Most protein-coding genes in higher eukaryotes contain introns, parts of the primary transcript that are removed during splicing. During splicing, a macromolecular complex called the spliceosome assembles on the pre-mRNA and catalyzes intron removal.

The splicing reaction is one of the most interesting – and beautiful – in biology. It occurs via two transesterification reactions that attach the 3′ end of the upstream exon to the 5′ end of its downstream neighbor, liberating the intervening intron. Many introns are very long, sometimes thousands of bases. Why these sequences exist in the first place is one of the most compelling mysteries of molecular biology.

The core spliceosome includes five ribonucleoproteins (U1, U2, U5, and U4/6 snRNPs) that depend on accessory proteins (SR proteins, hnRNP proteins) to determine intron boundaries. Differential expression and activity of accessory proteins influence splice site selection and also allow different sites to be selected.

This variability in splice site selection is called alternative splicing, a fascinating phenomenon that happens in near every species where introns occur. Alternative splicing makes it possible for one gene to produce multiple mRNA species and thus encode different – but similar – proteins. In animals, many processes use alternative splicing as a regulatory mechanism; two of the best studied are sex determination in invertebrates and neuronal cell differentiation in mammals. But the ability to splice also introduces risks – many genetic disorders in humans are due to splicing errors. Examples of splicing-related disorders include premature aging (progeria), muscular dystrophy, some forms of cancer, and many others. Many excellent research groups are hard at work studying the relationship between splicing and human disease.

In plants, we know far less about how splicing occurs and how it is regulated. And yet, studying splicing in plants could yield novel insights into this nearly universal process. Plants live their entire lives in one location and must acclimate to daily and seasonal fluctuations in temperature, water availability, and sunlight. Studying how the splicing machinery in plants adapts to environmental challenges will increase knowledge of splicing regulation and catalysis in all organisms, including humans.

In the Loraine Lab, we are studying how alternative splicing is regulated, focusing on how the splicing machinery in plants responds to stresses in the environment. Our approach involves studying RNA-Seq data sets from diverse plants and sample types to identify conditions and treatments that trigger changes in splicing.

Thus far, we’ve learned that for most genes that contain alternative splice sites, one major splice site predominates. We also observed that the relative abundance of splice forms is remarkably stable across many different data sets, suggesting that minor and major forms are co-expressed and that the splicing machinery is well-adapted to maintaining stable isoform abundance across diverse conditions. Lastly, our studies of splicing in pollen, which contains exactly two cell types (sperm cell and vegetative cell) suggests that in plants, splice variants arising from the same gene are co-expressed. That is, if one form is produced, the other forms are also produced, and the relative abundance of these forms is the same across many different sample types.

However, there are some genes for which splice site choice is highly variable, and many of these genes encode proteins with predicted or known roles in splicing. We call these genes “super-splicers” because they are able to generate many diverse splice forms in different relative amounts depending on the site of expression, time of day, and many other factors. We hypothesize that that highly variable alternative splicing of spliceosomal components and accessory proteins maintains splicing stability of other genes despite daily and seasonal changes in environmental conditions. We are testing this and other hypotheses using genetic, genomic, computational, and molecular approaches in Arabidopsis, rice, and other plants.