Combinatorial control of a neuron-specific exon.

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.     

Alternative splicing of U12-dependent introns in vivo responds to purine-rich enhancers.

Alternative splicing increases the coding capacity of genes through the production of multiple protein isoforms by the conditional use of splice sites and exons. Many alternative splice sites are regulated by the presence of purine-rich splicing enhancer elements (ESEs) located in the downstream exon. Although the role of ESEs in alternative splicing of the major class U2-dependent introns is well established, no alternatively spliced minor class U12-dependent introns have so far been described. Although in vitro studies have shown that ESEs can stimulate splicing of individual U12-dependent introns, there is no direct evidence that the U12-dependent splicing system can respond to ESEs in vivo. To investigate the ability of U12-dependent introns to use alternative splice sites and to respond to ESEs in an in vivo context, we have constructed two sets of artificial minigenes with alternative splicing pathways and evaluated the effects of ESEs on their alternative splicing patterns. In minigenes with alternative U12-dependent 3' splice sites, a purine-rich ESE promotes splicing to the immediately upstream 3' splice site. As a control, a mutant ESE has no stimulatory effect. In minigene constructs with two adjacent U12-dependent introns, the predominant in vivo splicing pattern results in the skipping of the internal exon. Insertion of a purine-rich ESE into the internal exon promotes the inclusion of the internal exon. These results show that U12-dependent introns can participate in alternative splicing pathways and that U12-dependent splice sites can respond to enhancer elements in vivo.     

Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a component of an intronic splicing enhancer complex that activates the splicing of the alternative exon 6A from chicken beta-tropomyosin pre-mRNA

Splicing of the chicken beta-tropomyosin exon 6A is stimulated, both in vivo and in vitro, by an intronic pyrimidine-rich element (S4) located 37 nucleotides downstream of exon 6A. Several pyrimidine-rich sequences are able to substitute for the natural S4 enhancer with various stimulatory effects. We show that the different enhancer sequences recruit U1 small nuclear ribonucleoprotein (SnRNP) to the exon 6A 5' splice site, with an efficiency that correlates with the splicing activation. By using RNA affinity and two-dimensional gel electrophoresis, we characterized several proteins that bind to the different enhancer sequences. Heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP I (polypyrimidine track-binding protein, PTB) exhibit a higher level of interaction with the strong enhancer sequences (S4) than with the weakest enhancers. Functional analysis shows that hnRNP K is a component of the enhancer complex that promotes exon 6A splicing through the wild-type S4 sequence. The addition of recombinant hnRNP K to nuclear extracts preincubated with poly(rC) RNA competitor completely restores splicing efficiency to the original level. hnRNP I (PTB) was also found associated with the strong enhancer sequences. Its function in the splicing of exon 6A is discussed.     

Regulated splicing of an alternative exon of beta-tropomyosin pre-mRNAs in myogenic cells depends on the strength of pyrimidine-rich intronic enhancer elements.

Alternative splicing of chicken beta-tropomyosin (beta-TM) pre-mRNAs ensures that in nonmuscle cells, only exon 6A is expressed, whereas in skeletal muscle, exon 6B is utilized preferentially. We have previously shown that efficient splicing of the nonmuscle exon 6A requires two pyrimidine-rich splicing enhancers (S4 and I5Y) that are present in the introns flanking exon 6A. Here, we examined the function of the S4 and I5Y elements by replacing them within beta-TM minigenes by other pyrimidine- and purine-rich sequence elements and analyzing splicing in transfected quail nonmuscle and muscle cells. Several features of these splicing regulatory elements were revealed by this study. First, a wide variety of pyrimidine-rich sequences can replace the intronic S4 splicing enhancer, indicating that pyrimidine composition, rather than sequence specificity, determines activity for this element. Second, one type of purine-rich sequence (GARn), normally found within exons, can also replace the S4 splicing enhancer. Third, the diverse elements tested exhibit differential activation of the splice sites flanking exon 6A and different positional constraints. Fourth, the strength of the S4 splicing enhancer is appropriately set to obtain proper regulation of the transition from exon 6A splicing in myoblasts to exon 6B splicing in myotubes, but this splicing regulatory element is not the target for cell-type-specific splicing factors.     

