Dysregulation of cellular ribose uptake can be indicative of metabolic abnormalities or tumorigenesis. However, analytical methods are currently limited for quantifying ribose concentration in ...complex biological samples. Here, we utilize the highly specific recognition of ribose by ribose-binding protein (RBP) to develop a single-protein ribose sensor detectable
a sensitive NMR technique known as hyperpolarized
Xe chemical exchange saturation transfer (hyper-CEST). We demonstrate that RBP, with a tunable ribose-binding site and further engineered to bind xenon, enables the quantitation of ribose over a wide concentration range (nM to mM). Ribose binding induces the RBP "closed" conformation, which slows Xe exchange to a rate detectable by hyper-CEST. Such detection is remarkably specific for ribose, with the minimal background signal from endogenous sugars of similar size and structure, for example, glucose or ribose-6-phosphate. Ribose concentration was measured for mammalian cell lysate and serum, which led to estimates of low-mM ribose in a HeLa cell line. This highlights the potential for using genetically encoded periplasmic binding proteins such as RBP to measure metabolites in different biological fluids, tissues, and physiologic states.
Dysregulation of cellular ribose uptake can be indicative of metabolic abnormalities or tumorigenesis. However, analytical methods are currently limited for quantifying ribose concentration in ...complex biological samples. Here, we utilize the highly specific recognition of ribose by ribose-binding protein (RBP) to develop a single-protein ribose sensor detectable via a sensitive NMR technique known as hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST). We demonstrate that RBP, with a tunable ribose-binding site and further engineered to bind xenon, enables the quantitation of ribose over a wide concentration range (nM to mM). Ribose binding induces the RBP “closed” conformation, which slows Xe exchange to a rate detectable by hyper-CEST. Such detection is remarkably specific for ribose, with the minimal background signal from endogenous sugars of similar size and structure, for example, glucose or ribose-6-phosphate. Ribose concentration was measured for mammalian cell lysate and serum, which led to estimates of low-mM ribose in a HeLa cell line. This highlights the potential for using genetically encoded periplasmic binding proteins such as RBP to measure metabolites in different biological fluids, tissues, and physiologic states.
Expansions of CAG trinucleotide repeats cause several rare neurodegenerative diseases. The disease-causing repeats are translated in multiple reading frames and without an identifiable initiation ...codon. The molecular mechanism of this repeat-associated non-AUG (RAN) translation is not known. We find that expanded CAG repeats create new splice acceptor sites. Splicing of proximal donors to the repeats produces unexpected repeat-containing transcripts. Upon splicing, depending on the sequences surrounding the donor, CAG repeats may become embedded in AUG-initiated open reading frames. Canonical AUG-initiated translation of these aberrant RNAs may account for proteins that have been attributed to RAN translation. Disruption of the relevant splice donors or the in-frame AUG initiation codons is sufficient to abrogate RAN translation. Our findings provide a molecular explanation for the abnormal translation products observed in CAG trinucleotide repeat expansion disorders and add to the repertoire of mechanisms by which repeat expansion mutations disrupt cellular functions.
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•Expanded CAG repeats act as splice acceptor sites•Splicing frequency increases with the number of CAG repeats•Splicing may place CAG repeats in RNA in unexpected sequence contexts•AUG-initiated translation of mis-spliced RNAs can account for aberrant proteins
Anderson et al. show that expanded CAG trinucleotide repeats produce 3′ splice sites. Proximal donors splice into the repeats in a repeat-length-dependent manner generating unexpected repeat-containing mRNAs. This splicing may place repeats in AUG-initiated frames, potentially explaining aberrant, out-of-frame protein products.
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