Alternative splicing of eukaryotic messenger RNA precursors represents a common mechanism for generating multiple transcripts from a single gene. Although there has been increasing information concerning the sequence requirements and the biochemical mechanisms involved in the constitutive splicing of primary RNA transcripts, very little is known about the sequences or mechanisms which determine alternative RNA-processing events in complex transcription units. The calcitonin/calcitonin gene-related peptide (CGRP) primary RNA transcript undergoes tissue-specific alternative processing, resulting in the differential production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons of the central and peripheral nervous systems. To elucidate the molecular mechanisms underlying these alternative RNA processing events, we have examined the nucleotide sequences involved in the production of calcitonin and CGRP mRNAs. Analyses of HeLa and F9 cell lines transfected with a variety of mutant calcitonin/CGRP transcription units have demonstrated that alternative splice-site selection is primarily regulated by cis-active element(s) near the calcitonin-specific 3'-splice junction. We suggest that the tissue-specific pattern of alternative RNA processing is conferred by sequence information at the calcitonin-specific acceptor which serves to inhibit the production of calcitonin transcripts in CGRP-producing cells.
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...
The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery.Alternative processing of primary transcripts plays a central role in the control of gene expression in higher eukaryotes. Alternative splicing often occurs in developmentally regulated and tissue-specific patterns and can lead to the production of functionally distinct protein isoforms from a single primary transcript. The number of genes for which alternative splicing has been demonstrated has expanded greatly in recent years, emphasizing the critical role of this posttranscriptional event in normal cellular regulation. Although increasing information has accumulated concerning the sequence requirements and mechanisms involved in constitutive splicing, the cis-active sequences and transacting factors regulating alternative splicing in mammalian gene expression remain largely unknown (32,46,48,51,54,61).In one of the first examples of mammalian alternative splicing, calcitonin and calcitonin gene-related peptide (CGRP) arise from a single genomic locus in which the primary RNA transcript undergoes tissue-specific processing, resulting in the differential production of calcitonin and CGRP mRNAs (5, 6, 64-66). The rat calcitonin/CGRP gene is organized in six exons (Fig. la); calcitonin mRNA is produced by splicing of the first three exons to the fourth exon, accompanied by cleavage and polyadenylation at the 3' end of the fourth exon. CGRP mRNA production results from the splicing of the first three exons to the fifth and sixth exons and the use of a distal polyadenylation signal at the 3' end of the sixth exon (5). Calcitonin is synthesized by the parafollicular (C) cells of the thyroid gland and ser...
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