Insights into nuclear organization in plants as revealed by the dynamic distribution of Arabidopsis SR splicing factors

Serine/arginine-rich (SR) proteins are splicing regulators that share a modular structure consisting of one or two N-terminal RNA recognition motif domains and a C-terminal RS-rich domain. We investigated the dynamic localization of the Arabidopsis thaliana SR protein RSZp22, which, as we showed previously, distributes in predominant speckle-like structures and in the nucleolus. To determine the role of RSZp22 diverse domains in its nucleolar distribution, we investigated the subnuclear localization of domain-deleted mutant proteins. Our results suggest that the nucleolar localization of RSZp22 does not depend on a single targeting signal but likely involves different domains/motifs. Photobleaching experiments demonstrated the unrestricted dynamics of RSZp22 between nuclear compartments. Selective inhibitor experiments of ongoing cellular phosphorylation influenced the rates of exchange of RSZp22 between the different nuclear territories, indicating that SR protein mobility is dependent on the phosphorylation state of the cell. Furthermore, based on a leptomycin B- and fluorescence loss in photobleaching-based sensitive assay, we suggest that RSZp22 is a nucleocytoplasmic shuttling protein. Finally, with electron microscopy, we confirmed that RSp31, a plant-specific SR protein, is dynamically distributed in nucleolar cap-like structures upon phosphorylation inhibition. Our findings emphasize the high mobility of Arabidopsis SR splicing factors and provide insights into the dynamic relationships between the different nuclear compartments.     

Plant serine/arginine-rich proteins and their role in pre-mRNA splicing

Pre-messenger RNA (pre-mRNA) splicing, a process by which mature mRNAs are generated by excision of introns and ligation of exons, is an important step in the regulation of gene expression in all eukaryotes. Selection of alternative splice sites in a pre-mRNA generates multiple mRNAs from a single gene that encode structurally and functionally distinct proteins. Alternative splicing of pre-mRNAs contributes greatly to the proteomic complexity of plants and animals and increases the coding potential of a genome. However, the mechanisms that regulate constitutive and alternative splicing of pre-mRNA are not understood in plants. A serine/arginine-rich (SR) family of proteins is implicated in constitutive and alternative splicing of pre-mRNAs. Here I review recent progress in elucidating the roles of serine/arginine-rich proteins in pre-mRNA splicing.     

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.     

Identification and characterization of three members of the human SR family of pre-mRNA splicing factors.

SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C-terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta-thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.     

A single ancient origin for prototypical serine/arginine-rich splicing factors

Eukaryotic precursor mRNA splicing is a process involving a very complex RNA-protein edifice. Serine/arginine-rich (SR) proteins play essential roles in precursor mRNA constitutive and alternative splicing and have been suggested to be crucial in plant-specific forms of developmental regulation and environmental adaptation. Despite their functional importance, little is known about their origin and evolutionary history. SR splicing factors have a modular organization featuring at least one RNA recognition motif (RRM) domain and a carboxyl-terminal region enriched in serine/arginine dipeptides. To investigate the evolution of SR proteins, we infer phylogenies for more than 12,000 RRM domains representing more than 200 broadly sampled organisms. Our analyses reveal that the RRM domain is not restricted to eukaryotes and that all prototypical SR proteins share a single ancient origin, including the plant-specific SR45 protein. Based on these findings, we propose a scenario for their diversification into four natural families, each corresponding to a main SR architecture, and a dozen subfamilies, of which we profile both sequence conservation and composition. Finally, using operational criteria for computational discovery and classification, we catalog SR proteins in 20 model organisms, with a focus on green algae and land plants. Altogether, our study confirms the homogeneity and antiquity of SR splicing factors while establishing robust phylogenetic relationships between animal and plant proteins, which should enable functional analyses of lesser characterized SR family members, especially in green plants.     

A novel SR-related protein is required for the second step of Pre-mRNA splicing

The SR family proteins and SR-related polypeptides are important regulators of pre-mRNA splicing. A novel SR-related protein of an apparent molecular mass of 53 kDa was isolated in a gene trap screen that identifies proteins which localize to the nuclear speckles. This novel protein possesses an arginine- and serine-rich domain and was termed SRrp53 (for SR-related protein of 53 kDa). In support for a role of this novel RS-containing protein in pre-mRNA splicing, we identified the mouse ortholog of the Saccharomyces cerevisiae U1 snRNP-specific protein Luc7p and the U2AF65-related factor HCC1 as interacting proteins. In addition, SRrp53 is able to interact with some members of the SR family of proteins and with U2AF35 in a yeast two-hybrid system and in cell extracts. We show that in HeLa nuclear extracts immunodepleted of SRrp53, the second step of pre-mRNA splicing is blocked, and recombinant SRrp53 is able to restore splicing activity. SRrp53 also regulates alternative splicing in a concentration-dependent manner. Taken together, these results suggest that SRrp53 is a novel SR-related protein that has a role both in constitutive and in alternative splicing.     

The relative strengths of SR protein-mediated associations of alternative and constitutive exons can influence alternative splicing.

