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.     

Protein splicing and auto-cleavage of bacterial intein-like domains lacking a C'-flanking nucleophilic residue

Bacterial intein-like (BIL) domains are newly identified homologs of intein protein-splicing domains. The two known types of BIL domains together with inteins and hedgehog (Hog) auto-processing domains form the Hog/intein (HINT) superfamily. BIL domains are distinct from inteins and Hogs in sequence, phylogenetic distribution, and host protein type, but little is known about their biochemical activity. Here we experimentally study the auto-processing activity of four BIL domains. An A-type BIL domain from Clostridium thermocellum showed both protein-splicing and auto-cleavage activities. The splicing is notable, because this domain has a native Ala C'-flanking residue rather than a nucleophilic residue, which is absolutely necessary for intein protein splicing. B-type BIL domains from Rhodobacter sphaeroides and Rhodobacter capsulatus cleaved their N' or C' ends. We propose an alternative protein-splicing mechanism for the A-type BIL domains. After an initial N-S acyl shift, creating a thioester bond at the N' end of the domain, the C' end of the domain is cleaved by Asn cyclization. The resulting amino end of the C'-flank attacks the thioester bond next at the N' end of the domain. This aminolysis step splices the two flanks of the domain. The B-type BIL domain cleavage activity is explained in the context of the canonical intein protein-splicing mechanism. Our results suggest that the different HINT domains have related biochemical activities of proteolytic cleavages, ligation and splicing. Yet the predominant reactions diverged in each HINT type according to their specific biological roles. We suggest that the BIL domain cleavage and splicing reactions are mechanisms for post-translationally generating protein variability, particularly in extracellular bacterial proteins.     

Horizontal gene transfers as metagenomic gene duplications

While it is well accepted that horizontal gene transfer plays an important role in the evolution and the diversification of prokaryotic genomes, many questions remain open regarding its functional mechanisms of action and its interplay with the extant genome. This study addresses the relationship between proteome innovation by horizontal gene transfer and genome content in Proteobacteria. We characterize the transferred genes, focusing on the protein domain compositions and their relationships with the existing protein domain superfamilies in the genome. In agreement with previous observations, we find that the protein domain architectures of horizontally transferred genes are significantly shorter than the genomic average. Furthermore, protein domains that are more common in the total pool of genomes appear to have a proportionally higher chance to be transferred. This suggests that transfer events behave as if they were drawn randomly from a cross-genomic community gene pool, much like gene duplicates are drawn from a genomic gene pool. Finally, horizontally transferred genes carry domains of exogenous families less frequently for larger genomes, although they might do it more than expected by chance.     

The multifunctional peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along with the entire 3'-untranslated region. Analysis of hybrid cell panels indicates that the human PAM gene is situated on the long arm of chromosome 5.     

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.     

Analysis of the functional specificity of RS domains in vivo.

A number of splicing factors contain extensive regions that are rich in arginine and serine (RS domains). These domains are thought to facilitate protein-protein interactions that are critical in the regulation of alternative splicing. Using a domain swap strategy, we have tested the ability of RS domains from several proteins to substitute in vivo for an essential RS domain in the Drosophila splicing regulator TRA-2. By several criteria, RS domains were found to vary significantly in their ability to support the splicing regulation functions of TRA-2. The RS domain of dU2AF50 functioned efficiently, while that of the dSRp55 protein did not. Moreover, we find similar differences in the ability of RS domains to direct fusion proteins to discrete subnuclear sites at which TRA-2 associates with spermatocyte chromosomes. These results indicate that RS domains are not all functionally equivalent in vivo.     

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.     

The splicing regulators Tra and Tra2 are unusually potent activators of pre-mRNA splicing

Sexual differentiation in Drosophila is regulated through alternative splicing of doublesex. Female-specific splicing is activated through the activity of splicing enhancer complexes assembled on multiple repeat elements. Each of these repeats serves as a binding platform for the cooperative assembly of a heterotrimeric complex consisting of the SR proteins Tra, Tra2 and 9G8. Using quantitative kinetic analyses, we demonstrate that each component of the enhancer complex is capable of recruiting the spliceosome. Surprisingly, Tra, Tra2 and 9G8 are much stronger splicing activators than other SR protein family members and their activation potential is significantly higher than expected from their serine/arginine content. 9G8 activates splicing not only through its RS domains but also through its RNA-binding domain. The RS domains of Tra and Tra2 are required but not sufficient for efficient complex assembly. Thus, the regulated assembly of the dsx enhancer complexes leads to the generation of an extended activation domain to guarantee the ‘all or none’ splicing switch that is required during Drosophila sexual differentiation.     

Alternative splicing and genetic diversity: silencers are more frequently modified by SNVs associated with alternative exon/intron borders

With the availability of a large amount of genomic data it is expected that the influence of single nucleotide variations (SNVs) in many biological phenomena will be elucidated. Here, we approached the problem of how SNVs affect alternative splicing. First, we observed that SNVs and exonic splicing regulators (ESRs) independently show a biased distribution in alternative exons. More importantly, SNVs map more frequently in ESRs located in alternative exons than in ESRs located in constitutive exons. By looking at SNVs associated with alternative exon/intron borders (by their common presence in the same cDNA molecule), we observed that a specific type of ESR, the exonic splicing silencers (ESSs), are more frequently modified by SNVs. Our results establish a clear association between genetic diversity and alternative splicing involving ESSs.     

Minimization of a eukaryotic mini-intein

Inteins are internal protein splicing elements that can autocatalytically self-excise from their host protein and ligate the protein flanks (exteins) with a peptide bond. Large inteins comprise independent protein splicing and endonuclease domains whereas mini-inteins lack the central endonuclease domain. To identify mini-intein domains that are essential for protein splicing, deletions were introduced at different sites of the 157-aa PRP8 mini-intein of Penicillium chrysogenum. The removal of eight and six amino acids at two different sites resulted in a functional eukaryotic mini-intein of only 143 aa.     

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.