Complex Evolutionary Relationships Among Four Classes of Modular RNA-Binding Splicing Regulators in Eukaryotes: The hnRNP, SR, ELAV-Like and CELF Proteins

Alternative RNA splicing in multicellular organisms is regulated by a large group of proteins of mainly unknown origin. To predict the functions of these proteins, classification of their domains at the sequence and structural level is necessary. We have focused on four groups of splicing regulators, the heterogeneous nuclear ribonucleoprotein (hnRNP), serine?Carginine (SR), embryonic lethal, abnormal vision (ELAV)-like, and CUG-BP and ETR-like factor (CELF) proteins, that show increasing diversity among metazoa. Sequence and phylogenetic analyses were used to obtain a broader understanding of their evolutionary relationships. Surprisingly, when we characterised sequence similarities across full-length sequences and conserved domains of ten metazoan species, we found some hnRNPs were more closely related to SR, ELAV-like and CELF proteins than to other hnRNPs. Phylogenetic analyses and the distribution of the RRM domains suggest that these proteins diversified before the last common ancestor of the metazoans studied here through domain acquisition and duplication to create genes of mixed evolutionary origin. We propose that these proteins were derived independently rather than through the expansion of a single protein family. Our results highlight inconsistencies in the current classification system for these regulators, which does not adequately reflect their evolutionary relationships, and suggests that a domain-based classification scheme may have more utility.     

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.     

The SR protein family

The processing of pre-mRNAs is a fundamental step required for the expression of most metazoan genes. Members of the family of serine/arginine (SR)-rich proteins are critical components of the machineries carrying out these essential processing events, highlighting their importance in maintaining efficient gene expression. SR proteins are characterized by their ability to interact simultaneously with RNA and other protein components via an RNA recognition motif (RRM) and through a domain rich in arginine and serine residues, the RS domain. Their functional roles in gene expression are surprisingly diverse, ranging from their classical involvement in constitutive and alternative pre-mRNA splicing to various post-splicing activities, including mRNA nuclear export, nonsense-mediated decay, and mRNA translation. These activities point up the importance of SR proteins during the regulation of mRNA metabolism.     

Regiospecific Phosphorylation Control of the SR Protein ASF/SF2 by SRPK1

SR proteins (splicing factors containing arginine-serine repeats) are essential factors that control the splicing of precursor mRNA by regulating multiple steps in spliceosome development. The prototypical SR protein ASF/SF2 (human alternative splicing factor) contains two N-terminal RNA recognition motifs (RRMs) (RRM1 and RRM2) and a 50-residue C-terminal RS (arginine-serine-rich) domain that can be phosphorylated at numerous serines by the protein kinase SR-specific protein kinase (SRPK) 1. The RS domain [C-terminal domain that is rich in arginine-serine repeats (residues 198-248)] is further divided into N-terminal [RS1: N-terminal portion of the RS domain (residues 198-227)] and C-terminal [RS2: C-terminal portion of the RS domain (residues 228-248)] segments whose modification guides the nuclear localization of ASF/SF2. While previous studies revealed that SRPK1 phosphorylates RS1, regiospecific and temporal-specific control within the largely redundant RS domain is not well understood. To address this issue, we performed engineered footprinting and single-turnover experiments to determine where and how SRPK1 initiates phosphorylation within the RS domain. The data show that local sequence elements in the RS domain control the strong kinetic preference for RS1 phosphorylation. SRPK1 initiates phosphorylation in a small region of serines (initiation box) in the middle of the RS domain at the C-terminal end of RS1 and then proceeds in an N-terminal direction. This initiation process requires both a viable docking groove in the large lobe of SRPK1 and one RRM (RRM2) on the N-terminal flank of the RS domain. Thus, while local RS/SR content steers regional preferences in the RS domain, distal contacts with SRPK1 guide initiation and directional phosphorylation within these regions.     

