Dephosphorylation-dependent sorting of SR splicing factors during mRNP maturation

SR proteins are a family of sequence-specific RNA binding proteins originally discovered as essential factors for pre-mRNA splicing and recently implicated in mRNA transport, stability, and translation. Here, we used a genetic complementation system derived from conditional knockout mice to address the function and regulation of SR proteins in vivo. We demonstrate that ASF/SF2 and SC35 are each required for cell viability, but, surprisingly, the effector RS domain of ASF/SF2 is dispensable for cell survival in MEFs. Although shuttling SR proteins have been implicated in mRNA export, prevention of ASF/SF2 from shuttling had little impact on mRNA export. We found that shuttling and nonshuttling SR proteins are segregated in an orderly fashion during mRNP maturation, indicating distinct recycling pathways for different SR proteins. We further showed that this process is regulated by differential dephosphorylation of the RS domain, thus revealing a sorting mechanism for mRNP transition from splicing to export.     

Mechanism of Dephosphorylation of the SR Protein ASF/SF2 by Protein Phosphatase 1

SR proteins are essential splicing factors whose function is controlled by multi-site phosphorylation of a C-terminal domain rich in arginine-serine repeats (RS domain). The protein kinase SRPK1 has been shown to polyphosphorylate the N-terminal portion of the RS domain (RS1) of the SR protein ASF/SF2, a modification that promotes nuclear entry of this splicing factor and engagement in splicing function. Later, dephosphorylation is required for maturation of the spliceosome and other RNA processing steps. While phosphates are attached to RS1 in a sequential manner by SRPK1, little is known about how they are removed. To investigate factors that control dephosphorylation, we monitored region-specific mapping of phosphorylation sites in ASF/SF2 as a function of the protein phosphatase PP1. We showed that 10 phosphates added to the RS1 segment by SRPK1 are removed in a preferred N-to-C manner, directly opposing the C-to-N phosphorylation by SRPK1. Two N-terminal RNA recognition motifs in ASF/SF2 control access to the RS domain and guide the directional mechanism. Binding of RNA to the RNA recognition motifs protects against dephosphorylation, suggesting that engagement of the SR protein with exonic splicing enhancers can regulate phosphoryl content in the RS domain. In addition to regulation by N-terminal domains, phosphorylation of the C-terminal portion of the RS domain (RS2) by the nuclear protein kinase Clk/Sty inhibits RS1 dephosphorylation and disrupts the directional mechanism. The data indicate that both RNA-protein interactions and phosphorylation in flanking sequences induce conformations of ASF/SF2 that increase the lifetime of phosphates in the RS domain.     

Nuclear export and retention signals in the RS domain of SR proteins

Splicing factors of the SR protein family share a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RS domain, which is extensively phosphorylated, promotes protein-protein interactions and directs subcellular localization and-in certain situations-nucleocytoplasmic shuttling of individual SR proteins. We analyzed mutant versions of human SF2/ASF in which the natural RS repeats were replaced by RD or RE repeats and compared the splicing and subcellular localization properties of these proteins to those of SF2/ASF lacking the entire RS domain or possessing a minimal RS domain consisting of 10 consecutive RS dipeptides (RS10). In vitro splicing of a pre-mRNA that requires an RS domain could take place when the mutant RD, RE, or RS10 domain replaced the natural domain. The RS10 version of SF2/ASF shuttled between the nucleus and the cytoplasm in the same manner as the wild-type protein, suggesting that a tract of consecutive RS dipeptides, in conjunction with the RRMs of SF2/ASF, is necessary and sufficient to direct nucleocytoplasmic shuttling. However, the SR protein SC35 has two long stretches of RS repeats, yet it is not a shuttling protein. We demonstrate the presence of a dominant nuclear retention signal in the RS domain of SC35.     

Deletion of the N-terminus of SF2/ASF permits RS-domain-independent pre-mRNA splicing

Serine/arginine-rich (SR) proteins are essential splicing factors with one or two RNA-recognition motifs (RRMs) and a C-terminal arginine- and serine-rich (RS) domain. SR proteins bind to exonic splicing enhancers via their RRM(s), and from this position are thought to promote splicing by antagonizing splicing silencers, recruiting other components of the splicing machinery through RS-RS domain interactions, and/or promoting RNA base-pairing through their RS domains. An RS domain tethered at an exonic splicing enhancer can function as a splicing activator, and RS domains play prominent roles in current models of SR protein functions. However, we previously reported that the RS domain of the SR protein SF2/ASF is dispensable for in vitro splicing of some pre-mRNAs. We have now extended these findings via the identification of a short inhibitory domain at the SF2/ASF N-terminus; deletion of this segment permits splicing in the absence of this SR protein's RS domain of an IgM pre-mRNA substrate previously classified as RS-domain-dependent. Deletion of the N-terminal inhibitory domain increases the splicing activity of SF2/ASF lacking its RS domain, and enhances its ability to bind pre-mRNA. Splicing of the IgM pre-mRNA in S100 complementation with SF2/ASF lacking its RS domain still requires an exonic splicing enhancer, suggesting that an SR protein RS domain is not always required for ESE-dependent splicing activation. Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing.     

