Two exonic elements in the flanking constitutive exons control the alternative splicing of the alpha exon of the ZO-1 pre-mRNA

The 240-bp alpha exon of the tight junction (TJ) protein ZO-1 pre-mRNA is alternatively spliced. Expression of both ZO-1alpha+/ZO-1alpha- isoforms results in hermetic TJs, and these become leaky when ZO-1alpha- expression prevails. The alpha exon inclusion/skipping mechanism was studied by in vivo RT-PCR splicing assays in neural and epithelial cells, utilizing a canine minigene construct containing the alpha exon, and the flanking introns and exons. Inclusion of the alpha exon always occurs in wild-type MDCK cells and it is detectable in transfected HeLa cells. However, the alpha exon is skipped in transfected neural cells. Accordingly, both 5' and 3' splice sites surrounding the alpha exon appear to be suboptimal and no cis-acting splicing control elements were found in this exon. Deletion analysis revealed an 83-bp splicing enhancer in the downstream exon and a 35-bp splicing silencer at the beginning of the upstream exon. In epithelial cells all constructs rendered alpha exon inclusion. We conclude that, in neural cells, skipping of the alpha exon depends on two antagonistic exonic elements located in the flanking constitutive exons.     

An exonic splicing silencer downstream of the 3' splice site A2 is required for efficient human immunodeficiency virus type 1 replication

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic mRNA produces more than 40 unique viral mRNA species, of which more than half remain incompletely spliced within an HIV-1-infected cell. Regulation of splicing at HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation of splicing occurs through binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A2, which produces vpr mRNA and promotes inclusion of HIV-1 exon 3, is repressed by the hnRNP A/B-dependent exonic splicing silencer ESSV. Here we show that ESSV activity downstream of 3'ss A2 is localized to a 16-nucleotide element within HIV-1 exon 3. HIV-1 replication was reduced by 95% when ESSV was inactivated by mutagenesis. Reduced replication was concomitant with increased inclusion of exon 3 within spliced viral mRNA and decreased accumulation of unspliced viral mRNA, resulting in decreased cell-associated p55 Gag. Prolonged culture of ESSV mutant viruses resulted in two independent second-site reversions disrupting the splice sites that define exon 3, 3'ss A2 and 5' splice site D3. Either of these changes restored both HIV-1 replication and regulated viral splicing. Therefore, inhibition of HIV-1 3'ss A2 splicing is necessary for HIV-1 replication.     

Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.     

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.     

An exonic splicing silencer in the testes-specific DNA ligase III beta exon

Alternative pre-mRNA splicing of two terminal exons (α and β) regulates the expression of the human DNA ligase III gene. In most tissues, the α exon is expressed. In testes and during spermatogenesis, the β exon is used instead. The α exon encodes the interaction domain with a scaffold DNA repair protein, XRCC1, while the β exon-encoded C-terminal does not. Sequence elements regulating the alternative splicing pattern were mapped by in vitro splicing assays in HeLa nuclear extracts. Deletion of a region beginning in the β exon and extending into the downstream intron derepressed splicing to the β exon. Two silencing elements were found within this 101 nt region: a 16 nt exonic splicing silencer immediately upstream of the β exon polyadenylation signal and a 45 nt intronic splicing silencer. The exonic splicing silencer inhibited splicing, even when the polyadenylation signal was deleted or replaced by a 5′ splice site. This element also enhanced polyadenylation under conditions unfavourable to splicing. The splicing silencer partially inhibited assembly of spliceosomal complexes and functioned in an adenoviral pre-mRNA context. Silencing of splicing by the element was associated with cross-linking of a 37 kDa protein to the RNA substrate. The element exerts opposite functions in splicing and polyadenylation.     

Promoter architecture modulates CFTR exon 9 skipping

Using hybrid minigene experiments, we have investigated the role of the promoter architecture on the regulation of two alternative spliced exons, cystic fibrosis transmembrane regulator (CFTR) exon 9 and fibronectin extra domain-A (EDB). A specific alternative splicing pattern corresponded to each analyzed promoter. Promoter-dependent sensitivity to cotransfected regulatory splicing factor SF2/ASF was observed only for the CFTR exon 9, whereas that of the EDB was refractory to promoter-mediated regulation. Deletion in the CFTR minigene of the downstream intronic splicing silencer element binding SF2/ASF abolished the specific promoter-mediated response to this splicing factor. A systematic analysis of the regulatory cis-acting elements showed that in the presence of suboptimal splice sites or by deletion of exonic enhancer elements the promoter-dependent sensitivity to splicing factor-mediated inhibition was lost. However, the basal regulatory effect of each promoter was preserved. The complex relationships between the promoter-dependent sensitivity to SF2 modulated by the exon 9 definition suggest a kinetic model of promoter-dependent alternative splicing regulation that possibly involves differential RNA polymerase II elongation.     

