SR proteins in vertical integration of gene expression from transcription to RNA processing to translation.

SR proteins have been studied extensively as a family of RNA-binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and colocalize with genes that are engaged in specific intra- and interchromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings, therefore, highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell-cycle progression in higher eukaryotic cells.

Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones.

Phosphorylation is essential for the SR family of splicing factors/regulators to function in constitutive and regulated pre-mRNA splicing; yet both hypo- and hyperphosphorylation of SR proteins are known to inhibit splicing, indicating that SR protein phosphorylation must be tightly regulated in the cell. However, little is known how SR protein phosphorylation might be regulated during development or in response to specific signaling events. Here, we report that SRPK1, a ubiquitously expressed SR protein-specific kinase, directly binds to the cochaperones Hsp40/DNAjc8 and Aha1, which mediate dynamic interactions of the kinase with the major molecular chaperones Hsp70 and Hsp90 in mammalian cells. Inhibition of the Hsp90 ATPase activity induces dissociation of SRPK1 from the chaperone complexes, which can also be triggered by a stress signal (osmotic shock), resulting in translocation of the kinase from the cytoplasm to the nucleus, differential phosphorylation of SR proteins, and alteration of splice site selection. These findings connect the SRPK to the molecular chaperone system that has been implicated in numerous signal transduction pathways and provide mechanistic insights into complex regulation of SR protein phosphorylation and alternative splicing in response to developmental cues and cellular signaling.

Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly.

It has been widely accepted that the early spliceosome assembly begins with U1 small nuclear ribonucleoprotein (U1 snRNP) binding to the 5' splice site (5'SS), which is assisted by the Ser/Arg (SR)-rich proteins in mammalian cells. In this process, the RS domain of SR proteins is thought to directly interact with the RS motif of U1-70K, which is subject to regulation by RS domain phosphorylation. Here we report that the early spliceosome assembly event is mediated by the RNA recognition domains (RRM) of serine/arginine-rich splicing factor 1 (SRSF1), which bridges the RRM of U1-70K to pre-mRNA by using the surface opposite to the RNA binding site. Specific mutation in the RRM of SRSF1 that disrupted the RRM-RRM interaction also inhibits the formation of spliceosomal E complex and splicing. We further demonstrate that the hypo-phosphorylated RS domain of SRSF1 interacts with its own RRM, thus competing with U1-70K binding, whereas the hyper-phosphorylated RS domain permits the formation of a ternary complex containing ESE, an SR protein, and U1 snRNP. Therefore, phosphorylation of the RS domain in SRSF1 appears to induce a key molecular switch from intra- to intermolecular interactions, suggesting a plausible mechanism for the documented requirement for the phosphorylation/dephosphorylation cycle during pre-mRNA splicing.

Regulated cellular partitioning of SR protein-specific kinases in mammalian cells.

Reversible phosphorylation of the SR family of splicing factors plays an important role in pre-mRNA processing in the nucleus. Interestingly, the SRPK family of kinases specific for SR proteins is localized in the cytoplasm, which is critical for nuclear import of SR proteins in a phosphorylation-dependent manner. Here, we report molecular dissection of the mechanism involved in partitioning SRPKs in the cytoplasm. Common among all SRPKs, the bipartite kinase catalytic core is separated by a unique spacer sequence. The spacers in mammalian SRPK1 and SRPK2 share little sequence homology, but they function interchangeably in restricting the kinases in the cytoplasm. Removal of the spacer in SRPK1 had little effect on the kinase activity, but it caused a quantitative translocation of the kinase to the nucleus and consequently induced aggregation of splicing factors in the nucleus. Rather than carrying a nuclear export signal as suggested previously, we found multiple redundant signals in the spacer that act together to anchor the kinase in the cytoplasm. Interestingly, a cell cycle signal induced nuclear translocation of the kinase at the G2/M boundary. These findings suggest that SRPKs may play an important role in linking signaling to RNA metabolism in higher eukaryotic cells.

