An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM

We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations.     

Structural and functional conservation of the Drosophila doublesex splicing enhancer repeat elements.

We have compared the RNA sequences and secondary structures of the Drosophila melanogaster and Drosophila virilis doublesex (dsx) splicing enhancers. The sequences of the two splicing enhancers are highly divergent except for the presence of nearly identical 13-nt repeat elements (six in D. melanogaster and four in D. virilis) and a stretch of nucleotides at the 5' and 3' ends of the enhancers. In vitro RNA structure probing of the two enhancers revealed that the 13-nt repeats are predominantly single-stranded. Thus, both the primary sequences and single-stranded nature of the repeats are conserved between the two species. The significance of the primary sequence conservation was demonstrated by showing that the two enhancers are functionally interchangeable in Tra-/Tra2-dependent in vitro splicing. In addition, inhibition of splicing enhancer activity by antisense oligonucleotides complementary to the repeats demonstrated the importance of the conserved single-stranded structure of the repeats. In vitro binding studies revealed that Tra2 interacts with each of the D. melanogaster repeat elements, except for repeat 2, with affinities that are indistinguishable, whereas Tra binds nonspecifically to the enhancer. Taken together, these observations indicate that the organization of sequences within the dsx splicing enhancers of D. melanogaster and D. virilis results in a structure in which each of the repeat elements is single-stranded and therefore accessible for specific recognition by the RNA-binding domain of Tra2.     

A complex intronic splicing enhancer from the c-src pre-mRNA activates inclusion of a heterologous exon.

The mouse c-src gene contains a short neuron-specific exon, N1. To characterize the sequences that regulate N1 splicing, we used a heterologous gene, derived from the human beta-globin gene, containing a short internal exon that is usually skipped by the splicing machinery. Various fragments from the src gene were inserted into the globin substrate to measure their effects on the splicing of the test exon. These clones were transiently expressed in neuronal and nonneuronal cell lines, and the level of exon inclusion was measured by primer extension. Several sequences from the N1 exon region induced the splicing of the heterologous exon. The most powerful effect was seen with a sequence from the intron downstream of the N1 exon. This sequence acted as a strong splicing enhancer, activating splicing of the test exon when placed in the intron downstream. The enhancer was strongest in neuronal LA-N-5 cells but also activated splicing in nonneuronal HEK293 cells. Deletion and linker scanning mutagenesis indicate that the enhancer is made up of multiple smaller elements that must act in combination. One of these elements was identified as the sequence UGCAUG. Three copies of this element can strongly activate splicing of the test exon in LA-N-5 neuroblastoma cells. These component elements of the src splicing enhancer are also apparently involved in the splicing of other short cassette exons.     

An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene.

Regulation of calcitonin (CT)/calcitonin gene-related peptide (CGRP) RNA processing involves the use of alternative 3' terminal exons. In most tissues and cell lines, the CT terminal exon is recognized. In an attempt to define regulatory sequences involved in the utilization of the CT-specific terminal exon, we performed deletion and mutation analyses of a mini-gene construct that contains the CT terminal exon and mimics the CT processing choice in vivo. These studies identified a 127-nucleotide intron enhancer located approximately 150 nucleotides downstream of the CT exon poly(A) cleavage site that is required for recognition of the exon. The enhancer contains an essential and conserved 5' splice site sequence. Mutation of the splice site resulted in diminished utilization of the CT-specific terminal exon and increased skipping of the CT exon in both the mini-gene and in the natural CT/CGRP gene. Other components of the intron enhancer modified utilization of the CT-specific terminal exon and were necessary to prevent utilization of the 5' splice site within the intron enhancer as an actual splice site directing cryptic splicing. Conservation of the intron enhancer in three mammalian species suggests an important role for this intron element in the regulation of CT/CGRP processing and an expanded role for intronic 5' splice site sequences in the regulation of RNA processing.     

A unique intronic splicing enhancer controls the inclusion of the agrin Y exon.