Elements of the rat tropoelastin gene associated with alternative splicing

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.     

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.     

SRp55 is a regulator of calcitonin/CGRP alternative RNA splicing

Alternative splicing is an important mechanism for the regulation of gene expression. The mammalian calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively spliced in a tissue-specific manner, leading to the production of calcitonin mRNA containing exons 1-4 in thyroid C cells and CGRP mRNA containing exons 1-3, 5, and 6 in neurons. The calcitonin-specific fourth exon contains an exonic splice enhancer (ESE) that binds SRp55. We define the RNA binding site of SRp55 in the ESE and demonstrate that base changes that decrease the level of SRp55 binding decrease the level of calcitonin splicing in vitro and calcitonin mRNA production in vivo. Base changes that increase the affinity of SRp55 for the ESE increase the level of calcitonin splicing in vitro and calcitonin mRNA levels in 293 cells. We also observe that SRp55 levels in different cell types correlate with the levels of calcitonin mRNA produced in these cells. Finally, we show that increasing the level of cellular expression of SRp55 stimulates calcitonin mRNA production in vivo. These observations suggest that SRp55 binding to a suboptimal RNA binding site in the calcitonin/CGRP pre-mRNA ESE is required for calcitonin mRNA production. Differential amounts of SRp55 present in different cell types would then control calcitonin/CGRP alternative splicing.     

An exonic splicing enhancer offsets the atypical GU-rich 3' splice site of human apolipoprotein A-II exon 3

Human apolipoprotein A-II (apoA-II) intron 2/exon 3 junction shows a peculiar tract of alternating pyrimidines and purines (GU tract) that makes the acceptor site deviate significantly from the consensus. However, apoA-II exon 3 is constitutively included in mRNA. We have studied this unusual exon definition by creating a construct with the genomic fragment encompassing the whole gene from apoA-II and its regulatory regions. Transient transfections in Hep3B cells have shown that deletion or replacement of the GU repeats at the 3' splice site resulted in a decrease of apoA-II exon 3 inclusion, indicating a possible role of the GU tract in splicing. However, a 3' splice site composed of the GU tract in heterologous context, such as the extra domain A of human fibronectin or cystic fibrosis transmembrane conductance regulator exon 9, resulted in total skipping of the exons. Next, we identified the exonic cis-acting elements that may affect the splicing efficiency of apoA-II exon 3 and found that the region spanning from nucleotide 87 to 113 of human apoA-II exon 3 is essential for its inclusion in the mRNA. Overlapping deletions and point mutations (between nucleotides 91 and 102) precisely defined an exonic splicing enhancer (ESEwt). UV cross-linking assays followed by immunoprecipitation with anti-SR protein monoclonal antibodies showed that ESEwt, but not mutated ESE RNA, was able to bind both alternative splicing factor/splicing factor 2 and SC35. Furthermore, overexpression of both splicing factors enhanced exon 3 inclusion. These results show that this protein-ESE interaction is able to promote the incorporation of exon 3 in mRNA and suggest that they can rescue the splicing despite the noncanonical 3' splice site.     

A novel intronic cis element, ISE/ISS-3, regulates rat fibroblast growth factor receptor 2 splicing through activation of an upstream exon and repression of a downstream exon containing a noncanonical branch point sequence

Mutually exclusive splicing of fibroblast growth factor receptor 2 (FGFR2) exons IIIb and IIIc yields two receptor isoforms, FGFR2-IIIb and -IIIc, with distinctly different ligand binding properties. Several RNA cis elements in the intron (intron 8) separating these exons have been described that are required for splicing regulation. Using a heterologous splicing reporter, we have identified a new regulatory element in this intron that confers cell-type-specific inclusion of an unrelated exon that mirrors its ability to promote cell-type-specific inclusion of exon IIIb. This element promoted inclusion of exon IIIb while at the same time silencing exon IIIc inclusion in cells expressing FGFR2-IIIb; hence, we have termed this element ISE/ISS-3 (for "intronic splicing enhancer-intronic splicing silencer 3"). Silencing of exon IIIc splicing by ISE/ISS-3 was shown to require a branch point sequence (BPS) using G as the primary branch nucleotide. Replacing a consensus BPS with A as the primary branch nucleotide resulted in constitutive splicing of exon IIIc. Our results suggest that the branch point sequence constitutes an important component that can contribute to the efficiency of exon definition of alternatively spliced cassette exons. Noncanonical branch points may thus facilitate cell-type-specific silencing of regulated exons by flanking cis elements.     

