Feedback translational regulation of the creatine transporter SCL6A8 implicated in X-linked intellectual disability

Authors

• IP Ivanov
• C Cao
• TE Dever

Abstract

SCL6A8 is a high-affinity sodium- and chloride-dependent creatine transporter. Mutations in SLC6A8 are the second most common cause of X-linked intellectual disability (XLID), and these mutations have been linked to creatine deficiency in the brain. In a genome-wide analysis of protein synthesis, we identified SCL6A8 in a subset of genes exhibiting high levels of translational regulation. Comparative sequence analysis of SLC6A8 among vertebrates identified an evolutionarily conserved upstream open reading frame (uORF) in the 5’ leader of the SLC6A8 mRNA. According to the scanning model of eukaryotic translation initiation, the 40S ribosome is loaded on an mRNA near the 5’ cap and scans in a 3’ direction in search of a start codon. Typically, translation initiates at the first encountered AUG codon in optimal context (with a purine at the -3 position relative to the A of the AUG codon being the strongest context determinant). Many eukaryotic mRNAs contain uORFs, and, as a consequence of ribosome scanning, most of these uORFs have stochastic cis-acting inhibitory effects on main ORF translation. A small subset of uORFs are used for gene regulation by modulation of their inhibitory function by environmental cues, often a small molecule. Although a uORF regulated by a cellular metabolite (arginine) was first discovered nearly 30 years ago in yeast, few mammalian metabolite-regulated uORFs have been described. Using an SLC6A8-luciferase fusion, we found that creatine inhibits SLC6A8 expression. This regulation was dependent on the conserved uORF as the regulation was lost upon mutation of the uORF start codon or by introducing mutations that alter the reading frame of the uORF. Thus, the encoded uORF peptide, nearly identical from lamprey to human, is critical for creatine control of SLC6A8 expression. We propose that creatine interaction with the nascent peptide in the ribosome exit tunnel causes an elongation pause during translation of the uORF and prevents ribosomes from accessing and translating the SLC6A8 ORF. Accordingly, high levels of creatine feedback inhibit expression of the creatine transporter, providing tight homeostatic control of cellular creatine levels. We believe that our studies provide a paradigm for metabolite-sensing uORFs to regulate gene expression and in the case of SLC6A8 are linked to a human hereditary disease.

Scientific Focus Area: Molecular Biology and Biochemistry

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