New proteins related to the Ser-Arg family of splicing factors.

A family of six highly conserved proteins that contain domains rich in alternating serine/arginine residues (SR proteins) function in the regulation of splice site selection and are required for splicing. Using a selective precipitation method, more than 35 proteins were detected in nuclear extracts of HeLa cells that co-fractionate with the defined SR family. Many of these proteins were recognized by three monoclonal antibodies that bind to distinct phosphoepitopes on SR proteins. Two of these SR-related proteins were identified as the nuclear matrix antigens B1C8 and B4A11, which previously have been implicated in splicing. A subset of SR proteins, in their phosphorylated state, are associated with spliceosome complexes through both steps of the splicing reaction, remaining preferentially bound to complexes containing the exon-product. In contrast, other SR-related proteins appear to remain specifically associated with the intron-Iariat complex. The results indicate the existence of a potentially large group of SR-related proteins, and also suggest possible additional functions of SR proteins at a post-splicing level.     

Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of C. elegans

BACKGROUND: SR family and SR-related proteins assemble on exonic splicing enhancer (ESE) sequences to promote both constitutive and regulated splicing. The SRm160 splicing coactivator, an SR-related nuclear matrix protein of 160 kDa, is important for the splicing of specific constitutive and ESE-dependent pre-mRNAs. RESULTS: In the present study, we show that SRm160 is required to promote pre-mRNA splicing mediated by a large population of functional ESE sequences within a randomized 18 nucleotide sequence. This suggests that it functions as a general coactivator by interacting with different SR family/SR-related proteins bound to different ESE sequences. Consistent with this, several SR family and SR-related proteins coimmunoprecipitated specifically with SRm160 in the presence of low salt. We used RNA interference (RNAi) in Caenorhabditis elegans to determine whether interactions between CeSRm160 and different CeSR family proteins are important in a whole-organism context. Previously we showed that RNAi of CeSRm160 and individual CeSR family genes other than CeSF2/ASF results in no obvious phenotype, which is indicative of gene redundancy. In the present study, we demonstrate that RNAi of CeSRm160 in combination with any CeSR family gene results in the production of unfertilized oocytes by the injected mother. CONCLUSIONS: The observation that simultaneous suppression of CeSRm160 and individual CeSR family proteins results in a distinct phenotype is indicative of critical functional interactions between these factors. Our results provide biochemical and genetic evidence indicating that interactions between SRm160 and multiple SR family proteins are important for both optimal splicing activity and for proper development.     

Advances in the study of SR protein family

The name of SR proteins is derived from their typical RS domain that is rich in serine (Ser, S) and arginine (Arg, R). They are conserved in evolution. Up to now, 10 members of the SR protein family have been identified in humans. SR proteins contain one or two RNA binding motifs aside from the RS domain, and also possess special biochemical and immunological features. As to the functions of SR proteins, they facilitate the recruitment of the components of splicesome via protein-protein interaction to prompt the assembly of early splicesome; while in alternative splicing, tissue-specifically expressed SR protein along with the relative ratio of SR protein and heterogeneous nuclear ribonucleoprotein (hnRNP) is composed of two main regulative mechanisms for alternative splicing. Almost all of the biochemical functions are regulated by reversible phosphorylation.     

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.     

Arginine/serine repeats are sufficient to constitute a splicing activation domain

SR proteins are essential pre-mRNA splicing factors that have been shown to bind a number of exonic splicing enhancers where they function to stimulate the splicing of adjacent introns. Members of the SR protein family contain one or two N-terminal RNA binding domains, as well as a C-terminal arginine–serine (RS) rich domain. The RS domains mediate protein–protein interactions with other RS domain containing proteins and are essential for many, but not all, SR protein functions. Hybrid proteins containing an RS domain fused to the bacteriophage MS2 coat protein are sufficient to activate enhancer-dependent splicing in HeLa cell nuclear extract when bound to the pre-mRNA. Here we report progress towards determining the protein sequence requirements for RS domain function. We show that the RS domains from non-SR proteins can also function as splicing activation domains when tethered to the pre-mRNA. Truncation experiments with the RS domain of the human SR protein 9G8 identified a 29 amino acid segment, containing 26 arginine or serine residues, that is sufficient to activate splicing when fused to MS2. We also show that synthetic domains composed solely of RS dipeptides are capable of activating splicing, although their potency is proportional to their size.     

Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins

Serine/arginine-rich (SR) splicing factors play an important role in constitutive and alternative splicing as well as during several steps of RNA metabolism. Despite the wealth of functional information about SR proteins accumulated to-date, structural knowledge about the members of this family is very limited. To gain a better insight into structure-function relationships of SR proteins, we performed extensive sequence analysis of SR protein family members and combined it with ordered/disordered structure predictions. We found that SR proteins have properties characteristic of intrinsically disordered (ID) proteins. The amino acid composition and sequence complexity of SR proteins were very similar to those of the disordered protein regions. More detailed analysis showed that the SR proteins, and their RS domains in particular, are enriched in the disorder-promoting residues and are depleted in the order-promoting residues as compared to the entire human proteome. Moreover, disorder predictions indicated that RS domains of SR proteins were completely unstructured. Two different classification methods, the charge-hydropathy measure and the cumulative distribution function (CDF) of the disorder scores, were in agreement with each other, and they both strongly predicted members of the SR protein family to be disordered. This study emphasizes the importance of the disordered structure for several functions of SR proteins, such as for spliceosome assembly and for interaction with multiple partners. In addition, it demonstrates the usefulness of order/disorder predictions for inferring protein structure from sequence.     

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.     

Unique and redundant functions of SR proteins, a conserved family of splicing factors, in Caenorhabditis elegans development

Serine/arginine-rich proteins (SR proteins) constitute a family of RNA-binding proteins conserved throughout metazoans. The SR proteins are essential for constitutive pre-mRNA splicing and also affect regulated pre-mRNA splicing. We identified five putative genes encoding SR proteins (referred to as srp genes) in Caenorhabditis elegans, examined their expression using the gfp gene as a reporter, and suppressed their functions by double-stranded RNA-mediated interference (RNAi). The srp::gfp fusion genes were expressed in the nuclei of most somatic cells and showed no obvious tissue- or stage-specific expression. Simultaneous RNAi of the five srp genes resulted in embryonic lethality, whereas RNAi of individual srp genes caused no obvious morphological abnormality in the F1 progeny, indicating functional redundancy of the SR proteins. However, RNAi of several combinations of srp genes caused various developmental abnormalities, such as abnormal somatic gonad structures, delayed shift of the germ cell sexual differentiation, and abnormal spermatogenesis. Our results suggest that individual SR proteins have unique but somewhat redundant functions in C. elegans development.     

Blastocyst formation is blocked in mouse embryos lacking the splicing factor SRp20.

SRp20 is a splicing factor belonging to the highly conserved family of SR proteins [1] [2] [3] [4], which have multiple roles in the regulation of constitutive and alternative splicing in vivo. It has been suggested that SR proteins are involved in bringing together the splice sites during spliceosome assembly [5]. SR proteins show partial redundancy, as each single SR protein can restore splicing activity to a splicing-deficient cytoplasmic extract (termed S-100 extract). Nevertheless, several studies demonstrate that individual SR proteins have different effects on the selection of specific alternative splice sites, and they recognize distinct RNA sequences [6] [7] [8] [9] [10] [11] [12]. Also, inactivation of two SR proteins, B52/SRp55 in Drosophila [13] or ASF/SF2 in the chicken cell line DT40 [14], is lethal, indicating the existence of nonredundant functions. Here, using Cre-loxP-mediated recombination in mice to inactivate the SRp20 gene, we found that it is essential for mouse development. Mutant preimplantation embryos failed to form blastocysts and died at the morula stage. Immunofluorescent staining showed that SRp20 is present in oocytes and early stages of embryonic development. This is the first report of mice deficient for a member of the SR protein family. Our experiments confirm that, although similar in structure, the SR proteins are not functionally redundant.     

Over-expression of SR-cyclophilin, an interaction partner of nuclear pinin, releases SR family splicing factors from nuclear speckles

Pre-mRNA splicing takes place within a dynamic ribonucleoprotein particle called the spliceosome and occurs in an ordered pathway. Although it is known that spliceosome consists of five small nuclear RNAs and at least 50 proteins, little is known about how the interaction among the proteins changes during splicing. Here we identify that SR-cyp, a Moca family of nuclear cyclophilin, interacts and colocalizes with nuclear pinin (pnn), a SR-related protein involving in pre-mRNA splicing. Nuclear pnn interacts with SR-cyp via its C-terminal RS domain. Upon SR-cyp over-expression, however, the subnuclear distribution of nuclear pnn is altered, resulting in its redistribution from nuclear speckles to a diffuse nucleoplasmic form. The diffuse subnuclear distribution of nuclear pnn is not due to epitope masking, accelerated protein turnover or post-translational modification. Furthermore, we find that SR-cyp regulates the subnuclear distribution of other SR family proteins, including SC35 and SRm300, in a similar manner as it does on nuclear pnn. This result is significant because it suggests that SR-cyp plays a general role in modulating the distribution pattern of SR-like and SR proteins, similar to that of Clk (cdc2-like kinase)/STY on SR family splicing factors. SR-cyp might direct its effect via either alteration of protein folding/conformation or of protein-protein interaction and thus may add another control level of regulation of SR family proteins and modification of their functions.     