A novel splicing regulator shares a nuclear import pathway with SR proteins

Alternative splicing of precursor mRNA is often regulated by serine/arginine-rich proteins (SR proteins) and hnRNPs, and varying their concentration in the nucleus can be a mechanism for controlling splice site selection. To understand the nucleocytoplasmic transport mechanism of splicing regulators is of key importance. SR proteins are delivered to the nucleus by transportin-SRs (TRN-SRs), importin beta-like nuclear transporters. Here we identify and characterize a non-SR protein, RNA-binding motif protein 4 (RBM4), as a novel substrate of TRN-SR2. TRN-SR2 interacts specifically with RBM4 in a Ran-sensitive manner. TRN-SR2 indeed mediates the nuclear import of a recombinant protein containing the RBM4 C-terminal domain. This domain serves as a signal for both nuclear import and export, and for nuclear speckle targeting. Finally, both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre-mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection. Thus, a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus.     

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.     

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.     

The serine/arginine-rich protein family in rice plays important roles in constitutive and alternative splicing of pre-mRNA

Ser/Arg-rich (SR) proteins play important roles in the constitutive and alternative splicing of pre-mRNA. We isolated 20 rice (Oryza sativa) genes encoding SR proteins, of which six contain plant-specific characteristics. To determine whether SR proteins modulate splicing efficiency and alternative splicing of pre-mRNA in rice, we used transient assays in rice protoplasts by cotransformation of SR protein genes with the rice Waxy(b) (Wx(b))-beta-glucuronidase fusion gene. The results showed that plant-specific RSp29 and RSZp23, an SR protein homologous to human 9G8, enhanced splicing and altered the alternative 5' splice sites of Wx(b) intron 1. The resulting splicing pattern was unique to each SR protein; RSp29 stimulated splicing at the distal site, and RSZp23 enhanced splicing at the proximal site. Results of domain-swapping experiments between plant-specific RSp29 and SCL26, which is a homolog of human SC35, showed the importance of RNA recognition motif 1 and the Arg/Ser-rich (RS) domain for the enhancement of splicing efficiencies. Overexpression of plant-specific RSZ36 and SRp33b, a homolog of human ASF/SF2, in transgenic rice changed the alternative splicing patterns of their own pre-mRNAs and those of other SR proteins. These results show that SR proteins play important roles in constitutive and alternative splicing of rice pre-mRNA.     

Role of SR protein modular domains in alternative splicing specificity in vivo

The SR proteins constitute a family of nuclear phosphoproteins which are required for constitutive splicing and also influence alternative splicing regulation. They have a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal domain, rich in arginine and serine residues. The functional role of the different domains of SR proteins in constitutive splicing activity has been extensively studied in vitro; however, their contribution to alternative splicing specificity in vivo has not been clearly established. We sought to address how the modular domains of SR proteins contribute to alternative splicing specificity. The activity of a series of chimeric proteins consisting of domain swaps between different SR proteins showed that splice site selection is determined by the nature of the RRMs and that RRM2 of SF2/ASF has a dominant role and can confer specificity to a heterologous protein. In contrast, the identity of the RS domain is not important, as the RS domains are functionally interchangeable. The contribution of the RRMs to alternative splicing specificity in vivo suggests that sequence-specific RNA binding by SR proteins is required for this activity.     

Serine-arginine (SR) protein-like factors that antagonize authentic SR proteins and regulate alternative splicing.

We have characterized two RNA-binding proteins, of apparent molecular masses of approximately 40 and 35 kDa, which possess a single N-terminal RNA-recognition motif (RRM) followed by a C-terminal domain rich in serine-arginine dipeptides. Their primary structures resemble the single-RRM serine-arginine (SR) protein, SC35; however their functional effects are quite distinctive. The 40-kDa protein cannot complement SR protein-deficient HeLa cell S100 extract and showed a dominant negative effect in vitro against the authentic SR proteins, SF2/ASF and SC35. Interestingly, the 40- and 35-kDa proteins antagonize SR proteins and activate the most distal alternative 5' splice site of adenovirus E1A pre-mRNA in vivo, an activity that is similar to that characterized previously for the heterogeneous nuclear ribonucleoprotein particles A/B group of proteins. A series of recombinant chimeric proteins consisting of domains from these proteins and SC35 in various combinations showed that the RRM, but not the C-terminal domain rich in serine-arginine dipeptides, has a dominant role in this activity. Because of the similarity to SR proteins we have named these proteins SRrp40 and SRrp35, respectively, for SR-repressor proteins of approximately 40 and approximately 35 kDa. Both factors show tissue- and cell type-specific patterns of expression. We propose that these two proteins are SR protein-like alternative splicing regulators that antagonize authentic SR proteins in the modulation of alternative 5' splice site choice.     