A Short Sequence within Two Purine-Rich Enhancers Determines 5′ Splice Site Specificity

Purine-rich enhancers are exon sequences that promote inclusion of alternative exons, usually via activation of weak upstream 3′ splice sites. A recently described purine-rich enhancer from the caldesmon gene has an additional activity by which it directs selection of competing 5′ splice sites within an alternative exon. In this study, we have compared the caldesmon enhancer with another purine-rich enhancer from the chicken cardiac troponin T (cTNT) gene for the ability to regulate flanking splice sites. Although similar in sequence and length, the two enhancers demonstrated strikingly different specificities towards 5′ splice site choice when placed between competing 5′ splice sites in an internal exon. The 32-nucleotide caldesmon enhancer caused effective usage of the exon-internal 5′ splice site, whereas the 30-nucleotide cTNT enhancer caused effective usage of the exon-terminal 5′ splice site. Both enhancer-mediated splicing pathways represented modulation of the default pathway in which both 5′ splice sites were utilized. Each enhancer is multipartite, consisting of two purine-rich sequences of a simple (GAR)_n repeat interdigitated with two enhancer-specific sequences. The entire enhancer was necessary for maximal splice site selectivity; however, a 5- to 7-nucleotide region from the 3′ end of each enhancer dictated splice site selectivity. Mutations that interchanged this short region of the two enhancers switched specificity. The portion of the cTNT enhancer determinative for 5′ splice site selectivity was different than that shown to be maximally important for activation of a 3′ splice site, suggesting that enhancer environment can have a major impact on activity. These results are the first indication that individual purine-rich enhancers can differentiate between flanking splice sites. Furthermore, localization of the specificity of splice site choice to a short region within both enhancers indicates that subtle differences in enhancer sequence can have profound effects on the splicing pathway.     

The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element.

We have characterized a novel positive-acting splicing element within the developmentally regulated alternative exon (exon 5) of the cardiac troponin T (cTNT) gene. The exon splicing element (ESE) is internal to the exon portions of the splice sites and is required for splicing to the 3' splice site but not the 5' splice site flanking the exon. Sequence comparisons between cTNT exon 5 and other exons that contain regions required for splicing reveal a common purine-rich motif. Sequence within cTNT exon 5 or a synthetic purine-rich motif facilitates splicing of heterologous alternative and constitutive splice sites in vivo. Interestingly, the ESE is not required for the preferential inclusion of cTNT exon 5 observed in primary skeletal muscle cultures. Our results strongly suggest that the purine-rich ESE serves as a general splicing element that is recognized by the constitutive splicing machinery.     

Determinants of 4-repeat tau expression. Coordination between enhancing and inhibitory splicing sequences for exon 10 inclusion

Mutations in the tau gene are pathogenic causing autosomal dominant frontotemporal dementia with Parkinsonism-chromosome 17 type (FTDP-17). Some mutations in tau exon 10 (E10) and immediately adjacent sequences cause disease by altering E10 splicing. To determine the mechanism of normal E10 splicing regulation and how FTDP-17 mutations alter splicing, mutational analysis of E10 was performed. The results show that E10 contains a complex array of both enhancer and inhibitor cis-acting elements that modulate usage of a weak 5' splice site. The 5' end of E10 contains a previously unrecognized multipartite exon splicing enhancer (ESE) composed of an SC35-like binding sequence, a purine-rich sequence, and an AC-rich element. Downstream of this ESE is a purine-rich exon splicing inhibitor. Intronic sequences immediately downstream of E10 also are inhibitory. The results support an alternative model in which I10 inhibitory sequences appear to function as a linear sequence. The cis-elements described are not redundant, and all appear required for normal E10 splicing. Results with double mutations demonstrate that the ESE and the intronic inhibitory element collaborate to regulate splicing. Thus splicing of tau E10 is regulated by a complex set of cis-acting elements that span nearly the entire exon and also include intronic sequences.     