Depolarization and CaM kinase IV modulate NMDA receptor splicing through two essential RNA elements

Alternative splicing controls the activity of many proteins important for neuronal excitation, but the signal-transduction pathways that affect spliced isoform expression are not well understood. One particularly interesting system of alternative splicing is exon 21 (E21) of the NMDA receptor 1 (NMDAR1 E21), which controls the trafficking of NMDA receptors to the plasma membrane and is repressed by Ca⁺⁺/calmodulin-dependent protein kinase (CaMK) IV signaling. Here, we characterize the splicing of NMDAR1 E21. We find that E21 splicing is reversibly repressed by neuronal depolarization, and we identify two RNA elements within the exon that function together to mediate the inducible repression. One of these exonic elements is similar to an intronic CaMK IV–responsive RNA element (CaRRE) originally identified in the 3′ splice site of the BK channel STREX exon, but not previously observed within an exon. The other element is a new RNA motif. Introduction of either of these two motifs, called CaRRE type 1 and CaRRE type 2, into a heterologous constitutive exon can confer CaMK IV–dependent repression on the new exon. Thus, either exonic CaRRE can be sufficient for CaMK IV–induced repression. Single nucleotide scanning mutagenesis defined consensus sequences for these two CaRRE motifs. A genome-wide motif search and subsequent RT-PCR validation identified a group of depolarization-regulated alternative exons carrying CaRRE consensus sequences. Many of these exons are likely to alter neuronal function. Thus, these two RNA elements define a group of co-regulated splicing events that respond to a common stimulus in neurons to alter their activity.     

Identification of regulatory cis-acting elements for alternative splicing of presenilin 2 exon 5 under hypoxic stress conditions

An alternatively spliced form of the presenilin 2 (PS2) gene lacking exon 5 (PS2V) was found in human brains with sporadic Alzheimer's disease. PS2V was induced by hypoxic stress in human neuroblastoma SK-N-SH cells, indicating that hypoxic stress affects the splicing machineries for PS2 exon 5. Here, we identified the critical cis-acting element (sec 2) on the PS2 pre-mRNA responsible for the aberrant splicing of PS2 exon 5 under hypoxic stress conditions. The element was composed of 23 nucleotides in exon 5 and RNA structural analyses showed a stem-loop structure in this sequence. Treatment with an antisense oligonucleotide directed toward the cis-acting element caused an increase in exon 5 inclusion. These results indicate that the sec 2 identified in this study is a novel regulatory element for exon 5 splicing under stress conditions and that trans-acting factors could specifically bind to the element to skip exon 5 of PS2.     

Specific binding of an exonic splicing enhancer by the pre-mRNA splicing factor SRp55.

Purine-rich exonic splicing enhancers (ESEs) have been identified in many alternatively spliced exons. Alternative splicing of several ESE-containing exons has been shown to depend on subsets of the SR protein family of pre-mRNA splicing factors. In this report, we show that purified SR protein family member SRp55 by itself binds a 30-nt ESE-containing exon, the alternatively spliced exon 5 of avian cardiac troponin T. We show that purified SRp55 binds specifically to this RNA sequence with an apparent Kd of 60 nM as assayed by gel mobility retardation experiments. Mutations in the exon 5 sequence that increase or decrease exon 5 inclusion in vivo and in vitro have correspondingly different affinities for SRp55 in our assays. The exon 5 sequence contains two purine-rich motifs, common to many ESEs, and both are required for SRp55 binding. Hill plot analysis of binding titration reactions indicates that there is a cooperative binding of at least two SRp55 proteins to the exon sequence. Chemical modification interference studies using kethoxal show that SRp55 binding to exon 5 requires the N1 and/or the N2 of almost every G residue in the exon. Dimethylsulfate modification interference studies indicate that none of the N1 positions of A residues in the exon are important for binding. We postulate that SRp55 may recognize both primary sequence and RNA secondary structural elements within pre-mRNA.