Hypophosphorylated ASF/SF2 binds TAP and is present in messenger ribonucleoproteins

Serine/arginine-rich proteins (SR proteins) function in precursor mRNA (pre-mRNA) splicing and may also act as adaptors for mRNA export. SR proteins are dynamically phosphorylated in their RS domain, and differential phosphorylation modulates their splicing activity and subcellular localization. In this study, we investigated the influence of phosphorylation on the function of SR proteins in events occurring during mRNA maturation. Immunoprecipitation experiments showed that the mRNA export receptor TAP associates preferentially with the hypophosphorylated form of shuttling SR proteins, including ASF/SF2. Overexpression of ASF induced subnuclear relocalization of TAP to SR protein-enriched nuclear speckles, suggesting their interaction in vivo. Moreover, the ASF found in a nucleoplasmic fraction rich in heterogeneous nuclear ribonucleoprotein (hnRNP) complexes is hyperphosphorylated, whereas mature messenger RNP (mRNP)-bound ASF is hypophosphorylated. Therefore, hypophosphorylation of ASF in mRNPs coincides with its higher affinity for TAP, suggesting that dephosphorylation of ASF promotes both its incorporation into mRNPs and recruitment of TAP for mRNA export. Thus, the phosphorylation state of RS domains may modulate the function of mammalian shuttling SR proteins during mRNA maturation or export.     

Distinct functions of the closely related tandem RNA-recognition motifs of hnRNP A1.

hnRNP A1 regulates alternative splicing by antagonizing SR proteins. It consists of two closely related, tandem RNA-recognition motifs (RRMs), followed by a glycine-rich domain. Analysis of variant proteins with duplications, deletions, or swaps of the RRMs showed that although both RRMs are required for alternative splicing function, each RRM plays distinct roles, and their relative position is important. Surprisingly, RRM2 but not RRM1 could support this function when duplicated, despite their very similar structure. Specific RNA binding and annealing are not sufficient for hnRNP A1 alternative splicing function. These observations, together with phylogenetic and structural data, suggest that the two RRMs are quasi-symmetric but functionally nonequivalent modules that evolved as components of a single bipartite domain.     

A novel family of plant splicing factors with a Zn knuckle motif: examination of RNA binding and splicing activities

An important group of splicing factors involved in constitutive and alternative splicing contain an arginine/serine (RS)-rich domain. We have previously demonstrated the existence of such factors in plants and report now on a new family of splicing factors (termed the RSZ family) from Arabidopsis thaliana which additionally harbor a Zn knuckle motif similar to the human splicing factor 9G8. Although only around 20 kDa in size, members of this family possess a multi-domain structure. In addition to the N-terminal RNA recognition motif (RRM), a Zn finger motif of the CCHC-type is inserted in an RGG-rich region; all three motifs are known to contribute to RNA binding. The C-terminal domain has a characteristic repeated structure which is very arginine-rich and centered around an SP dipeptide. One member of this family, atRSZp22, has been shown to be a phosphoprotein with properties similar to SR proteins. Furthermore, atRSZp22 was able to complement efficiently splicing deficient mammalian S100 as well as h9G8-depleted extracts. RNA binding assays to selected RNA sequences indicate an RNA binding specificity similar to the human splicing factors 9G8 and SRp20. Taken together, these result show that atRSZp22 is a true plant splicing factor which combines structural and functional features of both h9G8 and hSRp20.     

Adaptable molecular interactions guide phosphorylation of the SR protein ASF/SF2 by SRPK1

The SR (arginine-serine rich) protein ASF/SF2 (also called human alternative splicing factor), an essential splicing factor, contains two functional modules consisting of tandem RNA recognition motifs (RRMs; RRM1-RRM2) and a C-terminal arginine-serine repeat region (RS domain, a domain rich in arginine-serine repeats). The SR-specific protein kinase (SRPK) 1 phosphorylates the RS domain at multiple serines using a directional (C-terminal-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-turnover and multiturnover 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 the processive phosphorylation of SR proteins.