Most plant primary transcripts undergo alternative splicing (AS), and its impact on protein diversity is a subject of intensive investigation. Several studies have uncovered various mechanisms of how particular protein splice isoforms operate. However, the common principles behind the AS effects on protein function in plants have rarely been surveyed. Here, on the selected examples, we highlight diverse tissue expression patterns, subcellular localization, enzymatic activities, abilities to bind other molecules and other relevant features. We describe how the protein isoforms mutually interact to underline their intriguing roles in altering the functionality of protein complexes. Moreover, we also discuss the known cases when these interactions have been placed inside the autoregulatory loops. This review is particularly intended for plant cell and developmental biologists who would like to gain inspiration on how the splice variants encoded by their genes of interest may coordinately work.

Infertility in cattle is a major concern of farmers worldwide and despite the enormous improvements in assisted reproduction technologies, the success rates of pregnancies are still low. Embryonic loss is considered one of the main factors of infertility in cattle. As such, the identification of genetic markers for embryo quality and development can help elucidate the molecular mechanisms involved in the formation of embryos with the highest developmental potential. In a previous study, using next-generation RNA sequencing, we identified novel transcripts and alternatively spliced genes that were associated with embryo quality. The objectives of this study were to characterize these transcripts and validate their expression in new biological replications of embryos using quantitative real-time PCR. Two types of embryos differing in morphological and developmental statuses (blastocysts and degenerate embryos) were produced using in vitro fertilization. Quantitative expression of eight novel transcripts revealed a range of 2.5- to 90-fold difference in expression between degenerate embryos and blastocysts. Some of these novel transcripts showed sequence similarity to human and cattle genes known to affect differentiation, growth and development. In addition, expression analysis of alternative splicing isoforms of five genes (MYL6, NOP10, RNF187, RPS24 and RPS28) revealed significant differential expression of these isoforms in the different embryo types. Thus, results of this study suggest that novel transcripts and alternatively spliced genes, found to be differentially expressed between blastocysts and degenerate embryos, can be used as markers for blastocyst formation and development.

Alternative splicing is an important cellular mechanism that increases the diversity of gene products. The study of alternatively spliced genes reported so far in plants is far less documented than that in mammals, but considerable results have been reported, showing the role of these genes in regulating mechanisms, influencing factors, and specificities and function of alternative splicing in plants. This review summarizes briefly the major progress made on alternative splicing in plants.

Stage-specific alternative splicing of the heat-shock transcription factor of Schistosoma mansoni (SmHSF) generates isoforms with structural diversity that may modulate the activity of SmHSF at different life-stages, and thus may regulate the expression of different genes at different developmental stages. RT-PCR, cloning and DNA-sequence analyses showed stage-specific alternative splicing inside the DNA-binding domain (DBD) involving introns I1 and I2, and beyond the DBD involving introns I4a and I7. Retention of introns I2 and I4a would inactivate SmHSF since they contain termination codons. Retention of intron I1 would add 11 amino acids inside the DBD and may change the DNA-binding specificity of SmHSF; intron I7 would add 13 amino acids to the effector region of HSF. Retention of introns was more pronounced in cercariae (larval stage living in water) than in adult worms (parasitic form in mammals). The isoforms were expressed in bacteria, but functional evaluation was not feasible, because only the isoform lacking introns was soluble while isoforms with introns were insoluble. However, stage-specific alternative splicing that changed HSF function in vivo was evidenced in intact cercariae. The cercarial SmHSF mRNA was enriched with introns I2 and I4a that contain termination codons. Therefore, translation of the SmHSF mRNA was impaired, and the SmHSF protein was undetectable. Consequently, the HSP70 gene could not be transcribed, and the HSP70 mRNA was missing. Alternative splicing was observed for short DNA segments (33–45 bp) bound by splice signals, located in the coding region. These are not bona fida exons since they are not flanked by introns. Yet, they are not regular introns since they are often found in mature mRNA. Alternative splicing of these DNA segments caused structural diversity that could modulate the function of the gene product.

High-affinity binding sites for the hnRNP A1 protein stimulate the use of a distal 5′ splice site in mammalian pre-mRNAs. Notably, strong A1-mediated shifts in splice site selection are not accompanied by equivalent changes in the assembly of U1 snRNP-containing complexes on competing 5′ splice sites. To explain the above results, we have proposed that an interaction between hnRNP A1 molecules bound to high-affinity sites loops out the internal 5′ splice site. Here, we present additional evidence in support of the looping out model. First, replacing A1 binding sites with sequences that can generate a loop through RNA duplex formation activates distal 5′ splice site usage in an equivalent manner. Second, increasing the distance between the internal 5′ splice site and flanking A1 binding sites does not compromise activation of the distal 5′ splice site. Similar results were obtained with pre-mRNAs carrying inverted repeats. Using a pre-mRNA containing only one 5′ splice site, we show that splicing is repressed when flanked by two high-affinity A1 binding sites or by inverted repeats, and that inactivation of the internal 5′ splice site is sufficient to elicit a strong increase in the use of the distal donor site. Our results are consistent with the view that the binding of A1 to high-affinity sites promotes loop formation, an event that would repress the internal 5′ splice site and lead to distal 5′ splice site activation.

