CAPS1 RNA Editing Promotes Dense Core Vesicle Exocytosis

Abstract

SUMMARY Calcium-dependent activator protein for secretion 1 (CAPS1) plays a distinct role in the priming step of dense core vesicle (DCV) exocytosis. CAPS1 pre-mRNA is known to undergo adenosine-to-inosine RNA editing in its coding region, which results in a glutamate-to-glycine conversion at a site in its C-terminal region. However, the physiological significance of CAPS1 RNA editing remains elusive. Here, we created mutant mice in which edited CAPS1 was solely expressed. These mice were lean due to increased energy expenditure caused by physical hyperactivity. Electrophysiological and biochemical analyses demonstrated that the exocytosis of DCVs was upregulated in the chromaffin cells and neurons of these mice. Furthermore, we showed that edited CAPS1 bound preferentially to the activated form of syntaxin-1A, a component of the exocytotic fusion complex. These findings suggest that RNA editing regulates DCV exocytosis in vivo, affecting physical activity.

A comparative analysis among ADAR mutant mice reveals site-specific regulation of RNA editing

“…Tissue lysates from mouse cerebral cortex and spleen were prepared and stored at −80ºC until use as described previously (Miyake et al 2016). Lysates were then separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) and immunoblotted with primary antibodies using a SNAP i.d ® 2.0 Protein Detection System (Merck Millipore, Burlington, MA, USA) as previously described (Nakahama et al 2018).…”

“…The editing ratio was analysed by Sanger sequencing as described previously with minor modifications (Miyake et al 2016). In brief, 100 ng of each total RNA was incubated with 0.1 U/µl DNase I (Thermo Fisher Scientific) at 37°C for 20 min and then the denatured RNAs were reverse transcribed into cDNAs using the SuperScript™ III First-Strand Synthesis System (Thermo Fisher Scientific) with random hexamers.…”

“…Given that inosine is recognized as if it were guanosine by the translational machinery, RNA editing in CDS leads to re-coding events that can potentially affect the functions of the corresponding proteins (Pullirsch and Jantsch 2010). Indeed, mutant mice expressing either an edited or unedited protein alone exhibit abnormal phenotypes, such as being more prone to seizures, hyperactivity, depression and dysregulated vascular contractions (Higuchi et al 2000;Kawahara et al 2008a;Mombereau et al 2010;Miyake et al 2016;Jain et al 2018). In addition, dysregulated RNA editing in CDS is linked to human Pedro Henrique Costa Cruz 4 diseases such as cancer and amyotrophic lateral sclerosis (Kawahara et al 2004;Slotkin and Nishikura 2013;Peng et al 2018).…”

Functions of the RNA Editing Enzyme ADAR1 and Their Relevance to Human Diseases

“…A new target for ADAR1 was identified recently in the human CADPS gene, resulting in a E1250G substitution [81]. The encoded CAPS1 protein belongs to a family of proteins that regulate the exocytosis of dense-core vesicles, which are formed during the secretion of insulin and neurotransmitters.…”

“…The E1250G mutation causes increased catecholamine secretion. While this mutation has been shown in mice to result in increased locomotion and leaner bodies, the CAPS protein family has also been implicated in autism and learning disabilities, and further studies are needed to determine if editing by ADAR1 also affects the development of these disorders [81]. …”