The 2.9 A crystal structure of the core SRPK1:ASF/SF2 complex reveals that the N-terminal half of the basic RS domain of ASF/SF2, which is destined to be phosphorylated, is bound to an acidic docking groove of SRPK1 distal to the active site. Phosphorylation of ASF/SF2 at a single site in the C-terminal end of the RS domain generates a primed phosphoserine that binds to a basic site in the kinase. Biochemical experiments support a directional sliding of the RS peptide through the docking groove to the active site during phosphorylation, which ends with the unfolding of a beta strand of the RRM domain and binding of the unfolded region to the docking groove. We further suggest that the priming of the first serine facilitates directional substrate translocation and efficient phosphorylation.

SR proteins promote spliceosome formation by recognizing exonic splicing enhancers (ESEs) during pre-mRNA splicing. Each SR protein binds diverse ESEs using strategies that are yet to be elucidated. Here, we show that the RNA-binding domain (RBD) of SRSF1 optimally binds to decameric purine rich ESE sequences although locations of purines are not stringently specified. The presence of uracils either within or outside of the recognition site is detrimental for binding with SRSF1. The entire RBD, comprised of two RRMs and a glycine-rich linker, is essential for ESE binding. Mutation within each segment reduced or nearly abolished binding, suggesting that these segments mediate cooperative binding. The linker plays a decisive role in organizing ESE binding. The flanking basic regions of the linker appear to communicate with each other in bringing the two RRMs close together to form the complex with RNA. Our study thus suggests semi-conservative adaptable interaction between ESE and SRSF1, and such binding mode is not only essential for the recognition of plethora of physiological ESE sequences but may also be essential for the interaction with various factors during the spliceosome assembly.

The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and ϳ12 of these serines are processively phosphorylated by the SR protein kinase 1 (SRPK1). We have recently shown that a docking motif in ASF/SF2 specifically interacts with a groove in SRPK1, and this interaction is necessary for processive phosphorylation. We previously showed that SRPK1 and its yeast ortholog Sky1p maintain their active conformations using diverse structural strategies. Here we tested if the mechanism of ASF/SF2 phosphorylation by SRPK is evolutionarily conserved. We show that Sky1p forms a stable complex with its heterologous mammalian substrate ASF/SF2 and processively phosphorylates the same sites as SRPK1. We further show that Sky1p utilizes the same docking groove to bind yeast SR-like protein Gbp2p and phosphorylates all three serines present in a contiguous RS dipeptide stretch. However, the mechanism of Gbp2p phosphorylation appears to be non-processive. Thus, there are physical attributes of SR and SR-like substrates that dictate the mechanism of phosphorylation, whereas the ability to processively phosphorylate substrates is inherent to SR protein kinases.Pre-mRNA splicing and mRNA export are complex processes, which involve a large number of protein and RNA factors (1-4). SR proteins, a class of non-small nuclear ribonucleoprotein splicing factors, participate in every step in the spliceosome assembly and catalysis (5). SR proteins have unique domain architecture. The N-terminal domain contains one or two RNA recognition motifs (RRMs) 3 and is responsible for RNA binding, and the C-terminal domain, known as the RS domain, is rich in long stretches of serine-arginine/arginineserine (SR/RS) dipeptides (6). The RS domains of SR proteins are extensively phosphorylated. Although many kinases have been demonstrated to target SR proteins, members of the SR protein kinase (SRPK) family have been established as the predominant kinase (6 -10). Mammalian SR protein kinase 1 (SRPK1) and the yeast enzyme, Sky1p, are the two most studied SRPKs. Members of the SRPK family display strict substrate specificity, preferring to phosphorylate only serine residues flanked by arginines. One of the well studied mammalian SR proteins, ASF/SF2, contains 20 serines within its 50-residuelong RS domain (see Fig. 1a). SRPK1 phosphorylates ϳ10 -12 of these serines in the N-terminal RS1 region of the RS domain (11). This multisite phosphorylation occurs in a processive manner where the kinase remains bound to the substrate until all sites are phosphorylated (12). Phosphorylation of RS1 is essential for the nuclear import of ASF/SF2.In yeast, Sky1p regulates the phosphorylation and nuclear import of SR-like proteins . However, unlike the mammalian SR proteins, the yeast proteins do not have a classic RS domain with long stretches of arginine/ serine repeats (6, 16) (Fig. 1a). In the case of Npl3p, eight isolated RS dipeptides are present within the RGG domain, and the C-terminal most RS dipeptide is...

ASF/SF2, a member of the serine-arginine (SR) protein family, has two RRM domains (RRM1 and RRM2) and a C-terminus domain rich in RS dipeptides. The SR protein kinase 1 (SRPK1) phosphorylates approximately 12 of these serines using a semi-processive mechanism. The x-ray structure of the ASF/SF2:SRPK1 complex revealed several features of the complex that raised intriguing questions of how the substrate is phosphorylated by the kinase: The part of the RS domain destined to be phosphorylated at later stages of the reaction docks to a kinase groove distal to the active site while the neighboring RRM2 binds near the active site (1). In this study we investigated the interplay between the RS domain and RRM2 for stable association and phosphorylation of ASF/SF2. Despite several contacts in the enzyme-substrate complex, free RRM2 does not bind efficiently to SRPK1 unless the docking groove is occupied by the RS domain. This domain cross-talk enhances the processive phosphorylation of the RS domain. The RRM:SRPK1 contact residues control the folding of a critical beta strand in RRM2. Unfolding of this structural element may force the N-terminal serines of the RS domain into the active site for sequential phosphorylation. Thus, ASF/SF2 represents a new class of substrates that use unique primary sequence to induce allosteric binding, processive phosphorylation, and product release.The SR protein family plays essential roles in pre-mRNA splicing. These proteins are thought to be involved in every step during the assembly of the spliceosome, the catalyst for splicing reactions (2,3). SR proteins are modular and contain one or two RNA recognition motifs (RRMs) at the N-terminus and an arginine-serine dipeptide rich domain at the Cterminus. One of the most well studied members of the SR protein family, ASF/SF2, contains two N-terminal RRMs followed by a 50-residue C-terminus RS domain. The RS domain can be divided into two modules, RS1 and RS2, whose sequence and structural properties allow them to be distinctly recognized and regulated by functionally unique kinases. Of the eighteen serines in the RS domain, twelve are present as every other residue, primarily as RS dipeptides within a 24 residue-stretch, which is referred to as the RS1 motif † This work is supported by NIH grants (GM067969 to JAA and GM084277 to GG). * Corresponding authors: Joseph Adams, j2adams@ucsd.edu; Gourisankar Ghosh, gghosh@ucsd.edu. || Current Address: Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong SAR, People's Republic of China Supporting Information Available: Data showing non-specific RNA contaminants has no role in kinase-substrate interaction. Crosslinking data shows the interaction between β4 of ASF/SF2 and the docking groove of the kinase. This material is available free of charge via the Internet at