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.     

A novel protein factor is required for use of distal alternative 5'' splice sites in vitro.

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.     

High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5' splice site selection in support of a common looping out and repression mechanism

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

The cap and the 3' splice site similarly affect polyadenylation efficiency.

The 5' cap of a mammalian pre-mRNA has been shown to interact with splicing components at the adjacent 5' splice site for processing of the first exon and the removal of the first intron (E. Izaurralde, J. Lewis, C. McGuigan, M. Jankowska, E. Darzynkiewicz, and I.W. Mattaj, Cell 78:657-668, 1994). Likewise, it has been shown that processing of the last exon and removal of the last intron involve interaction between splicing components at the 3' splice site and the polyadenylation complex at the polyadenylation signal (M. Niwa, S. D. Rose, and S.M. Berget, Genes Dev. 4:1552-1559, 1990; M. Niwa and S. M. Berget, Genes Dev. 5:2086-2095, 1991). These findings suggest that the cap provides a function in first exon processing which is similar to the function of the 3' splice site at last exon processing. To determine whether caps and 3' splice sites function similarly, we compared the effects of the cap and the 3' splice site on the in vitro utilization of the simian virus 40 late polyadenylation signal. We show that the presence of a m7GpppG cap, but not a cap analog, can positively affect the efficiency of polyadenylation of a polyadenylation-only substrate. Cap analogs do not stimulate polyadenylation because they fail to bind titratable cap-binding factors. The failure of cap analogs to stimulate polyadenylation can be overcome if a 3' splice site is present upstream of the polyadenylation signal. These data indicate that factors interacting with the cap or the 3' splice site function similarly to affect polyadenylation signal, along with m7GpppG cap, is inhibitory to polyadenylation. This finding suggests that the interaction between the cap-binding complexes and splicing components at the 5' splice site may form a complex which is inhibitory to further processing if splicing of an adjacent intron is not achieved.     

A novel mutation in the neurofibromatosis type 1 (NF1) gene promotes skipping of two exons by preventing exon definition

Using a protein truncation assay, we have identified a new mutation in the neurofibromatosis type 1 (NF1) gene that causes a severe defect in NF1 pre-mRNA splicing. The mutation, which consists of a G to A transition at position +1 of the 5' splice site of exon 12a, is associated with the loss of both exons 11 and 12a in the NF1 mRNA. Through the use of in vivo and in vitro splicing assays, we show that the mutation inactivates the 5' splice site of exon 12a, and prevents the definition of exon 12a, a process that is normally required to stimulate the weak 3' splice site of exon 12a. Because the 5' splice site mutation weakens the interaction of splicing factors with the 3' splice site of exon 12a, we propose that exon 11/exon 12a splicing is also compromised, leading to the exclusion of both exons 11 and 12a. Our results provide in vivo support for the importance of the exon definition model during NF1 splicing, and suggest that the NF1 region containing exons 11 and 12a plays an important role in the activity of neurofibromin.     

The U1 snRNP base pairs with the 5' splice site within a penta-snRNP complex

Recognition of the 5' splice site is an important step in mRNA splicing. To examine whether U1 approaches the 5' splice site as a solitary snRNP or as part of a multi-snRNP complex, we used a simplified in vitro system in which a short RNA containing the 5' splice site sequence served as a substrate in a binding reaction. This system allowed us to study the interactions of the snRNPs with the 5' splice site without the effect of other cis-regulatory elements of precursor mRNA. We found that in HeLa cell nuclear extracts, five spliceosomal snRNPs form a complex that specifically binds the 5' splice site through base pairing with the 5' end of U1. This system can accommodate RNA-RNA rearrangements in which U5 replaces U1 binding to the 5' splice site, a process that occurs naturally during the splicing reaction. The complex in which U1 and the 5' splice site are base paired sediments in the 200S fraction of a glycerol gradient together with all five spliceosomal snRNPs. This fraction is functional in mRNA spliceosome assembly when supplemented with soluble nuclear proteins. The results argue that U1 can bind the 5' splice site in a mammalian preassembled penta-snRNP complex.     

Exonic splicing enhancer-dependent selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site can be rescued in a cell lacking splicing factor ASF/SF2 through activation of the phosphatidylinositol 3-kinase/Akt pathway

Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5' splice site alternatively splices to a proximal 3' splice site (at nucleotide 3225) to express L2 or to a distal 3' splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3' splice sites and have been identified as regulating alternative 3' splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3' splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3' splice site while increasing use of the distal 3' splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3' splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3' splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.