St John's wort induces both cytochrome P450 3A4-catalyzed sulfoxidation and 2C19-dependent hydroxylation of omeprazole.

OBJECTIVE: St John's wort, an extract of the medicinal plant Hypericum perforatum, is widely used as an herbal antidepressant. Although the ability of St John's wort to induce cytochrome P450 (CYP) 3A4-mediated reaction has been well established, the effect on CYP2C19 is still not determined. Thus the objective of this study was to determine the impact of St John's wort on the pharmacokinetic profiles of omeprazole and its metabolites. METHODS: Twelve healthy adult men (6 CYP2C19*1/CYP2C19*1, 4 CYP2C19*2/CYP2C19*2 and 2 CYP2C19*2/CYP2C19*3) were enrolled in a 2-phase randomized crossover design. In each phase the volunteers received placebo or a 300-mg St John's wort tablet 3 times daily for 14 days. Then all subjects took a 20-mg omeprazole capsule orally. Blood samples were collected up to 12 hours after omeprazole administration. Omeprazole and its metabolites were quantified by use of HPLC with ultraviolet detection. RESULTS: Omeprazole and its metabolites all exhibit CYP2C19 genotype-dependent pharmacokinetic profiles. After a 14-day treatment with St John's wort, substantial decreases in plasma concentrations of omeprazole were observed. The peak plasma concentration (C(max)) significantly decreased by 37.5% +/- 13.3% (P =.001) in CYP2C19*2/CYP2C19*2 or *3 and by 49.6% +/- 20.7% (P =.017) in CYP2C19*1/CYP2C19*1; the area under the concentration-time curve extrapolated to infinity [AUC(0- infinity )] decreased by 37.9% +/- 21.3% (P =.014) and 43.9% +/- 23.7% (P =.011) in CYP2C19 mutant and wild genotypes, respectively. Moreover, the C(max) and AUC(0- infinity ) of omeprazole sulfone increased by 160.3% +/- 45.5% (P =.001) and by 136.6% +/- 84.6% (P =.014), 155.5% +/- 58.8% (P =.001), and 158.7% +/- 101.4% (P =.017) in mutant and wild genotypes, respectively. St John's wort increased the C(max) of 5-hydroxyomeprazole by 38.1% +/- 30.5% (P =.028) and the AUC(0- infinity ) by 37.2% +/- 26% (P =.005) in CYP2C19 wild-type subjects, whereas it did not produce any significant alterations to the corresponding pharmacokinetic parameters in subjects with variant genotypes. CONCLUSION: St John's wort induces both CYP3A4-catalyzed sulfoxidation and CYP2C19-dependent hydroxylation of omeprazole and enormously decreases the plasma concentrations of omeprazole. Clinically relevant interactions with other drugs may occur and must be taken into account when St John's wort is being taken.

Mass spectrometric and kinetic analysis of ASF/SF2 phosphorylation by SRPK1 and Clk/Sty.

Assembly of the spliceosome requires the participation of SR proteins, a family of splicing factors rich in arginine-serine dipeptide repeats. The repeat regions (RS domains) are polyphosphorylated by the SRPK and Clk/Sty families of kinases. The two families of kinases have distinct enzymatic properties, raising the question of how they may work to regulate the function of SR proteins in RNA metabolism in mammalian cells. Here we report the first mass spectral analysis of the RS domain of ASF/SF2, a prototypical SR protein. We found that SRPK1 was responsible for efficient phosphorylation of a short stretch of amino acids in the N-terminal portion of the RS domain of ASF/SF2 while Clk/Sty was able to transfer phosphate to all available serine residues in the RS domain, indicating that SR proteins may be phosphorylated by different kinases in a stepwise manner. Both kinases bind with high affinity and use fully processive catalytic mechanisms to achieve either restrictive or complete RS domain phosphorylation. These findings have important implications on the regulation of SR proteins in vivo by the SRPK and Clk/Sty families of kinases.