RNA Splicing Factor Mutations That Cause Retinitis Pigmentosa Result in Circadian Dysregulation.

Circadian clocks regulate multiple physiological processes in the eye, but their requirement for retinal health remains unclear. We previously showed that Drosophila homologs of spliceosome proteins implicated in human retinitis pigmentosa (RP), the most common genetically inherited cause of blindness, have a role in the brain circadian clock. In this study, we report circadian phenotypes in murine models of RP. We found that mice carrying a homozygous H2309P mutation in Pre-mRNA splicing factor 8 (Prpf8) display a lengthened period of the circadian wheel-running activity rhythm. We show also that the daily cycling of circadian gene expression is dampened in the retina of Prpf8-H2309P mice. Surprisingly, molecular rhythms are intact in the eye cup, which includes the retinal pigment epithelium (RPE), even though the RPE is thought to be the primary tissue affected in this form of RP. Downregulation of Prp31, another RNA splicing factor implicated in RP, leads to period lengthening in a human cell culture model. The period of circadian bioluminescence in primary fibroblasts of human RP patients is not significantly altered. Together, these studies link a prominent retinal disorder to circadian deficits, which could contribute to disease pathology.

Splicing the Clock to Maintain and Entrain Circadian Rhythms.

Circadian clocks drive daily rhythms of physiology and behavior in multiple organisms and synchronize these rhythms to environmental cycles of light and temperature. The basic mechanism of the clock consists of a transcription-translation feedback loop, in which key clock proteins negatively regulate their own transcription. Although much of the focus with respect to clock mechanisms has been on the regulation of transcription and on the stability and activity of clock proteins, it is clear that other regulatory processes also have to be involved to explain aspects of clock function. Here, we review the role of alternative splicing in circadian clocks. Starting with a discussion of the Drosophila clock and then extending to other major circadian model systems, we describe how the control of alternative splicing enables organisms to maintain their circadian clocks as well as to respond to environmental inputs, in particular to temperature changes.

Spliceosome factors target timeless (tim) mRNA to control clock protein accumulation and circadian behavior in Drosophila.

Transcription-translation feedback loops that comprise eukaryotic circadian clocks rely upon temporal delays that separate the phase of active transcription of clock genes, such as Drosophila period (per) and timeless (tim), from negative feedback by the two proteins. However, our understanding of the mechanisms involved is incomplete. Through an RNA interference screen, we found that pre-mRNA processing 4 (PRP4) kinase, a component of the U4/U5.U6 triple small nuclear ribonucleoprotein (tri-snRNP) spliceosome, and other tri-snRNP components regulate cycling of the molecular clock as well as rest:activity rhythms. Unbiased RNA-Sequencing uncovered an alternatively spliced intron in tim whose increased retention upon prp4 downregulation leads to decreased TIM levels. We demonstrate that the splicing of tim is rhythmic with a phase that parallels delayed accumulation of the protein in a 24 hr cycle. We propose that alternative splicing constitutes an important clock mechanism for delaying the daily accumulation of clock proteins, and thereby negative feedback by them. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Caffeine promotes wakefulness via dopamine signaling in Drosophila.

Caffeine is the most widely-consumed psychoactive drug in the world, but our understanding of how caffeine affects our brains is relatively incomplete. Most studies focus on effects of caffeine on adenosine receptors, but there is evidence for other, more complex mechanisms. In the fruit fly Drosophila melanogaster, which shows a robust diurnal pattern of sleep/wake activity, caffeine reduces nighttime sleep behavior independently of the one known adenosine receptor. Here, we show that dopamine is required for the wake-promoting effect of caffeine in the fly, and that caffeine likely acts presynaptically to increase dopamine signaling. We identify a cluster of neurons, the paired anterior medial (PAM) cluster of dopaminergic neurons, as the ones relevant for the caffeine response. PAM neurons show increased activity following caffeine administration, and promote wake when activated. Also, inhibition of these neurons abrogates sleep suppression by caffeine. While previous studies have focused on adenosine-receptor mediated mechanisms for caffeine action, we have identified a role for dopaminergic neurons in the arousal-promoting effect of caffeine.

Regulation of cuticle pigmentation in drosophila by the nutrient sensing insulin and TOR signaling pathways.

BACKGROUND: Insect pigmentation is a phenotypically plastic trait that plays a role in thermoregulation, desiccation tolerance, mimicry, and sexual selection. The extent and pattern of pigmentation of the abdomen and thorax in Drosophila melanogaster is affected by environmental factors such a growth temperature and access to the substrates necessary for melanin biosynthesis. This study aimed to determine the effect of nutritional status during development on adult pigmentation and test whether nutrient sensing through the Insulin/IGF and target of rapamycin (TOR) pathways regulates the melanization of adult cuticle in Drosophila. RESULTS: Flies reared on low quality food exhibit decreased pigmentation, which can be phenocopied by inhibiting expression of the Insulin receptor (InR) throughout the entire fly during mid to late pupation. The loss of Insulin signaling through PI3K/Akt and FOXO in the epidermis underlying the developing adult cuticle causes a similar decrease in adult pigmentation, suggesting that Insulin signaling acts in a cell autonomous manner to regulate cuticle melanization. In addition, TOR signaling increases pigmentation in a cell autonomous manner, most likely through increased S6K activity. CONCLUSION: These results suggest that nutrient sensing through the Insulin/IGF and TOR pathways couples cuticle pigmentation of both male and female Drosophila with their nutritional status during metamorphosis.

The microRNA miR-8 is a positive regulator of pigmentation and eclosion in Drosophila.

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally silence gene expression by binding to target mRNAs. Previous studies have identified the miRNA miR-8 as a pleiotropic regulator of Drosophila development, controlling body size and neuronal survival by targeting multiple mRNAs. In this study we demonstrate that miR-8 is also required for proper spatial patterning of pigment on the adult abdominal cuticle in females but not males. RESULTS: Female adult flies lacking miR-8 exhibit decreased pigmentation of the dorsal abdomen, with a pattern of pigmentation similar to wild type flies grown at higher temperatures. This pigmentation defect in miR-8 mutants is independent of the previously reported body size defect, and miR-8 acts directly in the developing cuticle to regulate pigmentation patterning. The decrease in pigmentation in miR-8 mutants was more pronounced in flies grown at higher temperatures. We also found that loss of miR-8 dramatically affected the ability to eclose at higher temperatures. CONCLUSION: Loss of miR-8 increased the sensitivity of Drosophila to higher temperatures for both pigmentation patterning and the ability to eclose. Together, these data suggest that miR-8 acts as a buffer to stabilize gene expression patterns in the midst of environmental variation.