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We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3' splice site of alpha-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates containing weak 3' splice sites or mutated 5' splice sites. However, they are unique in that they can activate splicing whether they are placed in an upstream or downstream exon, and the two central elements can function regardless of their position relative to one another. The presence of excess RNAs containing these enhancers could effectively inhibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incubation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to rescue splicing. Using gel mobility shift assays, we show that formation of stable enhancer-specific complexes on alpha-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the doublesex exon enhancer elements in Drosophila, our results suggest that assembly of mammalian exon enhancer complexes requires both SR and non-SR proteins to activate selection of weak splice sites.

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We have analyzed synonymous codon usage in the genome of A. tamarense CCMP 1598 for protein-coding sequences from 10865 expressed sequence tags (ESTs). We reconstructed a total of 4284 unigenes, including 74 ribosomal protein and 40 plastid-related genes, from ESTs using FrameDP, an open reading frame (ORF) prediction program. Correspondence analysis of A. tamarense genes based on codon usage showed that the GC content at the third base of synonymous codons (GC3s) was strongly correlated with the first axis (r = 0.93 with P < .001). On the other hand, the second axis discriminated between presumed highly and low expressed genes, with expression levels being confirmed by the analysis of EST frequencies (r = −0.89 with P < .001). Our results suggest that mutational bias is the major factor in shaping codon usage in A. tamarense genome, but other factors, namely, translational selection, hydropathy, and aromaticity, also appear to influence the selection of codon usage in this species.

No abstract