Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5′-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes.

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The aim of this study is to investigate the effects of ambient thermal environments on the development of swallowtail butterflies (Sericinus montela Gray). Developmental durations and survival rates of S. montela were examined at two crucial developmental stages, embryonic and larval development, at varying temperatures ranging from 15°C to 35°C. As expected, our results indicated that increasing temperatures decreased the developmental duration and survival rate of the eggs. However, the larvae and pupae showed maximum survival rates at 20.0°C and 25.0°C, and the represented durations were similar to those of the eggs. Larval development was stage-specific, revealing that the fourth and fifth instars at the later stages were more susceptible to temperature variation. When considering both parameters, the optimal development of S. montela occurred within the temperature range of 20.0-25.0°C. The lower threshold for the complete development of S. montela from eggs to eclosion of adults was calculated at 10.6°C by linear regression analysis. The estimated value is similar to that of other endemic insects distributed in temperate climate zones, which indicates that S. montela belongs to a small group of swallowtails adjusted to low ambient temperatures. From the results, we predict that the full development of S. montela could be achieved within the temperature range of 17.5-30.0°C. Embryonic development ceased at both test temperature extremes, and no further larval development proceeded after the third instar at 35.0 o C. These results suggest that embryogenesis can be significantly influenced by slight variations in the ambient thermal environment that fall below the optimal range.