Abstract
Although alternative pre-mRNA splicing (AS) significantly diversifies the neuronal proteome, the extent of AS is still unknown due in part to the large number of diverse cell types in the brain. To address this complexity issue, we used an annotation-free computational method to analyze and compare the AS profiles between small specific groups of Drosophila circadian neurons. The method, the Junction Usage Model (JUM), allows the comprehensive profiling of both known and novel AS events from specific RNA-seq libraries. The results show that many diverse and novel pre-mRNA isoforms are preferentially expressed in one class of clock neuron and also absent from the more standard Drosophila head RNA preparation. These AS events are enriched in potassium channels important for neuronal firing, and there are also cycling isoforms with no detectable underlying transcriptional oscillations. The results suggest massive AS regulation in the brain that is also likely important for circadian regulation.
The life of nearly all creatures on Earth follows the rhythm of day and night. For example, in fruit flies, darkness and light dictate when the insects feed, rest, move or mate. This is possible thanks to the circadian clock, an internal program which is synchronized with the environment to tell cells in the body when to perform certain roles.In fruit flies, the structure that keeps the body clock ticking is formed of about 150 ‘circadian neurons’, which are divided into several subgroups. In these cells, a complex genetic programis at work, with networks of genes being ‘switched on’ in a cyclical way. To understand how this program works, scientists need to know which genes are turned on and when, as well as which proteins are created based on the information contained in these genes.This can be difficult because one gene does not necessarily code for only one protein. Indeed, when a gene is turned on, it gets copied into a pre-messenger RNA (pre-mRNA), which is formed of several modules. The pre-mRNA can then go through a process called alternative splicing that shuffles or removes the different modules. This means that one gene can give rise to different pre-mRNA molecules that will each serve as a template to build a distinct protein. Until now, there have been few studies that examine the different types of pre-mRNAs found in circadian neurons, and how these change with the time of day.Here, Wang, Abruzzi et al. extract three subgroups of circadian neurons, and one subgroup of non-circadian neurons, from the brain of fruit flies. The pre-mRNAs are isolated, and then a new computational method, called JUM, identifies, counts and categorizes the pre-mRNA molecules in the different groups of neurons.This analysis reveals hundreds of previously unknown pre-mRNA molecules, many of which differed extensively between the types of brain cells. When comparing circadian and non-circadian neurons, Wang, Abruzzi et al. show that the circadian cells had more pre-mRNAs that code for proteins that help the cell communicate with other neurons. Finally, many genes in the circadian neurons use alternative splicing to turn on different types of pre-mRNA molecules at different times of the day in a cyclical way; this suggests that these pre-mRNAs might be participating in the genetic circadian program.Many human disorders, such as certain forms of insomnia, emerge when the circadian clock is thrown off balance. The results reported by Wang, Abruzzi et al. show that alternative splicing may be an overlooked mechanism that shapes this complex program.