SR蛋白家族在RNA剪接中的调控作用

SR蛋白家族成员都具有一个富含丝氨酸/精氨酸(S/R)重复序列的RS结构域,在RNA剪接体的组装和选择性剪接的调控过程中具有重要的作用。绝大多数SR蛋白是生存的必需因子,通过其RS结构域和特有的其他结构域,实现与前体mRNA的特异性序列或其他剪接因子的相互作用,协同完成剪接位点的正确选择或促进剪接体的形成。深入研究SR蛋白家族在RNA选择性剪接中的调控机制,可以促进以疾病治疗或害虫防治为目的的应用研究。该文总结了SR蛋白家族在基础研究和应用方面的进展。     

Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo

SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function.     

Alternative splicing during chondrogenesis: modulation of fibronectin exon EIIIA splicing by SR proteins

The alternative exon EIIIA of the fibronectin gene is included in mRNAs produced in undifferentiated mesenchymal cells but excluded from differentiated chondrocytes. As members of the SR protein family of splicing factors have been demonstrated to be involved in the alternative splicing of other mRNAs, the role of SR proteins in chondrogenesis-associated EIIIA splicing was investigated. SR proteins interacted with chick exon EIIIA sequences that are required for exon inclusion in a gel mobility shift assay. Addition of SR proteins to in vitro splicing reactions increased the rate and extent of exon EIIIA inclusion. Co-transfection studies employing cDNAs encoding individual SR proteins revealed that SRp20 decreased mRNA accumulation in HeLa cells, which make A+ mRNA, apparently by interfering with pre-mRNA splicing. Co-transfection studies also demonstrated that SRp40 increased exon EIIIA inclusion in chondrocytes, but not in HeLa cells, suggesting the importance of cellular context for SR protein activity. Immunoblot analysis did not reveal a relative depletion of SRp40 in chondrocytic cells. Possible mechanisms for regulation of EIIIA splicing in particular, and chondrogenesis associated splicing in general, are discussed.     

The Protein Kinase Clk/Sty Directly Modulates SR Protein Activity: Both Hyper- and Hypophosphorylation Inhibit Splicing

The splicing of mammalian mRNA precursors requires both protein phosphorylation and dephosphorylation, likely involving modification of members of the SR protein family of splicing factors. Several kinases have been identified that can phosphorylate SR proteins in vitro, and transfection assays have provided evidence that at least one of these, Clk/Sty, can modulate splicing in vivo. But evidence that a specific kinase can directly affect the splicing activity of SR proteins has been lacking. Here, by using purified recombinant Clk/Sty, a catalytically inactive mutant, and individual SR proteins, we show that Clk/Sty directly affects the activity of SR proteins, but not other essential splicing factors, in reconstituted splicing assays. We also provide evidence that both hyper- and hypophosphorylation inhibit SR protein splicing activity, repressing constitutive splicing and switching alternative splice site selection. These findings indicate that Clk/Sty directly and specifically influences the activity of SR protein splicing factors and, importantly, show that both under- and overphosphorylation of SR proteins can modulate splicing.     

Pnn and SR family proteins are differentially expressed in mouse central nervous system

Pinin (pnn) is an SR-related protein that is ubiquitously expressed in most cell types and functions in regulating pre-mRNA splicing and mRNA export. Previously, we demonstrated that pnn is expressed in all tissues during mouse embryonic development with highest levels of expression in the central nervous system (CNS). Here we show that pnn and other SR proteins including SC35 are differentially expressed in the adult mouse CNS, displaying cell type-specific distribution patterns. Immunohistochemical analysis of whole-brain sections showed that levels of pnn and SR proteins expression were very low or nonexistent in the corpus callosum and white matter of cerebellum and spinal cord. Double-immunostaining with antibodies specific to neuron or glial cells showed that most astrocytes and microglia expressed neither pnn nor SR proteins. In contrast, oligodendrocytes and neurons expressed moderate and high levels, respectively, of both pnn and SR proteins. These results suggest that astrocytes are unique among cell types of neuroblast origin in terms of expression SR family proteins. Our results pave the way for future studies of the functional roles of pnn and SR family proteins in adults.     

The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution.

Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region which is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrated that the sites on ASF/SF2 phosphorylated in vitro overlap with those phosphorylated in vivo. Immunofluorescence studies showed that a catalytically inactive form of Clk/Sty co-localized with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase caused a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors.     

Implementing a rational and consistent nomenclature for serine/arginine-rich protein splicing factors (SR proteins) in plants

Growing interest in alternative splicing in plants and the extensive sequencing of new plant genomes necessitate more precise definition and classification of genes coding for splicing factors. SR proteins are a family of RNA binding proteins, which function as essential factors for constitutive and alternative splicing. We propose a unified nomenclature for plant SR proteins, taking into account the newly revised nomenclature of the mammalian SR proteins and a number of plant-specific properties of the plant proteins. We identify six subfamilies of SR proteins in Arabidopsis thaliana and rice (Oryza sativa), three of which are plant specific. The proposed subdivision of plant SR proteins into different subfamilies will allow grouping of paralogous proteins and simple assignment of newly discovered SR orthologs from other plant species and will promote functional comparisons in diverse plant species.