The goal of functional genomics is to determine the function of each protein encoded by an organism. Typically, this is done by inactivating individual genes and, subsequently, analyzing the phenotype of the modified organisms. In higher eukaryotes, where a tremendous amount of alternative splicing occurs, such approaches are not feasible because they have the potential to simultaneously affect multiple proteins that could have quite distinct and important functions. Thus, it is necessary to develop techniques that inactivate only a subset of proteins synthesized from genes encoding alternatively spliced mRNAs. Here we demonstrate that RNA interference (RNAi) can be used to selectively degrade specific alternatively spliced mRNA isoforms in cultured Drosophila cells. This is achieved by treating the cells with double-stranded RNA corresponding to an alternatively spliced exon. This technique may prove to be a powerful tool to assess the function of proteins synthesized from alternatively spliced mRNAs. In addition, these results have implications regarding the mechanism of RNAi in Drosophila.

Alternative RNA splicing generates extensive proteomic diversity in the nervous system, yet few neural-specific RNA binding proteins have been implicated in splicing control. Here we show that the biochemical properties and spatial expression of mouse neuroblastoma apoptosis-related RNA-binding protein (NAPOR; also called NAPOR-1) are consistent with its roles in the regulation of the exon 5 and exon 21 splicing events of the N-methyl-D-aspartate (NMDA) receptor R1 transcript. NAPOR, which is closely related to CUG binding protein 2 (CUG-BP2), promotes exon 21 and represses exon 5 splicing in functional coexpression assays. These NMDA mRNA isoforms are distributed, in vivo, in a region-specific manner in rat brain, such that high levels of exon 21 selection and exon 5 skipping coincide with high NAPOR mRNA expression in the forebrain. Within the forebrain, this spatial correspondence is most striking in the visual cortex. In contrast, low NAPOR expression coincides with the reciprocal pattern of alternative splicing in the hindbrain. Complementary experiments demonstrate a tissue-specific distribution of NAPOR, CUG-BP, and other highly related proteins within the nervous system as assayed by probing forebrain and hindbrain nuclear extracts with monoclonal antibody, mAb 3B1. Thus, NAPOR may be one of a group of closely related proteins involved in splicing regulation within the brain. An intronic RNA element responsible for the silencing of exon 21 splicing is identified by mutational analysis and shown to bind directly to recombinant NAPOR protein, suggesting a model in which exon 21 selection is positively regulated by an antirepression mechanism of action.

SRrp86 is an 86-kDa member of the SR protein superfamily that is unique in that it can alter splice site selection by regulating the activity of other SR proteins. To study the function of SRrp86, inducible cell lines were created in which the concentration of SRrp86 could be varied and its effects on alternative splicing determined. Here, we show that SRrp86 can activate SRp20 and repress SC35 in a dose-dependent manner both in vitro and in vivo. These effects are apparently mediated through direct protein–protein interaction, as pull-down assays showed that SRrp86 interacts with both SRp20 and SC35. Consistent with the hypothesis that relatively modest changes in the concentration or activity of one or more splicing factors can combinatorially regulate overall splicing, protein expression patterns of SRrp86, SRp20, and SC35 reveal that each tissue maintains a unique ratio of these factors. Regulation of SR protein activity, coupled with regulated protein expression, suggest that SRrp86 may play a crucial role in determining tissue specific patterns of alternative splicing.

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays.

Polypyrimidine tract binding protein (PTB) is an RNA-binding protein that regulates splicing by repressing specific splicing events. It also has roles in 3′-end processing, internal initiation of translation, and RNA localization. PTB exists in three alternatively spliced isoforms, PTB1, PTB2, and PTB4, which differ by the insertion of 19 or 26 amino acids, respectively, between the second and third RNA recognition motif domains. Here we show that the PTB isoforms have distinct activities upon α-tropomyosin (TM) alternative splicing. PTB1 reduced the repression of TM exon 3 in transfected smooth muscle cells, whereas PTB4 enhanced TM exon 3 skipping in vivo and in vitro. PTB2 had an intermediate effect. The PTB4 > PTB2 > PTB1 repressive hierarchy was observed in all in vivo and in vitro assays with TM, but the isoforms were equally active in inducing skipping of α-actinin exons and showed the opposite hierarchy of activity when tested for activation of IRES-driven translation. These findings establish that the ratio of PTB isoforms could form part of a cellular code that in turn controls the splicing of various other pre-mRNAs.

