The 5ʹ-UTR of the actin-related protein 2/3 complex subunit 2 (ARPC2) mRNA exists in two variants. Using a bicistronic reporter construct, the present study demonstrates that the longer variant of the 5ʹ-UTR harbours an internal ribosome entry site (IRES) which is lacking in the shorter one. Multiple control assays confirmed that only this variant promotes cap-independent translation. Furthermore, it includes a guanine-rich region that is capable of forming a guanine-quadruplex (G-quadruplex) structure which was found to contribute to the IRES activity. To investigate the cellular function of the IRES element, we determined the expression level of ARPC2 at various cell densities. At high cell density, the relative ARPC2 protein level increases, supporting the presumed function of IRES elements in driving the expression of certain genes under stressful conditions that compromise cap-dependent translation. Based on chemical probing experiments and computer-based predictions, we propose a structural model of the IRES element, which includes the G-quadruplex motif exposed from the central stemloop element. Taken together, our study describes the functional relevance of two alternative 5ʹ-UTR splice variants of the ARPC2 mRNA, one of which contains an IRES element with a G-quadruplex as a central motif, promoting translation under stressful cellular conditions ARTICLE HISTORY
UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase (GNE) is the key enzyme for the biosynthesis of sialic acids. Sialic acids are terminal monosaccharides of glycoconjugates and gangliosides, which have an essential influence on various cell interactions. The sialylation of proteins varies during development, aging, and pathogenesis of degenerative diseases such as Morbus Alzheimer, diabetes mellitus type II, or myopathies. Mutation of methionine 743 in the GNE leads to a 30% reduction of the enzyme activity and is responsible for an aggressive form of GNE myopathy. GNE myopathy or hereditary inclusion body myopathy (HIBM) is an age‐dependent muscular dystrophy. Here, we analyzed the impact of the exchange of methionine to threonine at position 743 which introduces an additional potential phosphorylation/O‐GlcNAcylation site. We found increased O‐GlcNAcylation of the M743T variant compared to the wild‐type GNE. In addition, removal of the O‐GlcNAc of the M743T variant resulted in an increased activity comparable to activity of the wild‐type GNE. Furthermore, the half‐life of the M743T variant is two times longer than for the wild‐type GNE protein. This study provides that the balance of phosphorylation and O‐GlcNAcylation is decisive involved in efficiency and regulation of GNE.
DeniseK reuzmann, [a] RüdigerH orstkorte,* [a] GuidoK ohla, [b] Christoph Kannicht, [b] Dorit Bennmann, [a] AnnettT hate, [a] and Kaya Bork [a] This work is dedicated to Werner Reutter,apioneer in thebiosynthesis of sialic acids, who first purifiedand cloned GNE in 1997.
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