A UHM-ULM interface with unusual structural features contributes to U2AF2 and SF3B1 association for pre-mRNA splicing.

During spliceosome assembly, the 3' splice site is recognized by sequential U2AF2 complexes, first with Splicing Factor 1 (SF1) and second by the SF3B1 subunit of the U2 small nuclear ribonuclear protein particle. The U2AF2-SF1 interface is well characterized, comprising a U2AF homology motif (UHM) of U2AF2 bound to a U2AF ligand motif (ULM) of SF1. However, the structure of the U2AF2-SF3B1 interface and its importance for pre-mRNA splicing are unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8-Å resolution. We discovered a distinctive trajectory of the SF3B1 ULM across the U2AF2 UHM surface, which differs from prior UHM/ULM structures and is expected to modulate the orientations of the full-length proteins. We established that the binding affinity of the U2AF2 UHM for the cocrystallized SF3B1 ULM rivals that of a nearly full-length U2AF2 protein for an N-terminal SF3B1 region. An additional SF3B6 subunit had no detectable effect on the U2AF2-SF3B1 binding affinities. We further showed that key residues at the U2AF2 UHM-SF3B1 ULM interface contribute to coimmunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2-SF3B1 interface changed splicing of representative human transcripts. From analysis of genome-wide data, we found that many of the splice sites coregulated by U2AF2 and SF3B1 differ from those coregulated by U2AF2 and SF1. Taken together, these findings support distinct structural and functional roles for the U2AF2-SF1 and U2AF2-SF3B1 complexes during the pre-mRNA splicing process.

Histone H2A.X phosphorylation and Caspase-Initiated Chromatin Condensation in late-stage erythropoiesis.

BACKGROUND: Condensation of chromatin prior to enucleation is an essential component of terminal erythroid maturation, and defects in this process are associated with inefficient erythropoiesis and anemia. However, the mechanisms involved in this phenomenon are not well understood. Here, we describe a potential role for the histone variant H2A.X in erythropoiesis. RESULTS: We find in multiple model systems that this histone is essential for normal maturation, and that the loss of H2A.X in erythroid cells results in dysregulation in expression of erythroid-specific genes as well as a nuclear condensation defect. In addition, we demonstrate that erythroid maturation is characterized by phosphorylation at both S139 and Y142 on the C-terminal tail of H2A.X during late-stage erythropoiesis. Knockout of the kinase BAZ1B/WSTF results in loss of Y142 phosphorylation and a defect in nuclear condensation, but does not replicate extensive transcriptional changes to erythroid-specific genes observed in the absence of H2A.X. CONCLUSIONS: We relate these findings to Caspase-Initiated Chromatin Condensation (CICC) in terminal erythroid maturation, where aspects of the apoptotic pathway are invoked while apoptosis is specifically suppressed.

Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM-ULM interaction.

Stable recognition of the intron branchpoint (BP) by the U2 snRNP to form the pre-spliceosome is the first ATP-dependent step of splicing. Genetic and biochemical data from yeast indicate that Cus2 aids U2 snRNA folding into the stem IIa conformation prior to pre-spliceosome formation. Cus2 must then be removed by an ATP-dependent function of Prp5 before assembly can progress. However, the location from which Cus2 is displaced and the nature of its binding to the U2 snRNP are unknown. Here, we show that Cus2 contains a conserved UHM (U2AF homology motif) that binds Hsh155, the yeast homolog of human SF3b1, through a conserved ULM (U2AF ligand motif). Mutations in either motif block binding and allow pre-spliceosome formation without ATP. A 2.0 Å resolution structure of the Hsh155 ULM in complex with the UHM of Tat-SF1, the human homolog of Cus2, and complementary binding assays show that the interaction is highly similar between yeast and humans. Furthermore, we show that Tat-SF1 can replace Cus2 function by enforcing ATP dependence of pre-spliceosome formation in yeast extracts. Cus2 is removed before pre-spliceosome formation, and both Cus2 and its Hsh155 ULM binding site are absent from available cryo-EM structure models. However, our data are consistent with the apparent location of the disordered Hsh155 ULM between the U2 stem-loop IIa and the HEAT repeats of Hsh155 that interact with Prp5. We propose a model in which Prp5 uses ATP to remove Cus2 from Hsh155 such that extended base-pairing between U2 snRNA and the intron BP can occur.