Exon and Intron Sequences, Respectively, Repress and Activate Splicing of a Fibroblast Growth Factor Receptor 2 Alternative Exon

Abstract

Two alternative exons, BEK and K-SAM, code for part of the ligand binding site of fibroblast growth factor receptor 2. Splicing of these exons is mutually exclusive, and the choice between them is made in a tissuespecific manner. We identify here pre-mRNA sequences involved in controlling splicing of the K-SAM exon. The short K-SAM exon sequence 5-TAGGGCAGGC-3 inhibits splicing of the exon. This inhibition can be overcome by mutating either the exon's 5 or 3 splice site to make it correspond more closely to the relevant consensus sequence. Two separate sequence elements in the intron immediately downstream of the K-SAM exon, one of which is a sequence rich in pyrimidines, are both needed for efficient K-SAM exon splicing. This is no longer the case if either the exon's 5 or 3 splice site is reinforced. Furthermore, if the exon inhibitory sequence is removed, the intron sequences are not required for splicing of the K-SAM exon in a cell line which normally splices this exon. At least three elements are thus involved in controlling splicing of the K-SAM exon: suboptimal 5 and 3 splice sites, an exon inhibitory sequence, and intron activating sequences.Alternative splicing is a mechanism frequently used by cells for the production of several proteins from one gene (29,33). When subject to control, this mechanism allows the production of similar but distinct versions of a protein in a cell-typespecific manner. Alternative splicing of the fibroblast growth factor receptor 2 (FGFR-2) gene pre-mRNA is a particularly attractive example of this phenomenon (9,21,30,50). The FGFR-2 extracellular domain is composed of three immunoglobulin-like domains, the third of which is particularly important for ligand binding (30, 50). The carboxy-terminal moiety of the third immunoglobulin-like domain is encoded by two alternative exons (9, 30, 50), K-SAM (or IIIb) and BEK (or IIIc). Splicing of these exons is mutually exclusive and is controlled both in cell lines and during development (9,30,31). Epithelial cells use mainly the K-SAM exon and synthesize a receptor with high affinity for keratinocyte growth factor. Other cell types use mainly the BEK exon and synthesize a high-affinity receptor for basic fibroblast growth factor. That the splicing choice is linked to the cell type is illustrated by the observation that oncoprotein fos activation in epithelial cells induces an epithelio-mesenchymal conversion and changes the exon spliced from K-SAM to BEK (36). Controlling the splicing of the FGFR-2 pre-mRNA may be very important for the cell, as a change in the splicing pattern has been correlated with malignant progression of rat prostate cells (48).This control is presumably exerted at the level of construction of the spliceosome, which is composed of four small nuclear ribonucleoproteins (snRNPs) as well as many associated splicing factors (19,37). Early steps in spliceosome formation are the recognition of the 5Ј splice site sequence by U1 snRNP and the recognition of the branch point sequence, associated with the 3Ј splice site...

TIA-1 or TIAR Is Required for DT40 Cell Viability

“…RT-PCR was carried out as described elsewhere (34,35). Primers used were 5Ј-ggaagacgacgggcagcc-3Ј from tiar exon 1, with 5Ј-gcaaaggctgacttgata-3Ј from tiar exon 5; 5Ј-caatcacttccacgtgttcg-3Ј from tiar exon 5, with 5Ј-gttagcccagaagcaat-3Ј from tiar exon 8; 5Ј-gtactcatgggaggccag-3Ј from tiar exon 7 with 5Ј-aggctgagcaccaaatccacccat-3Ј from tiar exon 12.…”

A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA

“…In addition to the exonic, purine-rich elements described above, a number of splicing enhancers are found within introns (Gooding et al+, 1994;Huh & Hynes, 1994;Del Gatto & Breathnach, 1995;Gallego et al+, 1997;Wei et al+, 1997;Carstens et al+, 1998;Kosaki et al+, 1998;Lim & Sharp, 1998;Lou et al+, 1998;McCarthy & Phillips, 1998;Chou et al+, 1999)+ The sequences of intronic splicing enhancers identified thus far are diverse and often distinct from the purine-rich splicing enhancers+ Consistent with the differences in sequence, proteins bound to intronic splicing enhancers are usually different from the SR proteins that bind purine-rich enhancers+ The activity of splicing enhancers may be regulated by the nature and amount of specific RNAbinding proteins within the cell nucleus+ Other variables are also likely to be important, including the locations of multiple enhancer and repressor elements, and the strength and position of adjacent or competing processing sites+ Characterization of such regulatory elements is important for understanding the mechanisms regulating alternative pre-mRNA processing+…”

Combinatorial control of a neuron-specific exon

“…Alternative pre-mRNA splicing is a common means of gene regulation in eukaryotes that allows the generation of multiple protein isoforms from a single gene (for reviews see Wang & Manley, 1997;Y+ Wang et al+, 1997)+ The control of pre-mRNA splicing pattern can be very precise and there are many examples of exons in mammalian cells whose inclusion is highly tissue specific+ A variety of pre-mRNA sequence elements and regulatory proteins are known to affect the splicing patterns of different RNA transcripts+ Although these regulatory components must ultimately alter spliceosome assembly at certain splice sites, the interactions between the general splicing apparatus and its regulators are mostly unknown+ In particular, the molecular events that determine the tissue-specific use of an exon in mammalian cells have not been described+ One difficulty in understanding the precise nature of an exon's regulation is the diversity of mechanisms that can affect its splicing+ Splicing enhancer elements activate the use of splice sites that are otherwise ignored by the general splicing machinery (Hertel et al+, 1997;Wang & Manley, 1997), whereas repressor sequences are thought to block recognition of certain splice sites+ The tissue-specific expression of a particular splicing pattern often depends on a combination of positive and negative control+ Splicing enhancers can be classified by their location in either exons or introns+ These two types of enhancer are thought to be mechanistically different and to bind different types of regulatory proteins+ Exonic splicing enhancer sequences are often bound by a group of factors called SR proteins (Fu, 1995;Chabot, 1996;Manley & Tacke, 1996;Valcarcel & Green, 1996;Caceres & Krainer, 1997)+ Proteins in the SR family contain one or two RNA binding domains of the RRM type and a C-terminal domain rich in serine-arginine dipeptides+ SR proteins are essential for constitutive splicing and also play an important role in the selection of alternative splice sites+ In particular, SR proteins bound with other factors to exonic splicing enhancers are thought to promote spliceosome assembly to upstream 39 splice sites, although the mechanism for this effect on splicing is not yet clear (Wang et al+, 1995;Zuo & Maniatis, 1996; Rudner et al+, 1998)+ In contrast to ex-onic enhancers, intronic splicing enhancers are often found in introns downstream of regulated exons, and have not been shown to bind SR proteins (Balvay et al+, 1992;Black, 1992;Huh & Hynes, 1994;Del Gatto & Breathnach, 1995;Sirand-Pugnet et al+, 1995;Carlo et al+, 1996;Kawamoto, 1996;…”