The mammalian thyroid hormone receptor gene c-erbAα gives rise to two mRNAs that code for distinct isoforms, TRα1 and TRα2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRα1-specific polyadenylation site or TRα2-specific 5′ splice site. A previous investigation of TRα minigene expression defined a critical role for the TRα2 5′ splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEα2, enhance splicing of TRα2 in vitro as well as in vivo. Although SEα2 is located within the intron of TRα2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEα2 functions by binding trans-acting factors in HeLa nuclear extract. Protein–RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEα2. SEα2 also includes an element resembling a 5′ splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5′ splice site sequence have a dramatic effect on splicing and protein binding. Thus SEα2 and its associated factors are required for splicing of TRα2 pre-mRNA.

The alternative splicing of the last intron (intron D) of bovine growth hormone (bGH) pre-mRNA requires a downstream exonic splicing enhancer (FP/ESE). The presence of at least one SR protein has been shown to be essential for FP/ESE function and splicing of intron D in in vitro splicing assays. However, in vitro reconstitution of splicing using individual purified SR proteins may not accurately reflect the true complexity of alternative splicing in an intact nucleus, where multiple SR proteins in varying amounts are likely to be available simultaneously. Here, a panel of recombinant baculovirus-expressed SR proteins was produced and tested for the ability to activate FP/ESE-dependent splicing. Individual recombinant SR proteins differed significantly in their activity in promoting intron D splicing. Among the recombinant SR proteins tested, SRp55 was the most active, SC35 showed very little activity, and ASF/SF2 and 9G8 individually had intermediate activity. At least one SR protein (ASF/SF2) bound to the FP/ESE with characteristics of a cooperative interaction. Most interestingly, low concentrations of ASF/SF2 and 9G8 acted synergistically to activate intron D splicing. This was due in part to synergistic binding to the FP/ESE. Splicing of bGH intron D is inherently complex, and is likely controlled by an interaction of the FP/ESE with several trans-acting protein factors acting both independently and cooperatively. This level of complexity may be required for precise control of alternative splicing by an exon sequence, which simultaneously is constrained to maintain translational integrity of the mature mRNA.

Splicing factor SF1 contributes to the recognition of the 3′ splice site by interacting with U2AF65 and binding to the intron branch site during the formation of the early splicing complex E. These interactions and the essential functional domains of SF1 are highly conserved in Saccharomyces cerevisiae. We have isolated cDNAs encoding SF1 from Drosophila (Dm) and Caenorhabditis (Ce). The encoded proteins share the U2AF65 interaction domain, a hnRNP K homology domain, and one or two zinc knuckles required for RNA binding as well as Pro-rich C-terminal sequences with their yeast and mammalian counterparts. In contrast to SF1 in other species, DmSF1 and CeSF1 are characterized by an N-terminal region enriched in Ser, Arg, Lys, and Asp residues with homology to the RS domains of several splicing proteins. These domains mediate protein–protein or protein–RNA interactions, suggesting an additional role for DmSF1 and CeSF1 in pre-mRNA splicing. Human (Hs), fly, and worm SF1 interact equally well with HsU2AF65 or the Drosophila homolog DmU2AF50. Moreover, DmSF1 lacking its N terminus is functional in prespliceosome formation in a HeLa splicing system, emphasizing the conserved nature of interactions at an early step in spliceosome assembly. The Ce-SF1 gene is located in a polycistronic transcription unit downstream of the genes encoding U2AF35 (uaf-2) and a cyclophilin (cyp-13), implying the coordinate transcriptional regulation of these genes. Injection of double-stranded RNA into C. elegans results in embryonic lethality; thus, the SF1 gene is essential not only in yeast but also in at least one metazoan.

The mouse c-src gene contains a short neuron-specific exon, N1. N1 exon splicing is partly controlled by an intronic splicing enhancer sequence that activates splicing of a heterologous reporter exon in both neural and nonneural cells. Here we attempt to dissect all of the regulatory elements controlling the N1 exon and examine how these multiple elements work in combination. We show that the 3′ splice site sequence upstream of exon N1 represses the activation of splicing by the downstream intronic enhancer. This repression is stronger in nonneural cells and these two regulatory sequences combine to make a reporter exon highly cell-type specific. Substitution of the 3′ splice site of this test exon with sites from other exons indicates that activation by the enhancer is very dependent on the nature of the upstream 3′ splice site. In addition, we identify a previously uncharacterized purine-rich sequence within exon N1 that cooperates with the downstream intronic enhancer to increase exon inclusion. Finally, different regulatory elements were tested in multiple cell lines of both neuronal and nonneuronal origin. The individual splicing regulatory sequences from the src gene vary widely in their activity between different cell lines. These results demonstrate how a simple cassette exon is controlled by a variety of regulatory elements that only in combination will produce the correct tissue specificity of splicing.

