Antagonistic effects of the SRp30c protein and cryptic 5' splice sites on the alternative splicing of the apoptotic regulator Bcl-x

Alternative 5' splice site selection allows Bcl-x to produce two isoforms with opposite effects on apoptosis. The pro-apoptotic Bcl-x(S) variant is up-regulated by ceramide and down-regulated by protein kinase C through specific cis-acting exonic elements, one of which is bound by SAP155. Splicing to the Bcl-x(S) 5' splice site is also enforced by heterogeneous nuclear ribonucleoprotein (hnRNP) F/H proteins and by Sam68 in cooperation with hnRNP A1. Here, we have characterized exon elements that influence splicing to the 5' splice site of the anti-apoptotic Bcl-x(L) isoform. Within a 86-nucleotide region (B3) located immediately upstream of the Bcl-x(L) donor site we have identified two elements (ML2 and AM2) that stimulate splicing to the Bcl-x(L) 5' splice site. SRp30c binds to these elements and can shift splicing to the 5' splice site of Bcl-x(L) in an ML2/AM2-dependent manner in vitro and in vivo. The B3 region also contains an element that represses the use of Bcl-x(L). This element is bound by U1 small nuclear ribonucleoprotein and contains two 5' splice sites that can be used when the Bcl-x(L) 5' splice site is mutated or the ML2/AM2 elements are deleted. Conversely, mutating the cryptic 5' splice sites stimulates splicing to the Bcl-x(L) site. Thus, SRp30c stimulates splicing to the downstream 5' splice site of Bcl-x(L), thereby attenuating the repressive effect of upstream U1 snRNP binding sites.     

The Proto-oncogene PKCι regulates the alternative splicing of Bcl-x pre-mRNA

Two splice variants derived from the Bcl-x gene via alternative 5' splice site selection (5'SS) are proapoptotic Bcl-x(s) and antiapoptotic Bcl-x(L). Previously, our laboratory showed that apoptotic signaling pathways regulated the alternative 5'SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive prosurvival signaling pathways that regulate the 5'SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by using a number of small-molecule inhibitors of various mitogenic/survival pathways, we found that only treatment of non-small cell lung cancer (NSCLC) cell lines with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (50 μmol/L) or the pan-protein kinase C (PKC) inhibitor G?6983 (25 μmol/L) decreased the Bcl-x(L)/(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/(s) mRNA ratio. Additional studies showed that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/(s) mRNA ratio. Furthermore, downregulation of PKCι correlated with a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 5'SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss of SAP155 complex formation at ceramide-responsive RNA cis-element 1. Finally, forced expression of Bcl-x(L) "rescued" the loss of cell survival induced by PKCι siRNA. In summary, the PI3K/PKCι regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.     

Exonic splicing enhancers contribute to the use of both 3' and 5' splice site usage of rat beta-tropomyosin pre-mRNA

The rat beta-tropomyosin gene encodes two tissue-specific isoforms that contain the internal, mutually exclusive exons 6 (nonmuscle/smooth muscle) and 7 (skeletal muscle). We previously demonstrated that the 3' splice site of exon 6 can be activated by introducing a 9-nt polyuridine tract at its 3' splice site, or by strengthening the 5' splice site to a U1 consensus binding site, or by joining exon 6 to the downstream common exon 8. Examination of sequences within exons 6 and 8 revealed the presence of two purine-rich motifs in exon 6 and three purine-rich motifs in exon 8 that could potentially represent exonic splicing enhancers (ESEs). In this report we carried out substitution mutagenesis of these elements and show that some of them play a critical role in the splice site usage of exon 6 in vitro and in vivo. Using UV crosslinking, we have identified SF2/ASF as one of the cellular factors that binds to these motifs. Furthermore, we show that substrates that have mutated ESEs are blocked prior to A-complex formation, supporting a role for SF2/ASF binding to the ESEs during the commitment step in splicing. Using pre-mRNA substrates containing exons 5 through 8, we show that the ESEs within exon 6 also play a role in cooperation between the 3' and 5' splice sites flanking this exon. The splicing of exon 6 to 8 (i.e., 5' splice site usage of exon 6) was enhanced with pre-mRNAs containing either the polyuridine tract in the 3' splice site or consensus sequence in the 5' splice site around exon 6. We show that the ESEs in exon 6 are required for this effect. However, the ESEs are not required when both the polyuridine and consensus splice site sequences around exon 6 were present in the same pre-mRNA. These results support and extend the exon-definition hypothesis and demonstrate that sequences at the 3' splice site can facilitate use of a downstream 5' splice site. In addition, the data support the hypothesis that ESEs can compensate for weak splice sites, such as those found in alternatively spliced exons, thereby providing a target for regulation.     

Accurate selection of a 5' splice site requires sequences within fibronectin alternative exon B.

