The pre-mRNA splicing and transcription factor Tat-SF1 is a functional partner of the spliceosome SF3b1 subunit via a U2AF homology motif interface

Abstract

Edited by Karin Musier-ForsythThe transcription elongation and pre-mRNA splicing factor Tat-SF1 associates with the U2 small nuclear ribonucleoprotein (snRNP) of the spliceosome. However, the direct binding partner and underlying interactions mediating the Tat-SF1-U2 snRNP association remain unknown. Here, we identified SF3b1 as a Tat-SF1-interacting subunit of the U2 snRNP. Our 1.1 Å resolution crystal structure revealed that Tat-SF1 contains a U2AF homology motif (UHM) protein-protein interaction module. We demonstrated that Tat-SF1 preferentially and directly binds the SF3b1 subunit compared with other U2AF ligand motif (ULM)-containing splicing factors, and further established that SF3b1 association depends on the integrity of the Tat-SF1 UHM. We next compared the Tat-SF1-binding affinities for each of the five known SF3b1 ULMs and then determined the structures of representative high-and low-affinity SF3b1 ULM complexes with the Tat-SF1 UHM at 1.9 Å and 2.1 Å resolutions, respectively. These structures revealed a canonical UHM-ULM interface, comprising a Tat-SF1 binding pocket for a ULM tryptophan (SF3b1 Trp 338 ) and electrostatic interactions with a basic ULM tail. Importantly, we found that SF3b1 regulates Tat-SF1 levels and that these two factors influence expression of overlapping representative transcripts, consistent with a functional partnership of Tat-SF1 and SF3b1. Altogether, these results define a new molecular interface of the Tat-SF1-U2 snRNP complex for gene regulation.Tat stimulatory factor 1 (Tat-SF1) 4 was originally identified as a host cofactor that activates Tat-directed HIV-1 transcrip-tion (1, 2). Subsequent studies revealed that Tat-SF1 normally stimulates human transcription elongation (3, 4) as a complex with P-TEFb, SPT5, and RAP30 (2, 5-7). The Saccharomyces cerevisiae Tat-SF1 homologue, CUS2, promotes transcription elongation in combination with the RNA unwindases PRP5 and PRP11 (8). Beyond transcription, CUS2 is well-established as an assembly factor for the U2 small nuclear (sn)RNA in the early stages of pre-mRNA splicing (9 -13). Like CUS2, human Tat-SF1 influences pre-mRNA splicing (14 -18). Reduced Tat-SF1 levels typically promote intron retention rather than exon-skipping or alternative splice sites. Most recently, critical Tat-SF1 functions in pre-mRNA splicing have emerged for embryonic stem cell differentiation (17,18).Tat-SF1 co-immunoprecipitates with the U2 small nuclear ribonucleoprotein particle (17)(18)(19)(20), which anneals with the pre-mRNA branch point site in the early steps of spliceosome assembly. Yet, the direct interaction partner(s) of Tat-SF1 in the human spliceosome remains unknown. Clues arise from experimental observations coupled with the primary sequences of Tat-SF1 and the U2 snRNP components. Primary sequence analysis suggests that Tat-SF1 contains two modular domains, an RNA recognition motif (RRM) and a U2AF homology motif (UHM) (Fig. 1A). The UHM is a protein-interaction module with specialized features for recognizing short "U2AF ligand motifs...

U2 AF 65 assemblies drive sequence‐specific splice site recognition

“…U2AF 65 reinforced the recruitment of CAPERa to GST-SF3b155c but not that of PUF60 and SPF45 (Fig 2A). A similar effect was observed when adding a high-affinity complex of U2AF 65 with a minimal domain of U2AF 35 that should not allow the access of CAPERa to the ULM of U2AF 65 (Fig 2A, right panel and Appendix Fig S3) [36,37]. U2AF 65 addition did not lead to an efficient pulldown of CAPERa with GST-SF1c (GST-SF1_1-255; Fig EV1A).…”

“…These data extend a previous FRET analysis that demonstrated the proximity of overexpressed U2AF 65 and CAPERα , and support the possibility that pairs of UHMSFs have coordinated actions in splicing. A recent report has now established that the protein tat‐SF1 can also form a UHM–ULM interaction structure with SF3b155, which might extend the complexity of the network of UHMSF interactions in vivo .…”

“…Interactions with additional partners could also regulate RS domain-dependent U2AF 65 and CAPERa assemblies. In particular, U2AF 65 is generally thought to be tightly associated with U2AF 35 to form a U2AF heterodimer. U2AF 35 also possesses an RS domain.…”

“…In particular, U2AF 65 is generally thought to be tightly associated with U2AF 35 to form a U2AF heterodimer. U2AF 35 also possesses an RS domain. As it was shown that the presence of at least one of the U2AF RS domains is necessary for drosophila viability, we envisage that the RS domain of U2AF 35 Putative specific functions of U2AF 65 and CAPERa assemblies…”

“…Splicing of intervening sequences from eukaryotic pre-mRNAs is achieved by a macromolecular machinery called the spliceosome that assembles in a stepwise manner (reviewed in Refs [1,2]). In an early step of assembly, the U2AF small subunit (U2AF 35 ) binds the conserved AG dinucleotide just preceding the 3 0 splice site [3][4][5] (Fig 1A). The U2AF large subunit (U2AF 65 ) binds the polypyrimidine tract (PPT) through its tandem RNA recognition motifs (RRM) [3,[6][7][8].…”