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.

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.

RNA interference (RNAi) has great potential to treat human disease1–3. However, in vivo delivery of short interfering RNAs (siRNAs), which are negatively charged double-stranded RNA macromolecules, remains a major hurdle4–9. Current siRNA delivery has begun to move away from large lipid and synthetic nanoparticles to more defined molecular conjugates9. Here we address this issue by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing for delivery into cells. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses. siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses. Unlike siRNAs, siRNNs avidly bind serum albumin to positively influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice. We believe that siRNNs represent a technology that will open new avenues for development of RNAi therapeutics.

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.It is estimated that more than one-half of human genes are alternatively spliced (1). Although many of these genes may contain only a few splice variants, a few have been shown to possess thousands (1-3). RNA splicing occurs in the nucleus at a macromolecular complex composed of many RNA and protein molecules known as the spliceosome (4, 5). In the last decade it has become apparent that both the assembly of the spliceosome and selection of splice sites depend on the proper phosphorylation of a family of splicing factors known as SR proteins (1). The SR proteins are so named because they have long stretches of arginineserine dipeptide repeats in RS domains. Phosphorylation leads to translocation of the SR protein from the cytoplasm to the nucleus (6, 7) and recruitment of the SR proteins from sites of nuclear storage in speckles to nascent transcripts for splicing (8 -10). Phosphorylated SR proteins are believed to facilitate both 5Ј and 3Ј splice site recognition through interaction with the RS domain in U1-70 K (a component of the U1 small nuclear ribonucleoprotein) and the U2AF heterodimer (11-13). More recently, RS domains were shown to interact directly with critical cis-acting elements in pre-mRNA (14,15). In addition to their functions in the spliceosome, SR proteins are also shown to play a role in the export of processed mRNA from the nucleus to the cytoplasm for translation. Two shuttling SR proteins, ASF/SF2 6 and 9G8, have been shown to conduct the export of mRNA through interactions with a nuclear export factor, TAP (16 -18). Hypophosphorylation of these SR proteins promotes interaction with TAP suggesting that transport may require splicing factor dephosphorylation. These shuttling SR proteins were also found to play a direct role in translation in the cytoplasm (19).Although it is clear that SR proteins can be polyphosphorylated in the RS domain regions by the SRPK ...

SummaryThe SR protein ASF/SF2, an essential splicing factor, contains two functional modules consisting of tandem RNA recognition motifs (RRM1-RRM2) and a C-terminal arginine-serine repeat region (RS domain). The serine protein kinase SRPK1 phosphorylates the RS domain at multiple serines using a directional (C-to-N-terminal) and processive mechanism, a process that directs the SR protein to the nucleus and influences protein-protein interactions associated with splicing function. To investigate how SRPK1 accomplishes this feat, the enzyme-substrate complex was analyzed using single and multi-turnover kinetic methods. Deletion studies revealed that while recognition of the RS domain by a docking groove on SRPK1 is sufficient to initiate the processive and directional mechanism, continued processive phosphorylation in the presence of building repulsive charge relies on the fine-tuning of contacts with the RRM1-RRM2 module. An electropositive pocket in SRPK1 that stabilizes newly phosphorylated serines enhanced processive phosphorylation of later serines. These data indicate that SRPK1 uses stable, yet highly flexible, protein-protein interactions to facilitate both early and late phases of processive phosphorylation of SR proteins. Keywordskinase; kinetics; phosphorylation; splicing; SR proteinThe splicing of precursor (pre-mRNA) is essential for proteome diversity and many cellular regulatory processes but is also associated with numerous diseases when mistakes are propagated into the mature, spliced mRNA. 1; 2; 3; 4 Splicing occurs in a macromolecular complex known as the spliceosome, a dynamic assembly of five small nuclear ribonucleoproteins (snRNPs) and many protein cofactors. 5 An important family of splicing © 2008 Elsevier Ltd. All rights reserved.*To whom correspondence should be sent: Joseph A. Adams,; Email:joeadams@chem.ucsd.edu.. # These authors contributed equally.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptcofactors is the SR proteins so named because they contain a C-terminal domain composed largely of arginine-serine dipeptide repeats (RS domain). In addition to these repetitive sequences, SR proteins contain one or two N-terminal RNA recognition motifs (RRMs) that are essential for recognition of exonic enhancer sequences in pre-mRNA. 6; 7; 8 ¶ The SR proteins play roles in both constitutive and alternative splicing. 9; 10 While SR proteins are critical for the early stages of spliceosome assembly during the establishment of the proper 5′ and 3′ splice sites i...