An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon.

The average length of a vertebrate axon is approximately 130 nt. Decreasing the size of an internal axon to less than 51 nt induces axon skipping, implying a minimal size for exons. A few constitutively included internal exons, however, are extremely small. To investigate if such micro-exons require special mechanisms for their inclusion, we studied the sequences necessary for inclusion of a 6-nt axon from chicken cardiac troponin T (cTNT). In vivo, the cTNT micro-exon was not included in mRNA unless accompanied by a 134-nt sequence located next to the micro-exon in the downstream intron. Increasing the length of the micro-exon alleviated the requirement for the intron element, indicating that the lack of inclusion of the micro-exon in the absence of a facilitating sequence was due to its small size, rather than suboptimal splice sites. The intron element contained six copies of a G-rich 7-nt sequence. Multimers of the repeat supported exon inclusion, indicating that the repeat sequence is an important part of the intron element. The entire intron element activated inclusion of a heterologous 7-nt exon, suggesting that the intron element is a general enhancer for the splicing of micro-exons. In vitro, the intron element and the repeated sequence facilitated splicing of a heterologous exon. Because of the ability of the cTNT intron element to facilitate the splicing of heterologous exons, we have termed the element an intron splicing enhancer (ISE). Interestingly, the ISE demonstrated position independence in that it facilitated inclusion of the heterologous micro-exon when placed either upstream or downstream of the micro-exon. In vitro, the ISE or copies of the ISE G-rich repeat stimulated splicing of an adjacent intron. The ISE thus becomes one of only a few characterized ISEs containing a G-rich repeat and the first to work both upstream and downstream of a target axon.     

A 5' splice site-proximal enhancer binds SF1 and activates exon bridging of a microexon

Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3' and 5' splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5' splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3' splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon.     

Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors.

Human immunodeficiency virus type 1 (HIV-1) pre-mRNA splicing is regulated in order to maintain pools of unspliced and partially spliced viral RNAs as well as the appropriate levels of multiply spliced mRNAs during virus infection. We have previously described an element in tat exon 2 that negatively regulates splicing at the upstream tat 3' splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, Mol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicing enhancer (ESE) elements, we have termed this element an exon splicing silencer (ESS). We show evidence for another negative cis-acting region within tat-rev exon 3 of HIV-1 RNA that has sequence motifs in common with a 20-nt ESS element in tat exon 2. This sequence is juxtaposed to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream tat-rev 3' splice site. Inhibition of the splicing of substrates containing the ESS element in tat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing mediated by the ESS can be specifically abrogated by the addition of competitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and tat-rev exon 3.     

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.     

Strong RNA splicing enhancers identified by a modified method of cycled selection interact with SR protein.

A modified method of cycled selection was used to characterize splicing enhancers for exon inclusion from a pool of beta-globin-based three exon/two intron pre-mRNAs with a variable number of random nucleotides incorporated in the internal exon. The pre-mRNAs generated by this method contained random sequences ranging from 0 to 18 nucleotides in length. This method was used to isolate particular splicing enhancer motifs from a previously enriched pool of extremely diverse enhancers. After four cycles of selection for mRNA containing the internal exon, a distinct enhancer motif (GACGAC...CAGCAG) was highly enriched. This motif served as strong splicing enhancers in a heterogeneous exon. We have shown here that the selected enhancer motif promotes exon inclusion through specific interaction with SRp30. We have also shown that although present in many of our selected splicing enhancers conforming to this motif, a typical purine-rich enhancer sequence is dispensable for either enhancer activity or binding with SRp30.     