Alternative splicing of the agrin mRNA controls the ability of agrin protein to induce the clustering of acetylcholine receptors at the neuromuscular junction. Using a transfectable reporter gene, we show that one agrin alternative exon, the Y exon, is controlled by a regulatory sequence in the downstream intron. Portions of this intronic sequence have the properties of a splicing enhancer that can activate splicing of a heterologous exon when placed in the intron downstream. The regulatory region is complex in structure, containing several different elements capable of activating splicing. Individual enhancing elements differ in their cell-type specificity, and are not apparently synergistic, as two elements together induce lower splicing than either does separately. Essential nucleotides within these regulatory elements were identified by scanning mutagenesis across the active region. Interestingly, the elements do not appear similar to known intronic splicing enhancer elements. This Y exon enhancer and its components take part in an apparent combinatorial system of control where multiple regulatory elements of varying activity combine to produce a precisely cell-specific exon inclusion. As a major contributor to the regulation of the Y exon, the enhancer ultimately controls the properties of the agrin protein.     

Binding of PurH to a muscle-specific splicing enhancer functionally correlates with exon inclusion in vivo

Regulated alternative splicing of avian cardiac troponin T (cTNT) pre-mRNA requires multiple intronic elements called muscle-specific splicing enhancers (MSEs) that flank the alternative exon 5 and promote muscle-specific exon inclusion. To understand the function of the MSEs in muscle-specific splicing, we sought to identify trans-acting factors that bind to these elements. MSE3, which is located 66-81 nucleotides downstream of exon 5, assembles a complex that is both sequence- and muscle-specific. Purification and characterization of the MSE3 complex identified one component as 5-aminoimidazole-4-carboxamide ribonucleotideformyltransferase/IMP cyclohydrolase (PurH), an enzyme involved in de novo purine synthesis. Recombinant human PurH protein directly binds MSE3 RNA and PurH is the primary determinant of sequence-specific binding in the native complex. Furthermore, we show a direct correlation between the in vitro binding affinity of both the MSE3 complex and recombinant PurH with functional activation of exon inclusion in vivo. Together, these results strongly suggest that PurH performs a second function as a component of a complex that regulates MSE3-dependent exon inclusion.     

An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken beta-tropomyosin pre-mRNA.

Computer analysis of human intron sequences have revealed a 50 nucleotide (nt) GC-rich region downstream of the 5' splice site; the trinucleotide GGG occurs almost four times as frequently as it would in a random sequence. The 5' part of a beta-tropomyosin intron exhibits six repetitions of the motif (A/U)GGG. In order to test whether these motifs play a role in the splicing process we have mutated some or all of them. Mutated RNAs show a lower in vitro splicing efficiency when compared with the wild-type, especially when all six motifs are mutated (> 70% inhibition). Assembly of the spliceosome complex B and, to a lesser extent, of the pre-spliceosome complex A also appears to be strongly affected by this mutation. A 55 kDa protein within HeLa cell nuclear extract is efficiently cross-linked to the G-rich region. This protein is present in the splicing complexes and its cross-linking to the pre-mRNA requires the presence of one or several snRNP. Altogether our results suggest that the G-rich sequences present in the 5' part of introns may act as an enhancer of the splicing reaction at the level of spliceosome assembly.     

Group II intron domain 5 facilitates a trans-splicing reaction.

A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.     

An allosteric-feedback mechanism for protein-assisted group I intron splicing

The I-AniI maturase facilitates self-splicing of a mitochondrial group I intron in Aspergillus nidulans. Binding occurs in at least two steps: first, a specific but labile encounter complex rapidly forms and then this intermediate is slowly resolved into a native, catalytically active RNA/protein complex. Here we probe the structure of the RNA throughout the assembly pathway. Although inherently unstable, the intron core, when bound by I-AniI, undergoes rapid folding to a near-native state in the encounter complex. The next transition includes the slow destabilization and docking into the core of the peripheral stacked helix that contains the 5' splice site. Mutational analyses confirm that both transitions are important for native complex formation. We propose that protein-driven destabilization and docking of the peripheral stacked helix lead to subtle changes in the I-AniI binding site that facilitate native complex formation. These results support an allosteric-feedback mechanism of RNA-protein recognition in which proteins engaged in an intermediate complex can influence RNA structure far from their binding sites. The linkage of these changes to stable binding ensures that the protein and RNA do not get sequestered in nonfunctional complexes.     

In vivo characterization of a vertebrate ultraconserved enhancer

Genomic sequence comparisons among human, mouse, and pufferfish (Takifugu rubripes (Fugu)) have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2, located near the dachshund gene, which displays a human-Fugu identity of 84% over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence of a large block of sequence (144 bp) that is completely identical among human, mouse, chicken, zebrafish, and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424-bp Dc2-conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144-bp 100% conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16-bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144-bp 100% conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on the Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence.     

