Nucleotide substitutions within the cardiac troponin T alternative exon disrupt pre-mRNA alternative splicing.

The cardiac troponin T (cTNT) pre-mRNA contains a single alternative exon (exon 5) which is either included or excluded from the processed mRNA. Using transient transfection of cTNT minigenes, we have previously localized pre-mRNA cis elements required for exon 5 alternative splicing to three small regions of the pre-mRNA which include exons 4, 5, and 6. In the present study, nucleotide substitutions were introduced into the region containing exon 5 to begin to define specific nucleotides required for exon 5 alternative splicing. A mutation within the 5' splice site flanking the cTNT alternative exon that increases its homology to the consensus sequence improves splicing efficiency and leads to increased levels of mRNAs that include the alternative exon. Surprisingly, substitution of as few as four nucleotides within the alternative exon disrupts cTNT pre-mRNA alternative splicing and prevents recognition of exon 5 as a bona fide exon. These results establish that the cTNT alternative exon contains information in cis that is required for its recognition by the splicing machinery.     

Functional analysis of the polypyrimidine tract in pre-mRNA splicing.

The polypyrimidine tract is one of the important cis-acting sequence elements directing intron removal in pre-mRNA splicing. Progressive deletions of the polypyrimidine tract have been found to abolish correct lariat formation, spliceosome assembly and splicing. In addition, the polypyrimidine tract can alter 3'-splice site selection by promoting alternative branch site selection. However, there appears to be great flexibility in the specific sequence of a given tract. Not only the optimal composition of the polypyrimidine tract, but also the role of the tract in introns with no apparent polypyrimidine tracts or where changes in the tract are apparently harmless are uncertain. Accordingly, we have designed a series of cis-competition splicing constructs to test the functional competitive efficiency of a variety of systematically mutated polypyrimidine tracts. An RT/PCR assay was used to detect spliced product formation as a result of differential branch point selection dependent on direct competition between two opposing polypyrimidine tracts. We found that pyrimidine tracts containing 11 continuous uridines are the strongest pyrimidine tracts. In such cases, the position of the uridine stretch between the branch point and 3'-splice site AG is unimportant. In contrast, decreasing the continuous uridine stretch to five or six residues requires that the tract be located immediately adjacent to the AG for optimal competitive efficiency. The block to splicing with decreasing polypyrimidine tract strength is primarily prior to the first step of splicing. While lengthy continuous uridine tracts are the most competitive, tracts with decreased numbers of consecutive uridines and even tracts with alternating purine/pyrimidine residues can still function to promote branch point selection, but are far less effective competitors in 3'-splice site selection assays.     

An intronic polypyrimidine-rich element downstream of the donor site modulates cystic fibrosis transmembrane conductance regulator exon 9 alternative splicing

Two intronic elements, a polymorphic TGmTn locus at the end of intron 8 and an intronic splicing silencer in intron 9, regulate aberrant splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Previous studies (Pagani, F., Buratti, E., Stuani, C., Romano, M., Zuccato, E., Niksic, M., Giglio, L., Faraguna, D., and Baralle, F. E. (2000) J. Biol. Chem. 275, 21041-21047 and Buratti, E., Dork, T., Zuccato, E., Pagani, F., Romano, M., and Baralle, F. E. (2001) Embo J. 20, 1774-1784) have demonstrated that trans-acting factors that bind to these sequences, TDP43 and Ser/Arg-rich proteins, respectively, mediate splicing inhibition. Here, we report the identification of two polypyrimidine-binding proteins, TIA-1 and polypyrimidine tract-binding protein (PTB), as novel players in the regulation of CFTR exon 9 splicing. In hybrid minigene experiments, TIA-1 induced exon inclusion, whereas PTB induced exon skipping. TIA-1 bound specifically to a polypyrimidine-rich controlling element (PCE) located between the weak 5'-splice site (ss) and the intronic splicing silencer. Mutants of the PCE polypyrimidine motifs did not bind TIA-1 and, in a splicing assay, did not respond to TIA-1 splicing enhancement. PTB antagonized in vitro TIA-1 binding to the PCE, but its splicing inhibition was independent of its binding to the PCE. Recruitment of U1 small nuclear RNA to the weak 5'-ss by complementarity also induced exon 9 inclusion, consistent with the facilitating role of TIA-1 in weak 5'-ss recognition by U1 small nuclear ribonucleoprotein. Interestingly, in the presence of a high number of TG repeats and a low number of T repeats in the TGmTn locus, TIA-1 activated a cryptic exonic 3'-ss. This effect was independent of both TIA-1 binding to the PCE and U1 small nuclear RNA recruitment to the 5'-ss. Moreover, it was abolished by deletion of either the TG or T sequence. These data indicate that, in CFTR exon 9, TIA-1 binding to the PCE recruits U1 small nuclear ribonucleoprotein to the weak 5'-ss and induces exon inclusion. The TIA-1-mediated alternative usage of the 3'-splice sites, which depends on the composition of the unusual TGmTn element, represents a new mechanism of splicing regulation by TIA-1.     

Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin pre-mRNA.

Previous studies of alternative splicing of the rat beta-tropomyosin gene have shown that nonmuscle cells contain factors that block the use of the skeletal muscle exon 7 (Guo, W., Mulligan, G. J., Wormsley, S., and Helfman, D. M. (1991) Genes & Dev. 5, 2095-2106). Using an RNA mobility-shift assay we have identified factors in HeLa cell nuclear extracts that specifically interact with sequences responsible for exon blockage. Here we present the purification to apparent homogeneity of a protein that exhibits these sequence specific RNA binding properties. This protein is identical to the polypyrimidine tract binding protein (PTB) which other studies have suggested is involved in the recognition and efficient use of 3'-splice sites. PTB binds to two distinct functional elements within intron 6 of the beta-tropomyosin pre-mRNA: 1) the polypyrimidine tract sequences required for the use of branch points associated with the splicing of exon 7, and 2) the intron regulatory element that is involved in the repression of exon 7. Our results demonstrate that the sequence requirements for PTB binding are different than previously reported and shows that PTB binding cannot be predicted solely on the basis of pyrimidine content. In addition, PTB fails to bind stably to sequences within intron 5 and intron 7 of beta-TM pre-mRNA, yet forms a stable complex with sequences in intron 6, which is not normally spliced in HeLa cells in vitro and in vivo. The nature of the interactions of PTB within this regulated intron reveals several new details about the binding specificity of PTB and suggests that PTB does not function exclusively in a positive manner in the recognition and use of 3'-splice sites.     

Alternative splicing of fibronectin mRNAs in chondrosarcoma cells: role of far upstream intron sequences

The fibronectin (FN) gene encodes multiple mRNAs through the process of alternative splicing, and production of certain isoforms is characteristic of a given cell type. Chondrocytes produce FNs that completely lack alternative exon EIIIA, and loss of inclusion of the exon is tightly linked to chondrogenic condensation of mesenchymal cells. The inclusion of a second exon, EIIIB, is high in embryonic cartilage, but declines with age. Multiple exons are omitted to produce the (V + C)-form that is highly specific for cartilage and chondrocytes. A rat chondrosarcoma cell line, RCS, was identified that preserves key features of the cartilage-specific splicing phenotype. RCS cells, which exclude exon EIIIA, and HeLa cells, which include exon EIIIA similar to mesenchymal cells, were used to assess the contribution of intron sequences flanking exon EIIIA to splicing regulation. Deletion of most of the intron downstream of the exon had little effect on splicing in either cell type. However, deletions within upstream intron 32-A reduced inclusion of the alternative exon in both cell types. The sequences involved lie more than 200 nucleotides away from the exon, but could not be localized to a single region by deletion mapping. These intronic sequences contribute to the efficiency of exon EIIIA recognition, but not to cell-type specific regulation. The normally inhibitory factor polypyrimidine tract binding protein promotes exon EIIIA inclusion in a manner that is partially dependent on the regulatory sequences within intron 32-A.     

