Control of alternative pre-mRNA splicing by distributed pentameric repeats

Abstract

Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3 splice site in the rat calcitonin͞CGRP gene. Specific mutagenesis of these repeats in calcitonin͞CGRP pre-mRNA resulted in the loss of calcitoninspecific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.The search for regulatory sequences in alternatively spliced pre-mRNAs has led to the definition of a variety of sequence elements that are necessary for normal cell-specific splicing (for reviews, see refs. 1-4). These include constitutive sequences necessary for general splicing as well as novel sequences that apparently respond to specific trans-acting factors (5-7). Competition between alternative exons depends on the relative quality of the constitutive splicing signals, a balance that presumably can be shifted by cell-specific factors acting on regulatory sequences. Further, a cell-specific protein complex binding to a regulatory sequence in c-src pre-mRNA was shown to include ubiquitously expressed proteins, raising the possibility that cell-specific modifications in constitutive proteins also may contribute to alternative splicing regulation (7,8). However, in most cases, the molecular mechanisms underlying tissue-specific alternative splicing remain to be defined.Recently, a repeated hexanucleotide element, TGCATG, was shown to regulate the tissue-specific splicing of the fibronectin alternative exon EIIIB (9). The EIIIB exon is included in transfected F9 cells, but excluded in transfected HeLa cells. In fibronectin pre-mRNA, TGCATG repeats occur within the intron downstream of EIIIB. Deletion of intron sequences containing these elements caused EIIIB skipping in F9 cells, whereas replacement of these sequences with synthetic TGCATG repeats restored EIIIB inclusion in these cells. These elements may positively activate splicing of the 5Ј alternative exon (9); alternatively, they may act negatively by delaying splicing of the 3Ј constitutive exon, thus allowing a kinetic advantage for EIIIB recognition.Extensive mutatio...

Fox-2 Mediates Epithelial Cell-Specific Fibroblast Growth Factor Receptor 2 Exon Choice

“…Not all (U)GCAUG elements within a particular transcript have a demonstrable effect on splicing, and it is not immediately obvious what determines the difference. In the case of calcitonin/CGRP, it was shown that only two of the five (U) GCAUG elements, one intronic and one exonic, are needed for proper regulation (17). This mirrors the situation with FGFR2 splicing regulation where deletion of two (U)GCAUG elements within the loop region between IAS2 and ISAR core in FGFR2 transcripts has no effect on cell-type-specific exon inclusion (1,32).…”

“…Previously we had identified two overlapping (U)GCAUG elements downstream of ISAR core that were important for both exon IIIb activation and exon IIIc repression (1). Understanding that (U)GCAUG elements generally are present in multiple copies in the same pre-mRNA (8,17,19,20,23,26), we scanned the FGFR2 sequence for other (U)GCAUG elements and found a (U)GCAUG element present in exon IIIc. To determine if this sequence played a role in activating exon IIIb and repressing exon IIIc, we created minigenes where we mutated the overlapping GCAUG elements downstream of ISAR core (C10-18), the (U)GCAUG in exon IIIc (IIIc Mut), or both (C10-18 IIIc Mut) (Fig.…”

“…This change in FGFR2 splicing pattern is accompanied by other changes consistent with a mesenchyme-to-epithelium-like transition (A. P. VOL. 26,2006 Fox PROTEINS REGULATE FGFR2 ALTERNATIVE SPLICING 1219 on May 9, 2018 by guest http://mcb.asm.org/ 4.1R, calcitonin/CGRP, the mitochondrial ATP synthase ␥ subunit (F1␥), and ␣-actinin RNAs (8,17,19,20,23,26,28,30). Usually multiple copies of (U)GCAUG are found in the vicinity of regulated exons within these transcripts.…”

“…The GCAUG or UGCAUG sequence is a previously characterized splicing enhancer element that has been shown to be important for the proper splicing regulation of fibronectin, c-src, calcitonin/CGRP, nonmuscle myosin II heavy chain B (NMHC-B), and 4.1R transcripts (8,17,19,23,26,30). In addition, a computational study demonstrated an overrepresentation of UGCAUG hexamers in the downstream intron of neural and muscle-specific alternatively spliced exons (2,28).…”

Autoregulation of Fox protein expression to produce dominant negative splicing factors

“…These proteins each contain a single highly conserved RNA recognition motif (RRM) that binds the sequence (U)GCAUG (Jin et al 2003;Auweter et al 2006;Ponthier et al 2006;Yeo et al 2009). This element serves an important role in controlling the splicing of many alternative exons (Black 1992;Huh and Hynes 1994;Kawamoto 1996;Del Gatto et al 1997;Hedjran et al 1997;Modafferi and Black 1997;Lim and Sharp 1998;Deguillien et al 2001;Baraniak et al 2006). The Fox proteins typically act as splicing activators when bound downstream of an alternative exon, or as silencers when bound upstream (Underwood et al 2005;Zhang et al 2008;Yeo et al 2009).…”