43-OR: Post-transcriptional Regulation of Slc16a1 mRNA Is Essential for Maintaining Normal ß-Cell Function

Abstract

Slc16a1 gene belongs to a family of genes that are selectively silenced in mature β-cells as compared to other mammalian cell types and thereby named “β-cell disallowed genes.” Forced expression of Slc16a1 in β-cells interfered with insulin secretion and normal β-cell function. Slc16a1 is previously shown to be regulated by promoter methylation transcriptionally and miRNA post-transcriptionally. Here, we show deadenylation by CCR4-NOT complex is involved in silencing of Slc16a1 and maintaining normal β-cell function. We have previously shown that β-cell specific knockout of CNOT3 (CNOT3βKO), a CCR4-NOT complex and a positive modulator of its function causes upregulation of β-cell disallowed genes among them is Slc16a1 alongside with the decreased expression of β-cell specific genes and loss of normal β-cell function. In this study, we demonstrate the molecular mechanism by which CCR4-NOT complex is involved in silencing Slc16a1. Slc16a1 mRNA is stabilized in CNOT3βKO islets compared to wild type islets as assessed by calculating the relative amounts of introns and exons for Slc16a1 using total RNAseq data. RNA immunoprecipitation revealed Slc16a1 mRNA binds to CCR4-NOT complex. Expression of luciferase constructs bearing the 3′-untranslated regions (3’UTR) of Slc16a1 mRNA in wild type or CNOT3 knockdown HEK293T cells demonstrated that Slc16a1 mRNA is a direct target of CNOT3. CCR4-NOT complex binds to its target genes through interaction with RNA binding proteins that bind to special motifs in 3’UTR. We identified Roquin, an mRNA binding protein that binds to stem-loop structure in Slc16a1 3’UTR. Thus, CCR4-NOT complex -Roquin axis inhibits Slc16a1 expression in β-cells maintaining normal β-cell function. Disclosure D. Mostafa: None. A. Takahashi: None. T. Yamamoto: None.