Role of the U2AF Homology Motif in Human Tat‐SF1 as a Cofactor for HIV‐1 Splicing

The HIV‐1 life cycle relies on controlled ratios of unspliced and alternatively spliced mRNAs. Human Tat‐SF1 is a requisite host factor for the regulation of HIV‐1 RNA splicing. It is known that Tat‐SF1 associates with the U2 small nuclear ribonucloprotein particle (snRNP) of the spliceosome. However, the underlying mechanisms by which Tat‐SF1 affects HIV‐1 RNA splicing remain elusive. The core U2 snRNP protein subunit SF3b155 comprises five tryptophan‐containing motifs, termed U2AF ligand motifs (ULM) interspersed with regulated phosphorylation sites. Our lab has shown previously that the SF3b155 ULMs are recognized by splicing factors containing a small globular domain named U2AF homology motif (UHM). Based on sequence homology, we identified a putative UHM in Tat‐SF1. We hypothesize that this UHM provides a phosphorylation‐sensitive link between this alternative splicing factor and the splicing machinery via SF3b155. To investigate the Tat‐SF1 UHM interactions with SF3b155 and the role of phosphorylation, we determined the crystal structures of the Tat‐SF1 UHM in complex with a minimal phosphorylated SF3b155 ULM and for comparison the unphosphorylated and apo ‐forms at 1.7 Å, 1.9 Å, and 1.1 Å, respectively. The apo ‐Tat‐SF1 UHM displays a canonical UHM‐fold. The complex with the phosphorylated ULM reveals that the phosphorylation site is located closely above the plane of an aromatic residue. This suggests a reduced affinity of phosphorylated SF3b155 for the UHM due to electrostatic repulsion between the phosphate‐anion and the π‐cloud of the aromatic side chain. We are currently testing the phosphorylation‐sensitivity and significance of these Tat‐SF1 UHM/SF3b155 ULM‐interactions for HIV‐1 infectivity in vivo .