Distinct RNP complexes of shuttling hnRNP proteins with pre-mRNA and mRNA: candidate intermediates in formation and export of mRNA

Nascent pre-mRNAs associate with hnRNP proteins in hnRNP complexes, the natural substrates for mRNA processing. Several lines of evidence indicate that hnRNP complexes undergo substantial remodeling during mRNA formation and export. Here we report the isolation of three distinct types of pre-mRNP and mRNP complexes from HeLa cells associated with hnRNP A1, a shuttling hnRNP protein. Based on their RNA and protein compositions, these complexes are likely to represent distinct stages in the nucleocytoplasmic shuttling pathway of hnRNP A1 with its bound RNAs. In the cytoplasm, A1 is associated with its nuclear import receptor (transportin), the cytoplasmic poly(A)-binding protein, and mRNA. In the nucleus, A1 is found in two distinct types of complexes that are differently associated with nuclear structures. One class contains pre-mRNA and mRNA and is identical to previously described hnRNP complexes. The other class behaves as freely diffusible nuclear mRNPs (nmRNPs) at late nuclear stages of maturation and possibly associated with nuclear mRNA export. These nmRNPs differ from hnRNPs in that while they contain shuttling hnRNP proteins, the mRNA export factor REF, and mRNA, they do not contain nonshuttling hnRNP proteins or pre-mRNA. Importantly, nmRNPs also contain proteins not found in hnRNP complexes. These include the alternatively spliced isoforms D01 and D02 of the hnRNP D proteins, the E0 isoform of the hnRNP E proteins, and LRP130, a previously reported protein with unknown function that appears to have a novel type of RNA-binding domain. The characteristics of these complexes indicate that they result from RNP remodeling associated with mRNA maturation and delineate specific changes in RNP protein composition during formation and transport of mRNA in vivo.     

The shuttling SR protein 9G8 plays a role in translation of unspliced mRNA containing a constitutive transport element

The splicing regulatory SR protein, 9G8, has recently been proposed to function in mRNA export in conjunction with the export protein, Tap/NXF1. Tap interacts directly with the Mason-Pfizer monkey virus constitutive transport element (CTE), an element that enables export of unspliced, intron-containing mRNA. Based on our previous finding that Tap can promote polysome association and translation of CTE-RNA, we investigated the effect of 9G8 on cytoplasmic RNA fate. 9G8 was shown to enhance expression of unspliced RNA containing either the Mason-Pfizer monkey virus-CTE or the recently discovered Tap-CTE. 9G8 also enhanced polyribosome association of unspliced RNA containing a CTE. Hyperphosphorylated 9G8 was present in monosomes and small polyribosomes, whereas soluble fractions contained only hypophosphorylated protein. Our results are consistent with a model in which hypophosphorylated SR proteins remain stably associated with messenger ribonucleoprotein (mRNP) complexes during export and are released during translation initiation concomitant with increased phosphorylation. These results provide further evidence for crucial links between RNA splicing, export and translation.     

Functional characterization of SR and SR-related genes in Caenorhabditis elegans

The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR-related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development.     

Phosphorylation by Sky1p promotes Npl3p shuttling and mRNA dissociation

Mammalian SR proteins are currently thought to function in mRNA export as well as splicing. They contain multiple phosphorylated serine/arginine (RS/SR) dipeptides. Although SR domains can be phosphorylated by many kinases in vitro, the physiologically relevant kinase(s), and the role(s) of these modifications in vivo have remained unclear. Npl3 is a shuttling protein in budding yeast that we showed previously to be a substrate for the mammalian SR protein kinase, SRPK1, as well as the related yeast kinase, Sky1. Here we demonstrate that Sky1p phosphorylates only one of Npl3p's eight SR/RS dipeptides. Mutation of the C-terminal RS to RA, or deletion of SKY1, results in the cytoplasmic accumulation of Npl3p. The redistribution of Npl3p is accompanied by its increased association with poly(A)+ RNA and decreased association with its import receptor, Mtr10p, in vivo. We propose that phosphorylation of Npl3p by the cytoplasmically localized Sky1p is required for efficient release of mRNA upon termination of export.     

Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.     

Phosphorylation of Y14 modulates its interaction with proteins involved in mRNA metabolism and influences its methylation

The multicomponent exon junction complex (EJC) is deposited on the spliced mRNA during pre-mRNA splicing and is implicated in several post-splicing events, including mRNA export, nonsense-mediated mRNA decay (NMD), and translation control. This report is the first to identify potential post-translational modifications of the EJC core component Y14. We demonstrate that Y14 is phosphorylated at its repeated arginine/serine (RS) dipeptides, likely by SR protein-specific kinases. Phosphorylation of Y14 abolished its interaction with EJC components as well as factors that function downstream of the EJC. A non-phosphorylatable Y14 mutant was equivalent to the wild-type protein with respect to its association with spliced mRNA and its ability in NMD activation, but the mutant sequestered EJC and NMD factors on ribosome-containing mRNA ribonucleoproteins (mRNPs). We therefore hypothesize that phosphorylation of Y14 occurs upon completion of mRNA surveillance, leading to dissociation of Y14 from ribosome-containing mRNPs. Moreover, we found that Y14 is possibly methylated at multiple arginine residues in the carboxyl-terminal domain and that methylation of Y14 was antagonized by phosphorylation of RS dipeptides. This study reveals antagonistic post-translational modifications of Y14 that may be involved in the remodeling of Y14-containing mRNPs.     

