Involvement of Alternative Splicing Regulation in Adipose Tissue Thermogenic Induction

Abstract

[emg] Alternative splicing (AS) is a gene expression regulatory mechanism that enables generation of multiple mRNA isoforms from a single gene which commonly are translated into proteins. This mechanism is tightly regulated by cis- and trans-acting factors. Among the latter, there are splicing factors, which form part of spliceosome, and splicing regulators, which can enhance or inhibit spliceosome activity. Therefore, AS is a mechanism that can have a dramatic impact in the regulation of protein activity, which can affect other relevant cellular mechanisms and even tissue functions. AS has been studied extensively in some diseases but the knowledge in adipometabolic pathology is still scarce but growing evidence highlights AS as a regulator of gene expression in adipose tissue. Besides energy-storing white adipose tissue, AS is especially unexplored in brown adipose tissue (BAT), which can dissipate energy in form of heat and thus increase energy expenditure. Considering the potential therapeutic approach of using rediscovered BAT in human adults to assist in the treatment of obesity due to its special energy-consuming properties, we decided to study the possible involvement of AS in brown/beige adipose tissue thermogenic function.

We first determined the transcriptomic signature associated with the acquisition of a beige phenotype in human adipose tissue by analysing adipose browning occurring in patients affected by pheochromocytoma using deep RNAseq analysis and last generation bioinformatic tools. We identified a massive down regulation of splicing machinery- associated genes occurring in association with human adipose browning and in differentiating human beige cells in culture. In addition, these splicing modifications may act by means of direct modulation of the enzymatic machinery involved in key metabolic and thermogenic functions and/or more indirectly affecting the isoform expression of master regulators and functional molecular actors of browning. This affectation of splicing machinery-associated genes in adipose browning led us to investigate the possible role of two candidates: SF3B1, a key component of the spliceosome, and RBFOX2, a splicing factor/regulator of AS, in brown adipocytes.

We discovered that SF3B1 was up-regulated in vivo in murine BAT after thermogenic activation in response to acute cold, and in in vitro brown adipocytes in response to cAMP treatment, the main intracellular mediator of the noradrenergic stimuli. We further explored the effects of blocking SF3B1 using siRNA-mediated interference and a pharmacological inhibitor, pladienolide-B. Blockage of this splicing factor actions reduced both the expression of prominent thermogenesis-associated transcripts and respiration in brown adipocytes in vitro.

Next, we assessed the effects of silencing RBFOX2 in brown adipocytes in vitro through shRNA-mediated interference. Repression of RBFOX2 caused alterations in transcript levels of thermogenesis-related genes and in alternative splicing-driven mRNA isoforms involved in brown adipocyte function. Moreover, signs of impaired respiration and lipolysis, together with the aforementioned changes, suggested that RBFOX2 activity is required for appropriate brown adipocyte thermogenic function. Finally, we obtained preliminary results of adipose tissue-specific knock out of RBFOX2 in mice model which indicated mild effects on thermogenesis, with RBFOX2-KO mice showing enhanced cold exposure adaptation by increasing their body temperature faster that controls, but

further research is required to full-characterize the role of RBFOX2 in vivo during adaptative thermogenesis.

Taken together, our results indicate that AS is a critical process in brown adipose tissue functions that can have a deep impact in systemic energy homeostasis, suggesting further studies on the potential applications energy-consuming brown fat AS regulation- based therapies to counter obesity, type 2 diabetes and its associated comorbidties.