, and the proteasome. Studies are currently underway to determine the sites of interaction and functional significance of these associations.