Phosphorylation mechanism and structure of serine-arginine protein kinases

The splicing of mRNA requires a group of essential factors known as SR proteins, which participate in the maturation of the spliceosome. These proteins contain one or two RNA recognition motifs and a C-terminal domain rich in Arg-Ser repeats (RS domain). SR proteins are phosphorylated at numerous serines in the RS domain by the SR-specific protein kinase (SRPK) family of protein kinases. RS domain phosphorylation is necessary for entry of SR proteins into the nucleus, and may also play important roles in alternative splicing, mRNA export, and other processing events. Although SR proteins are polyphosphorylated in vivo, the mechanism underlying this complex reaction has only been recently elucidated. Human alternative splicing factor [serine/arginine-rich splicing factor 1 (SRSF1)], a prototype for the SR protein family, is regiospecifically phosphorylated by SRPK1, a post-translational modification that controls cytoplasmic-nuclear localization. SRPK1 binds SRSF1 with unusually high affinity, and rapidly modifies about 10-12 serines in the N-terminal region of the RS domain (RS1), using a mechanism that incorporates sequential, C-terminal to N-terminal phosphorylation and several processive steps. SRPK1 employs a highly dynamic feeding mechanism for RS domain phosphorylation in which the N-terminal portion of RS1 is initially bound to a docking groove in the large lobe of the kinase domain. Upon subsequent rounds of phosphorylation, this N-terminal segment translocates into the active site, and a β-strand in RNA recognition motif 2 unfolds and occupies the docking groove. These studies indicate that efficient regiospecific phosphorylation of SRSF1 is the result of a contoured binding cavity in SRPK1, a lengthy Arg-Ser repetitive segment in the RS domain, and a highly directional processing mechanism.     

Structural and functional analysis of RNA and TAP binding to SF2/ASF

The serine/arginine-rich (SR) protein splicing factor 2/alternative splicing factor (SF2/ASF) has a role in splicing, stability, export and translation of messenger RNA. Here, we present the structure of the RNA recognition motif (RRM) 2 from SF2/ASF, which has an RRM fold with a considerably extended loop 5 region, containing a two-stranded beta-sheet. The loop 5 extension places the previously identified SR protein kinase 1 docking sequence largely within the RRM fold. We show that RRM2 binds to RNA in a new way, by using a tryptophan within a conserved SWQLKD motif that resides on helix alpha1, together with amino acids from strand beta2 and a histidine on loop 5. The linker connecting RRM1 and RRM2 contains arginine residues, which provide a binding site for the mRNA export factor TAP, and when TAP binds to this region it displaces RNA bound to RRM2.     

SR splicing factors serve as adapter proteins for TAP-dependent mRNA export

The only mammalian RNA binding adapter proteins known to partner with TAP/NXF1, the primary receptor for general mRNA export, are members of the REF family. We demonstrate that at least three shuttling SR (serine/arginine-rich) proteins interact with the same domain of TAP/NXF1 that binds REFs. Included are 9G8 and SRp20, previously shown to promote the export of intronless RNAs. A peptide derived from the N terminus of 9G8 inhibits the binding of both REF and SR proteins to TAP/NXF1 in vitro, and this finding argues for competitive interactions. In Xenopus oocytes, the N terminus of 9G8 exhibits a dominant-negative effect on mRNA export from the nucleus, while addition of excess TAP/NXF1 overcomes this inhibition. Thus, multiple adapters including SR proteins most likely cooperate to recruit multiple copies of TAP/NXF1 for efficient mRNA export.     

Shuttling SR proteins: more than splicing factors

Serine-arginine (SR) proteins commonly designate a family of eukaryotic RNA binding proteins containing a protein domain composed of several repeats of the arginine-serine dipeptide, termed the arginine-serine (RS) domain. This protein family is involved in essential nuclear processes such as constitutive and alternative splicing of mRNA precursors. Besides participating in crucial activities in the nuclear compartment, several SR proteins are able to shuttle between the nucleus and the cytoplasm and to exert regulatory functions in the latter compartment. This review aims at discussing the properties of shuttling SR proteins with particular emphasis on their nucleo-cytoplasmic traffic and their cytoplasmic functions. Indeed, recent findings have unravelled the complex regulation of SR protein nucleo-cytoplasmic distribution and the diversity of cytoplasmic mechanisms in which these proteins are involved.     

The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation

The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.     

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.