At the interface: Physical basis for FIR/PUF60 to navigate between transcriptional regulation and pre-mRNA splicing

Accumulating evidence indicates that transcription and pre-mRNA splicing are either concurrent or coupled events for the cell to process genetic information efficiently. The far upstream element binding protein interacting repressor (FIR), also known as the poly(U)-binding splicing factor 60kDa (PUF60), is at this interface, regulating both transcription of an essential proto-oncogene, c-myc, and pre-mRNA splicing. However, it is unclear how nature uses a single protein architecture to modulate two biological processes with very different protein-DNA, protein-RNA, and protein-protein interaction networks. Here we delineate the functional and structural features of the core domains of FIR/PUF60---two central canonical RNA-Recognition Motifs (RRMs) and one C-terminal atypical RRM, known as the U2AF-Homology Motif (UHM). We show that the two central RRMs of FIR/PUF60 comprise the DNA/RNA interacting domain mediating the dimerization of FIR/PUF60 in response to nucleic-acid binding. The structure of the PUF60:AdML pre-mRNA 3' intron sequence shows a dimeric structure analogous to the FIR:FUSE DNA complex. Binding sites of the RRMs interact mainly with the bases of nucleic acids and accommodate either DNA or RNA. The UHM is critical for association with another splicing factor, SF1, but dispensable for its association with FBP, a partner protein in the regulation of c-myc. The extended linker between RRM2 and the UHM is likely conferring flexibility for the UHM to locate SF1. Using the accumulated information, we re-mapped the structure of FIR/PUF60 as a unique paradigm for multi-RRM proteins that are key players with single stranded DNA and RNA. Our study reveals the design of a protein architecture that can bridge transcription and pre-mRNA splicing, two essential genetic processing mechanisms that were conventionally considered separate.