Muscle-specific splicing enhancers regulate inclusion of the cardiac troponin T alternative exon in embryonic skeletal muscle.

The alternative exon 5 of the striated muscle-specific cardiac troponin T (cTNT) gene is included in mRNA from embryonic skeletal and cardiac muscle and excluded in mRNA from the adult. The embryonic splicing pattern is reproduced in primary skeletal muscle cultures for both the endogenous gene and transiently transfected minigenes, whereas in nonmuscle cell lines, minigenes express a default exon skipping pattern. Using this experimental system, we previously showed that a purine-rich splicing enhancer in the alternative exon functions as a constitutive splicing element but not as a target for factors regulating cell-specific splicing. In this study, we identify four intron elements, one located upstream,and three located downstream of the alternative exon, which act in a positive manner to mediate the embryonic splicing pattern of exon inclusion. Synergistic interactions between at least three of the four elements are necessary and sufficient to regulate splicing of a heterologous alternative exon and heterologous splice sites. Mutations in these elements prevent activation of exon inclusion in muscle cells but do not affect the default level of exon inclusion in nonmuscle cells. Therefore, these elements function as muscle-specific splicing enhancers (MSEs) and are the first muscle-specific positive-acting splicing elements to be described. One MSE located downstream from the alternative exon is conserved in the rat and chicken cTNT genes. A related sequence is found in a third muscle-specific gene, that encoding skeletal troponin T, downstream from an alternative exon with a developmental pattern of alternative splicing similar to that of rat and chicken cTNT. Therefore, the MSEs identified in the cTNT gene may play a role in developmentally regulated alternative splicing in a number of different genes.     

5'- and 3'-terminal nucleotides in the FGFR2 ISAR splicing element core have overlapping roles in exon IIIb activation and exon IIIc repression

The cell type-specific, mutually-exclusive alternative splicing of the fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is tightly regulated. A sequence termed ISAR (intronic splicing activator and repressor) has been implicated as an important cis regulatory element in both activation of exon IIIb and repression of exon IIIc splicing in epithelial cells. In order to better understand how this single sequence could have dual roles, we transfected minigenes containing a series of 2-bp mutations in the 18 3′-most nucleotides of ISAR that we refer to as the ISAR core. Transfection of cells with dual-exon (IIIb and IIIc) minigenes revealed that mutation of terminal sequences of the core led to decreased exon IIIb inclusion and increased exon IIIc inclusion. Transfection of cells with single-exon IIIb minigenes and single-exon IIIc minigenes revealed that mutation of terminal sequences of the ISAR core led to decreased exon IIIb inclusion and increased exon IIIc inclusion, respectively. Nucleotides of the ISAR core responsible for exon IIIb activation appear to overlap very closely with those required for exon IIIc repression. We describe a model in which ISAR and a 5′ intronic sequence known as IAS2 form a stem structure required for simultaneous exon IIIb activation and exon IIIc repression.     

Use of minigene systems to dissect alternative splicing elements

Pre-mRNA splicing is an essential step for gene expression in higher eukaryotes. The splicing efficiency of individual exons is determined by multiple features involving gene architecture, a variety of cis-acting elements within the exons and flanking introns, and interactions with components of the basal splicing machinery (called the spliceosome) and auxiliary regulatory factors which transiently co-assemble with the spliceosome. Both alternative and constitutive exons are recognized by multiple weak protein:RNA interactions and different exons differ in the interactions which are determinative for exon usage. Alternative exons are often regulated according to cell-specific patterns and regulation is mediated by specific sets of cis-acting elements and trans-acting factors. Transient expression of minigenes is a commonly used in vivo assay to identify the intrinsic features of a gene that control exon usage, identify specific cis-acting elements that control usage of constitutive and alternative exons, identify cis-acting elements that control cell-specific usage of alternative exons, and once regulatory elements have been identified, to identify the trans-acting factors that bind to these elements and modulate splicing. This chapter describes approaches and strategies for using minigenes to define the cis-acting elements that determine splice site usage and to identify and characterize the trans-acting factors that bind to these elements and regulate alternative splicing.