Exon͞intron architecture varies across the eukaryotic kingdom with large introns and small exons the rule in vertebrates and the opposite in lower eukaryotes. To investigate the relationship between exon and intron size in pre-mRNA processing, internally expanded exons were placed in vertebrate genes with small and large introns. Both exon and intron size inf luenced splicing phenotype. Intron size dictated if large exons were efficiently recognized. When introns were large, large exons were skipped; when introns were small, the same large exons were included. Thus, large exons were incompatible for splicing if and only if they were f lanked by large introns. Both intron and exon size became problematic at Ϸ500 nt, although both exon and intron sequence inf luenced the size at which exons and introns failed to be recognized. These results indicate that present-day gene architecture ref lects at least in part limitations on exon recognition. Furthermore, these results strengthen models that invoke pairing of splice sites during recognition of pre-mRNAs, and suggest that vertebrate consensus sequences support pairing across either introns or exons.Vertebrate genes are typically split into numerous small exons (average size, 134 nt) interrupted by much larger introns (1). Large introns present problems for initial splice site recognition via models that postulate interactions between factors that concertedly recognize the 5Ј and 3Ј splice sites located at intron termini. In vitro, interactions between the ends of introns have been observed during early spliceosome assembly both in yeast and in mammalian systems (reviewed in refs. 2-7). Most experiments with the mammalian system have utilized premRNAs with internally deleted introns. Extrapolation of results from experiments with precursor RNAs containing small introns to more natural pre-mRNAs with large introns is difficult. An alternate spliceosome assembly mode for premRNAs with small exons and large introns (reviewed in refs. 5-7), which we have termed exon definition, suggests that the earliest interactions between 3Ј and 5Ј splice sites occurs across exons rather than across introns. In vitro expansion of an internal exon to Ͼ300 nt severely inhibits the ability to detect ATP-dependent spliceosome formation (8), suggesting an experimental limit on exon size during exon definition that agreed well with known vertebrate exon sizes. Few experiments have addressed the ability of the in vivo vertebrate splicing machinery to recognize large exons.Large exons are only a rarity in vertebrates. Lower eukaryotes have an inverted exon͞intron architecture compared with higher eukaryotes such that many genes have small introns and large exons (1). Thus, many Schizosaccharomyces pombe or Caenorhabditis elegans genes have introns smaller than 100 nt, and Ϸ50% of the introns in Drosophila melanogaster are Ͻ100 nt. Expansion of such small introns in both S. pombe and D. melanogaster causes either a loss of splicing or incorrect splicing via utilization of cryptic spli...
Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3 ' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro.Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.All intron-containing pre-mRNAs contain specific conserved sequence elements at the 5' and 3' splice sites that are essential for accurate and efficient splicing. In vertebrates, the branch point, pyrimidine tract, 3' splice site, and 5' splice site are important determinants of splicing efficiency (recently reviewed in reference 27). Although each can diverge from consensus, the degree to which each matches the consensus is related to the efficiency of splicing of the adjacent exon (19,21,25,35,44,48,50,52,71,(73)(74)(75)(76). In certain differentially spliced genes, additional elements located upstream of the branch point (36, 58) or downstream of the 5' splice site (7) have been shown to influence recognition of the neighboring exon. In addition, splicing efficiency in a number of systems is influenced by exon sequences (5,6,11,16,18,20,22,23,28,29,36,37,40,51,59,(63)(64)(65)72). Thus, a region encompassing shortly upstream of the branch point to shortly downstream of the 5' splice site usually contains all of the sequences necessary for recognition of an exon. We have recently suggested that the exon is the unit of splice site recognition in vertebrates (53). This perspective suggests that successful exon recognition occurs when the balance of all of these intron and exon elements is sufficient to support the definition process.The length of the exon is also an important parameter in its recognition. The average size of vertebrate internal exons is 137 nucleotides (30). Experiments man...
We investigated DNA damage caused by carcinogenic metals in a murine sarcoma virus (MuSV)-based mutagenicity assay in which mutations targeted to v-mos expression can be selected. Nickel chloride treatment of NRK cells (termed 6m2 cells) infected with MuSVts110, a retrovirus conditionally defective in viral RNA splicing and cell transformation, caused the outgrowth of transformed "revertants" with changes in the MuSVts110 RNA splicing phenotype. Cadmium and chromium treatment of 6m2 cells resulted in the selection of a second class of revertants with what appeared to be frameshift mutations allowing the translation of a readthrough gag-mos protein. In both classes of metal-induced revertants, viral gene expression was distinct from that observed in revertants arising in untreated 6m2 cultures, arguing that metal treatment did not simply enhance the rate of spontaneous reversion. In one representative nickel revertant line the operative nickel-induced mutation affecting MuSVts110 RNA splicing was a duplication of 70 bases surrounding the 3' splice site. The effect of this mutation was to direct splicing to the most downstream of the duplicated 3' sites and concomitantly relax its characteristic thermosensitivity. These data establish the mutagenic potential of nickel and provide the first example of a defined nickel-induced mutation in a mammalian gene.
We investigated whether the MuSVtsllO gag gene product (P58gag) can regulate the novel growth temperature dependence of MuSVtsllO RNA splicing. MuSVtsllO mutants with either frameshifts or deletions in the gag gene were tested for their ability to maintain the MuSVts1lO splicing phenotype. Only small decreases in splicing efficiency and no changes in the thermosensitivity of viral RNA splicing were observed in MuSVtsllO gag gene frameshift mutants. Deletions within the gag gene, however, variably decreased MuSVtsllO splicing efficiency but had no effect on its thermosensitivity. Another class of MuSVtsllO splicing mutants generated by treatment of MuSVtsllO-infected cells with NiCl2 was also examined. In these "nickel revertants," P58gag is made, but splicing of the viral transcript is nearly complete at all growth temperatures. The splicing of "tagged" viral RNA transcribed from a modified MuSVtsllO DNA introduced into nickel revertant cells remained thermosensitive, arguing against trans effects of viral gene products on splicing efficiency. These experiments indicated that neither the MuSVtsllO P58gas protein nor any other viral gene product acts in trans to regulate MuSVtsllO splicing.
Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.
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