ASF/SF2 is a member of a conserved family of splicing factors known as SR proteins. These proteins, which are necessary for splicing in vitro, contain one or two amino-terminal RNP-type RNA-binding domains and an extensively phosphorylated carboxy-terminal region enriched in repeating Arg-Ser dipeptides (RS domains). Previous studies have suggested that RS domains participate in protein-protein interactions with other RS domain-containing proteins. Here we provide evidence that the RS domain of unphosphorylated recombinant ASF/SF2 is necessary, but not sufficient, for binding to the Ul snRNP-specific 70-kD protein (70K) in vitro. An apparent interaction of the isolated RS domain with 70K was observed if contaminating RNA was not removed, suggesting a nonspecific bridging between the basic RS domain, RNA, and 70K. In vitro phosphorylation of recombinant ASF/SF2 both significantly enhanced binding to 70K and also eliminated the RS domain-RNA interaction. Providing evidence that these interactions are relevant to splicing, ASF/SF2 can bind selectively to Ul snRNP in an RS domain-dependent, phosphorylation-enhanced manner. We also describe conditions that reveal for the first time a phosphorylation requirement for ASF/SF2 splicing activity in vitro.

Sphingosine kinase 1 (SphK1) is a lipid kinase that catalyzes the conversion of sphingosine to sphingosine-1-phosphate (S1P), which has been shown to play a role in lymphocyte trafficking, angiogenesis, and response to apoptotic stimuli. As a central enzyme in modulating the S1P levels in cells, SphK1 emerges as an important regulator for diverse cellular functions and a potential target for drug discovery. Here, we present the crystal structures of human SphK1 in the apo form and in complexes with a substrate sphingosine-like lipid, ADP, and an inhibitor at 2.0-2.3 Å resolution. The SphK1 structures reveal a two-domain architecture in which its catalytic site is located in the cleft between the two domains and a hydrophobic lipid-binding pocket is buried in the C-terminal domain. Comparative analysis of these structures with mutagenesis and kinetic studies provides insight into how SphK1 recognizes the lipid substrate and catalyzes ATP-dependent phosphorylation.

SR proteins are a conserved family of splicing factors that function in both constitutive and activated splicing. We reported previously that phosphorylation of the SR protein ASF/SF2 enhances its interaction with the U1 snRNP-specific 70K protein and is required for the protein to function in splicing, while other studies have provided evidence that subsequent dephosphorylation can also be required for SR protein function, at least in constitutive splicing. We now show that the phosphorylation status of ASF/SF2 can differentially affect several properties of the protein. In keeping with a dynamic cycle of phosphorylation-dephosphorylation during splicing, ASF/SF2 phosphorylation was found to affect interaction with several putative protein targets in different ways: positively, negatively or not at all. Extending these results, we also show that, in contrast to constitutive splicing, dephosphorylation is not required for ASF/SF2 to function as a splicing activator. We discuss these results with respect to the differential protein-protein interactions that must occur during constitutive and activated splicing.

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The SR proteins constitute a family of splicing factors, highly conserved in metazoans, that contain one or two amino-terminal RNA-binding domains (RBDs) and a region enriched in arginine/serine repeats (RS domain) at the carboxyl terminus. Previous studies have shown that SR proteins possess distinct RNA-binding specificities that likely contribute to their unique functions, but it is unclear whether RS domains have specific roles in vivo. Here, we used a genetic system developed in the chicken B cell line DT40 to address this question. Expression of chimeric proteins generated by fusion of the RS domains of heterologous SR proteins, or a human TRA-2 protein, with the RBDs of ASF/SF2 allowed cell growth following genetic inactivation of endogenous ASF/SF2, indicating that RS domains are interchangeable for all functions required to maintain cell viability. However, a chimera containing the RS domain from a related splicing factor, U2AF 65 , could not rescue viability and was inactive in in vitro splicing assays, suggesting that this domain performs a distinct function. We also used the DT40 system to show that depletion of ASF/SF2 affects splicing of specific transcripts in vivo. Although splicing of several simple constitutive introns was not significantly affected, the alternative splicing patterns of two model pre-mRNAs switched in a manner consistent with predictions from previous studies. Unexpectedly, ASF/SF2 depletion resulted in a substantial increase in splicing of an HIV-1 tat pre-mRNA substrate, indicating that ASF/SF2 can repress tat splicing in vivo. These results provide the first demonstration that an SR protein can influence splicing of specific pre-mRNAs in vivo.