Inclusion of fibronectin alternative exon B in mRNA is developmentally regulated. Here we demonstrate that exon B contains two unique purine-rich sequence tracts, PRE1 and PRE2, that are important for proper 5' splice site selection both in vivo and in vitro. Targeted mutations of both PREs decreased the inclusion of exon B in the mRNA by 50% in vivo. Deletion or mutation of the PREs reduced removal of the downstream intron, but not the upstream intron, and induced the activation of cryptic 5' splice sites in vitro. PRE-mediated 5' splice selection activity appears sensitive to position and sequence context. A well characterized exon sequence enhancer that normally acts on the upstream 3' splice site can partially rescue proper exon B 5' splice site selection. In addition, we found that PRE 5' splice selection activity was preserved when exon B was inserted into a heterologous pre-mRNA substrate. Possible roles of these unique activities in modulating exon B splicing are considered.     

The Drosophila SR protein RBP1 contributes to the regulation of doublesex alternative splicing by recognizing RBP1 RNA target sequences.

The SR proteins represent a family of splicing factors several of which have been implicated in the regulation of sex-specific alternative splicing of doublesex (dsx) pre-mRNA in Drosophila. The dsx gene is involved in Drosophila sex determination. We have identified two RNA target sequence motifs recognized by the SR protein RBP1 from Drosophila using an in vitro selection approach. Several copies of these RBP1 target sequences were found within two regions of the dsx pre-mRNA which are important for the regulation of dsx alternative splicing, the repeat region and the purine-rich polypyrimidine tract of the regulated female-specific 3' splice site. We show that RBP1 target sequences within the dsx repeat region are required for the efficient splicing of dsx pre-mRNA. Moreover, our studies reveal that RBP1 contributes to the activation of female-specific dsx splicing in vivo by recognizing the RBP1 target sequences within the purine-rich polypyrimidine tract of the female-specific 3' splice site.     

Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing

Biochemical components (splicing factors) interact with specific intron regions during pre-mRNA splicing in vitro. The pre-mRNA specifically associates with factors at both the branch point and the 5' splice site and these RNA-factor interactions are maintained in the intron-containing RNA processing products. The first detectable event, the ATP-dependent association of a factor (or factors) with the branch point, is mediated by at least one factor containing an essential nucleic acid component. Mutant RNA substrates that lack either the 5' splice site or the vast majority of exon sequences can still associate with the branch point binding factor(s). However, this branch point-factor interaction does not occur with a mutant RNA substrate that contains the branch point but that lacks the 3' splice site consensus sequence. These results suggest that selection of the 3' splice site accompanied by the association of a factor with the branch point may be the initial step in mammalian pre-mRNA splicing.     

Exon skipping in purine nucleoside phosphorylase mRNA processing leading to severe immunodeficiency.

We report a defect in splicing of precursor messenger RNA (pre-mRNA) resulting from a naturally occurring mutation of the gene encoding purine nucleoside phosphorylase (PNP) in a patient with PNP-deficient severe combined immunodeficiency. This defects results from a G to T transversion at the terminal nucleotide of exon 2 within the 5' splice site of intron 2 and causes skipping of exon 2 during processing of PNP pre-mRNA. Translation of the misspliced mRNA results in a reading frameshift at the exon 1-exon 3 junction. The predicted polypeptide encoded by the aberrant mRNA is severely truncated, terminating at 31 amino acids. Only 4 residues at the NH2 terminus of the polypeptide correspond to PNP amino acids. Otherwise the translation product of the misspliced mRNA differs completely from PNP in amino acid sequence and has no PNP activity. The finding of exon skipping in PNP is the first report of a splicing defect resulting in PNP-deficient severe combined immunodeficiency. Analysis of the genomic context of the G-1 to T mutation of the 5' splice site lends support for the exon definition model of pre-mRNA splicing and contributes to the understanding of splice site selection.     

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.     

A highly stable duplex structure sequesters the 5' splice site region of hnRNP A1 alternative exon 7B.

Exon 7B in the hnRNP A1 pre-mRNA is alternatively spliced to yield A1 and A1(B), two proteins that differ in their ability to modulate 5' splice site selection. Sequencing the murine intron downstream of exon 7B revealed the existence of several regions of similarity to the corresponding human intron. In vitro splicing assays indicate that an 84-nt region (CE6IO) decreases splicing to the proximal 5' splice site in a pre-mRNA carrying the 5' splice sites of exon 7 and 7B. In vivo, the CE6IO element promotes exon 7B skipping in pre-mRNAs expressed from a mini-gene containing the hnRNP A1 alternative splicing unit. Using oligonucleotide-targeted RNase H cleavage assays, we provide support for the existence of highly stable base pairing interactions between CE6IO and the 5' splice site region of exon 7B. Duplex formation occurs in naked pre-mRNA, resists incubation in splicing extracts, and is associated with a reduction in the assembly of U1 snRNP-dependent complexes to the 5' splice site of exon 7B. Our results demonstrate that pre-mRNA secondary structure plays an important role in promoting exon 7B skipping in the A1 pre-mRNA.     