CA- and Purine-Rich Elements Form a Novel Bipartite Exon Enhancer Which Governs Inclusion of the Minute Virus of Mice NS2-Specific Exon in Both Singly and Doubly Spliced mRNAs

The alternatively spliced 290-nucleotide NS2-specific exon of the parvovirus minute virus of mice (MVM), which is flanked by a large intron upstream and a small intron downstream, constitutively appears both in the R1 mRNA as part of a large 5′-terminal exon (where it is translated in open reading frame 3 [ORF3]), and in the R2 mRNA as an internal exon (where it is translated in ORF2). We have identified a novel bipartite exon enhancer element, composed of CA-rich and purine-rich elements within the 5′ and 3′ regions of the exon, respectively, that is required to include NS2-specific exon sequences in mature spliced mRNA in vivo. These two compositionally different enhancer elements are somewhat redundant in function: either element alone can at least partially support exon inclusion. They are also interchangeable: either element can function at either position. Either a strong 3′ splice site upstream (i.e., the exon 5′ terminus) or a strong 5′ splice site downstream (i.e., the exon 3′ terminus) is sufficient to prevent skipping of the NS2-specific exon, and a functional upstream 3′ splice site is required for inclusion of the NS2-specific exon as an internal exon into the mature, doubly spliced R2 mRNA. The bipartite enhancer functionally strengthens these termini: the requirement for both the CA-rich and purine-rich elements can be overcome by improvements to the polypyrimidine tract of the upstream intron 3′ splice site, and the purine-rich element also supports exon inclusion mediated through the downstream 5′ splice sites. In summary, a suboptimal large-intron polypyrimidine tract, sequences within the downstream small intron, and a novel bipartite exonic enhancer operate together to yield the balanced levels of R1 and R2 observed in vivo. We suggest that the unusual bipartite exonic enhancer functions to mediate proper levels of inclusion of the NS2-specific exon in both singly spliced R1 and doubly spliced R2.     

Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression

Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two majorcis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 20 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3 splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on thecis-element's identity and changes in cellular splicing factors under physiological or pathological conditions.     

Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.     

An intronic enhancer regulates splicing of the twintron of Drosophila melanogaster prospero pre-mRNA by two different spliceosomes

We have examined the alternative splicing of the Drosophila melanogaster prospero twintron, which contains splice sites for both the U2- and U12-type spliceosome and generates two forms of mRNA, pros-L (U2-type product) and pros-S (U12-type product). We find that twintron splicing is developmentally regulated: pros-L is abundant in early embryogenesis while pros-S displays the opposite pattern. We have established a Kc cell in vitro splicing system that accurately splices a minimal pros substrate containing the twintron and have examined the sequence requirements for pros twintron splicing. Systematic deletion and mutation analysis of intron sequences established that twintron splicing requires a 46-nucleotide purine-rich element located 32 nucleotides downstream of the U2-type 5' splice site. While this element regulates both splicing pathways, its alteration showed the severest effects on the U2-type splicing pathway. Addition of an RNA competitor containing the wild-type purine-rich element to the Kc extract abolished U2-type splicing and slightly repressed U12-type splicing, suggesting that a trans-acting factor(s) binds the enhancer element to stimulate twintron splicing. Thus, we have identified an intron region critical for prospero twintron splicing as a first step towards elucidating the molecular mechanism of splicing regulation involving competition between the two kinds of spliceosomes.     

SR proteins and hnRNP H regulate the splicing of the HIV-1 tev-specific exon 6D

A naturally arising point mutation in the env gene of HIV-1 activates the aberrant inclusion of the cryptic exon 6D into most viral messages, leading to inefficient viral replication. We set out to understand how a single nucleotide substitution could cause such a dramatic change in splicing. We have determined that the exon 6D mutation promotes binding of the SR protein SC35 to the exon. Mutant exon 6D sequences function as a splicing enhancer when inserted into an enhancer-dependent splicing construct. hnRNP H family proteins bind to the enhancer as well; their binding is dependent on the sequence GGGA located just downstream of the point mutation and depletion-- reconstitution studies show that hnRNP H is essential for enhancer activity. A polypurine sequence located further downstream in exon 6D binds SR proteins but acts as an exonic splicing silencer. hnRNP H is required for interaction of U1 snRNP with the enhancer, independent of the point mutation. We propose that SC35 binding to the point mutation region may convert the hnRNP H-U1 snRNP complex into a splicing enhancer.     

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.