Identification of an intronic splicing enhancer essential for the inclusion of FGFR2 exon IIIc

The ligand specificity of fibroblast growth factor receptor 2 (FGFR2) is determined by the alternative splicing of exons 8 (IIIb) or 9 (IIIc). Exon IIIb is included in epithelial cells, whereas exon IIIc is included in mesenchymal cells. Although a number of cis elements and trans factors have been identified that play a role in exon IIIb inclusion in epithelium, little is known about the activation of exon IIIc in mesenchyme. We report here the identification of a splicing enhancer required for IIIc inclusion. This 24-nucleotide (nt) downstream intronic splicing enhancer (DISE) is located within intron 9 immediately downstream of exon IIIc. DISE was able to activate the inclusion of heterologous exons rat FGFR2 IIIb and human beta-globin exon 2 in cell lines from different tissues and species and also in HeLa cell nuclear extracts in vitro. DISE was capable of replacing the intronic activator sequence 1 (IAS1), a known IIIb splicing enhancer and vice versa. This fact, together with the requirement for DISE to be close to the 5'-splice site and the ability of DISE to promote binding of U1 snRNP, suggested that IAS1 and DISE belong to the same class of cis-acting elements.     

A novel bipartite intronic splicing enhancer promotes the inclusion of a mini-exon in the AMP deaminase 1 gene

Alternative splicing of the 12-base exon 2 of the adenosine monophosphate deaminase (AMPD) gene is subject to regulation by both cis- and trans-regulatory signals. The extent of exon 2 inclusion is stage- and cell type-specific and is subject to the physiological state of the cell. In adult skeletal muscle, a cell type that regulates the activity of this allosteric enzyme at several levels, the exon 2-plus form of AMPD, predominates. We have performed a systematic analysis of the cis-acting regulatory sequences that reside in the intron immediately downstream of this mini-exon. A complex element comprising sequences that enhance exon 2 inclusion and sequences that counteract this effect resides in the middle of this intron. We demonstrate that the enhancing component is bipartite, with more than a kilobase of sequence separating the two functional sites. The presence of even minimal levels the mini-exon in the fully processed AMPD mRNA requires both of these sites, neither of which appears in any other published splicing enhancer. An RNA binding activity derived from a muscle cell line requires both of the enhancing sites. Mutations in either of the sites that eliminate exon 2 inclusion abrogate this binding activity.     

Getting to the heart of a splicing enhancer

Dinucleotide CA repeats of variable length in an intron of the human endothelial nitric oxide synthase gene act as regulatory signals for splicing of the gene's pre-mRNA. Repeat length-dependent binding of hnRNP L to the repeats provides an intriguing link between splicing efficiency, nitric oxide synthesis and cardiovascular disease.     

Polypurine sequences within a downstream exon function as a splicing enhancer.

We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin mu gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin mu gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection.     

An oligodendrocyte enhancer in a phylogenetically conserved intron region of the mammalian myelin gene Opalin

Opalin is a transmembrane protein detected specifically in mammalian oligodendrocytes. Opalin homologs are found only in mammals and not in the genome sequences of other animal classes. We first determined the nucleotide sequences of Opalin orthologs and their flanking regions derived from four prosimians, a group of primitive primates. A global comparison revealed that an evolutionarily conserved region exists in the first intron of Opalin. When the conserved domain was assayed for its enhancer activity in transgenic mice, oligodendrocyte-directed expression was observed. In an oligodendroglial cell line, Oli-neu, the conserved domain showed oligodendrocyte-directed expression. The conserved domain is composed of eight subdomains, some of which contain binding sites for Myt1 and cAMP-response element binding protein (CREB). Deletion analysis and cotransfection experiments revealed that the subdomains have critical roles in Opalin gene expression. Over-expression of Myt1, treatment of the cell with leukemia inhibitory factor (LIF), and cAMP analog (CREB activator) enhanced the expression of endogenous Opalin in Oli-neu cells and activated the oligodendrocyte enhancer. These results suggest that LIF, cAMP signaling cascades and Myt1 play significant roles in the differentiation of oligodendrocytes through their action on the Opalin oligodendrocyte enhancer.