We have characterized the functional role of SR protein-mediated exon/exon associations in the alternative splicing of exon 5 of chicken cardiac troponin T (cTnT). We have previously shown that SR proteins can promote the association of the alternative exon 5 with the flanking constitutive exon 6 of this pre-mRNA. In this study, we have shown that when exons 2, 3, and 4 of the cTnT pre-mRNA are spliced together, the composite exon 2/3/4 contains an additional SR protein binding site. Furthermore, we have found that SR proteins can also promote interactions between the pairs of exons 2/3/4-5 and 2/3/4-6. We then asked whether the SR protein binding sites in these exons play a role in cTnT alternative splicing in vivo. We found that the SR protein binding sites in exons 2/3/4 and 6 promote exon 5 skipping, and it has previously been shown that the SR protein binding site in exon 5 promotes exon 5 inclusion. Consistent with these results, we find that the SR protein-mediated association of exon 2/3/4 with 6 is preferred over associations involving exon 5, in that exons 2/3/4 and 6 are more efficient than exon 5 in competing an SR protein-mediated exon/exon association. We suggest that the relative strengths of SR protein-mediated associations of alternative and constitutive exons play a role in determining alternative splicing patterns.     

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.     

Substrate specificities of SR proteins in constitutive splicing are determined by their RNA recognition motifs and composite pre-mRNA exonic elements

We report striking differences in the substrate specificities of two human SR proteins, SF2/ASF and SC35, in constitutive splicing. beta-Globin pre-mRNA (exons 1 and 2) is spliced indiscriminately with either SR protein. Human immunodeficiency virus tat pre-mRNA (exons 2 and 3) and immunoglobulin mu-chain (IgM) pre-mRNA (exons C3 and C4) are preferentially spliced with SF2/ASF and SC35, respectively. Using in vitro splicing with mutated or chimeric derivatives of the tat and IgM pre-mRNAs, we defined specific combinations of segments in the downstream exons, which mediate either positive or negative effects to confer SR protein specificity. A series of recombinant chimeric proteins consisting of domains of SF2/ASF and SC35 in various combinations was used to localize trans-acting domains responsible for substrate specificity. The RS domains of SF2/ASF and SC35 can be exchanged without effect on substrate specificity. The RNA recognition motifs (RRMs) of SF2/ASF are active only in the context of a two-RRM structure, and RRM2 has a dominant role in substrate specificity. In contrast, the single RRM of SC35 can function alone, but its substrate specificity can be influenced by the presence of an additional RRM. The RRMs behave as modules that, when present in different combinations, can have positive, neutral, or negative effects on splicing, depending upon the specific substrate. We conclude that SR protein-specific recognition of specific positive and negative pre-mRNA exonic elements via one or more RRMs is a crucial determinant of the substrate specificity of SR proteins in constitutive splicing.     

DX16 is a novel SR protein phosphorylated by DOA

The serine-arginine-rich (SR) proteins belong to a conserved splicing factor family that not only is essential for constitutive pre-mRNA splicing, but also plays important roles in regulation of alternative splicing. Dx16 is a member of SR protein family in Drosophila. In order to get more insight of dx16 function, we identified the proteins interacting with DX16 through yeast two-hybrid and GST-pull down assays. DX16 interacts with the U1 snRNP subunit CG7564, the SR protein RBP1 and the SR protein kinase DOA. The first and second serine-and arginine-rich regions of DOA are required for the interaction between DOA and DX16. DX16 could be phosphorylated by DOA in vitro and DX16 is highly phosphorylated in vivo. Immunofluorescence microscopy results reveal that doa and dx16 are both highly expressed in embryonic central nervous system. These results suggest that DX16 could be a novel SR protein phosphorylated by DOA and it may participate in the formation of splicing complex through its interactions with other splicing related proteins.     

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.     

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.     

Distinct factor requirements for exonic splicing enhancer function and binding of U2AF to the polypyrimidine tract

Exonic splicing enhancer (ESE) sequences are important for the recognition of adjacent splice sites in pre-mRNA and for the regulation of splice site selection. It has been proposed that ESEs function by associating with one or more serine/arginine-repeat (SR) proteins which stabilize the binding of the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor (U2AF) to the polypyrimidine tract upstream of the 3' splice site. We have tested this model by analyzing the composition of splicing complexes assembled on an ESE-dependent pre-mRNA derived from the doublesex gene of Drosophila. Several SR proteins and hTra2beta, a human homolog of the Drosophila alternative splicing regulator Transformer-2, associate with this pre-mRNA in the presence, but not in the absence, of a purine-rich ESE. By contrast, the 65-kDa subunit of U2AF (U2AF-65 kDa) bound equally to the pre-mRNA in the presence and absence of the ESE. Time course experiments revealed differences in the levels and kinetics of association of individual SR proteins with the ESE-containing pre-mRNA, whereas U2AF-65 kDa bound prior to most SR proteins and hTra2beta and its level of binding did not change significantly during the course of the splicing reaction. Binding of U2AF-65 kDa to the ESE-dependent pre-mRNA was, however, dependent on U1 snRNP. The results indicate that an ESE promotes spliceosome formation through interactions that are distinct from those required for the binding of U2AF-65 kDa to the polypyrimidine tract.     

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.     

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。