Enhancer elements activate the weak 3' splice site of alpha-tropomyosin exon 2.

We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3' splice site of alpha-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates containing weak 3' splice sites or mutated 5' splice sites. However, they are unique in that they can activate splicing whether they are placed in an upstream or downstream exon, and the two central elements can function regardless of their position relative to one another. The presence of excess RNAs containing these enhancers could effectively inhibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incubation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to rescue splicing. Using gel mobility shift assays, we show that formation of stable enhancer-specific complexes on alpha-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the doublesex exon enhancer elements in Drosophila, our results suggest that assembly of mammalian exon enhancer complexes requires both SR and non-SR proteins to activate selection of weak splice sites.     

De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.

Previous studies have demonstrated that several splice variants are derived from both the caspase 9 and Bcl-x genes in which the Bcl-x splice variant, Bcl-x(L) and the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9. In a recent study, we showed that ceramide induces the dephosphorylation of SR proteins, a family of protein factors that regulate alternative splicing. In this study, the regulation of the alternative processing of pre-mRNA of both caspase 9 and Bcl-x(L) was examined in response to ceramide. Treatment of A549 lung adenocarcinoma cells with cell-permeable ceramide, D-e-C(6) ceramide, down-regulated the levels of Bcl-x(L) and caspase 9b mRNA and immunoreactive protein with a concomitant increase in the mRNA and immunoreactive protein levels of Bcl-x(s) and caspase 9 in a dose- and time-dependent manner. Pretreatment with calyculin A (5 nm), an inhibitor of protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A) blocked ceramide-induced alternative splicing in contrast to okadaic acid (10 nm), a specific inhibitor of PP2A at this concentrations in cells, demonstrating a PP1-mediated mechanism. A role for endogenous ceramide in regulating the alternative splicing of caspase 9 and Bcl-x was demonstrated using the chemotherapeutic agent, gemcitabine. Treatment of A549 cells with gemcitabine (1 microm) increased ceramide levels 3-fold via the de novo sphingolipid pathway as determined by pulse labeling experiments and inhibition studies with myriocin (50 nm), a specific inhibitor of serine palmitoyltransferase (the first step in de novo synthesis of ceramide). Treatment of A549 cells with gemcitabine down-regulated the levels of Bcl-x(L) and caspase 9b mRNA with a concomitant increase in the mRNA levels of Bcl-x(s) and caspase 9. Again, inhibitors of ceramide synthesis blocked this effect. We also demonstrate that the change in the alternative splicing of caspase 9 and Bcl-x occurred prior to apoptosis following treatment with gemcitabine. Furthermore, doses of D-e-C(6) ceramide that induce the alternative splicing of both caspase 9 and Bcl-x-sensitized A549 cells to daunorubicin. These data demonstrate a role for protein phosphatases 1 (PP1) and endogenous ceramide generated via the de novo pathway in regulating this mechanism. This is the first report on the dynamic regulation of RNA splicing of members of the Bcl-2 and caspase families in response to regulators of apoptosis.     

An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1.

The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5' splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5' splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5' splice site. RNase T1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5' splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site. In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5' splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5' splice sites.     

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 novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection.     

Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.     

Antisense-targeted immuno-EM localization of the pre-mRNA path in the spliceosomal C complex

A first step in understanding the architecture of the spliceosome is elucidating the positions of individual spliceosomal components and functional centers. Catalysis of the first step of pre-mRNA splicing leads to the formation of the spliceosomal C complex, which contains the pre-mRNA intermediates--the cleaved 5' exon and the intron-3' exon lariat. To topographically locate the catalytic center of the human C complex, we first determined, by DNA oligonucleotide-directed RNAse H digestions, accessible pre-mRNA regions closest to nucleotides of the cleaved 5' splice site (i.e., the 3' end of exon 1 and the 5' end of the intron) and the intron lariat branch point, which are expected to be at/near the catalytic center in complex C. For electron microscopy (EM) localization studies, C complexes were allowed to form, and biotinylated 2'-OMe RNA oligonucleotides were annealed to these accessible regions. To allow localization by EM of the bound oligonucleotide, first antibiotin antibodies and then protein A-coated colloidal gold were additionally bound. EM analyses allowed us to map the position of exon and intron nucleotides near the cleaved 5' splice site, as well as close to the anchoring site just upstream of the branch adenosine. The identified positions in the C complex EM map give first hints as to the path of the pre-mRNA splicing intermediates in an active spliceosomal C complex and further define a possible location for its catalytic center.