The activity of the SR protein family of splicing factors in constitutive or alternative splicing requires direct interactions with the pre-mRNA substrate. Thus it is important to define the high affinity targets of the various SR species and to evaluate their ability to discriminate between defined RNA targets. We have analyzed the binding specificity of the 30-kDa SR protein 9G8, which contains a zinc knuckle in addition to the RNA binding domain (RBD). Using a SELEX approach, we demonstrate that 9G8 selects RNA sequences formed by GAC triplets, whereas a mutated zinc knuckle variant selects different RNA sequences, centered around a (A/U)C(A/U)(A/U)C motif, indicating that the zinc knuckle is involved in the RNA recognition specificity of 9G8. In contrast, SC35 selects sequences composed of pyrimidine or purine-rich motifs. Analyses of RNA–protein interactions with purified recombinant 30-kDa SR proteins or in nuclear extracts, by means of UV crosslinking and immunoprecipitation, demonstrate that 9G8, SC35, and ASF/SF2 recognize their specific RNA targets with high specificity. Interestingly, the RNA sequences selected by the mutated zinc knuckle 9G8 variant are efficiently recognized by SRp20, in agreement with the fact that the RBD of 9G8 and SRp20 are similar. Finally, we demonstrate the ability of 9G8 and of its zinc knuckle variant, or SRp20, to act as efficient splicing transactivators through their specific RNA targets. Our results provide the first evidence for cooperation between an RBD and a zinc knuckle in defining the specificity of an RNA binding domain.

A series of HeLa cell lines which stably express β-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 5′ splice sites and activate a common 3′ cryptic splice site upstream leading to aberrantly spliced β-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 5′ splice sites affected sensitivity of both the 5′ and 3′ splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 5′ splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.

In this study, we demonstrate the ability of the polypyrimidine tract binding protein PTB to function as a coordinator of splicing regulation for a trio of neuron-specific exons that are subject to developmental splicing changes in the rat cerebellum. Three neuron-specific exons that show positive regulation are derived from the GABAA receptor γ2 subunit 24 nucleotide exon, clathrin light chain B exon EN, and N-methyl-D-aspartate receptor NR1 subunit exon 5 pre-mRNAs. The functional activity of splicing repressor signals located in the 3′ splice site regions adjacent to the neural exons is shown using an alternative splicing switch assay, in which these short RNA sequences function in trans to switch splicing to the neural pathway in HeLa splicing reactions. Parallel UV crosslinking/competition assays demonstrate selective binding of PTB in comparison to substantially lower binding at adjacent, nonneural 3′ splice sites. Substantially lower PTB binding and splicing switch activity is also observed for the 3′ splice site of NMDA exon 21, which is subject to negative regulation in cerebellum tissue in the same time frame. In splicing active neural extracts, the balance of control shifts to positive regulation, and this shift correlates with a PTB status that is predominantly the neural form. In this context, the addition of recombinant PTB is sufficient to switch splicing to the nonneural pathway. The neural extracts also reveal specific binding of the CUG triplet repeat binding protein to a subset of regulatory 3′ splice site regions. These interactions may interfere with PTB function or modulate splicing levels in a substrate-specific manner within neural tissue. Together these results strengthen the evidence that PTB is a splicing regulator with multiple targets and demonstrate its ability to discriminate among neural and nonneural substrates. Thus, a variety of mechanisms that counterbalance the splicing repressor function of PTB in neural tissue are capable of mediating developmental splicing control. Altered expression of PTB isoforms during cerebellar development, as documented by Western blot analysis, is proposed to be a contributing mechanism.

We analyzed the roles of the three domains of a Drosophila hnRNP A1 homolog by expression of wild-type and mutant versions of HRB87F/hrp36 in Drosophila melanogaster. HRB87F/hrp36 is one of two Drosophila proteins that is most similar to mammalian A1 hnRNP, and like A1, consists of two copies of the RNA-binding domain (RBD) motif followed by a glycine-rich domain (GRD). The role of the domains in nuclear localization and RNA binding to polytene chromosomal sites was determined. RBD-1 and the GRD were largely responsible for both the cellular location of the protein and for the typical chromosomal distribution pattern of the protein at sites of Pol II transcription. RBD-1 also provided a role in the exon-skipping activity of the protein that was not provided by RBD-2. On the other hand, RBD-2 and the GRD were responsible for the very limited chromosomal distribution pattern seen upon heat shock, when HRB87F/hrp36 is sequestered at heat-shock puff 93D, which encodes a long nucleus-restricted RNA. Thus, these studies indicate that the two RBDs function independently of each other but in concert with the GRD. In addition, the self-association property of the GRD was strikingly evident in these overexpressed proteins.

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.