GNE deficiency impairs Myogenesis in C2C12 cells and cannot be rescued by ManNAc supplementation.

GNE myopathy (GNEM) is a late-onset muscle atrophy, caused by mutations in the gene for the key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). With an incidence of one to nine cases per million it is an ultra-rare, so far untreatable, autosomal recessive disease. Several attempts have been made to treat GNEM patients by oral supplementation with sialic acid precursors (e.g. N-acetylmannosamine, ManNAc) to restore sarcolemmal sialylation and muscle strength. In most studies, however, no significant improvement was observed. The lack of a suitable mouse model makes it difficult to understand the exact pathomechanism of GNEM and many years of research have failed to identify the role of GNE in skeletal muscle due to the lack of appropriate tools. We established a CRISPR/Cas9-mediated Gne-knockout cell line using murine C2C12 cells to gain insight into the actual role of the GNE enzyme and sialylation in a muscular context. The main aspect of this study was to evaluate the therapeutic potential of ManNAc and N-acetylneuraminic acid (Neu5Ac). Treatment of Gne-deficient C2C12 cells with Neu5Ac, but not with ManNAc, showed a restoration of the sialylation level back to wild type levels-albeit only with long-term treatment, which could explain the rather low therapeutic potential. We furthermore highlight the importance of sialic acids on myogenesis, for C2C12 Gne-knockout myoblasts lack the ability to differentiate into mature myotubes.

Glycation Interferes with the Activity of the Bi-Functional UDP-N-Acetylglucosamine 2-Epimerase/N-Acetyl-mannosamine Kinase (GNE).

Mutations in the gene coding for the bi-functional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of the sialic acid biosynthesis, are responsible for autosomal-recessive GNE myopathy (GNEM). GNEM is an adult-onset disease with a yet unknown exact pathophysiology. Since the protein appears to work adequately for a certain period of time even though the mutation is already present, other effects appear to influence the onset and progression of the disease. In this study, we want to investigate whether the late onset of GNEM is based on an age-related effect, e.g., the accumulation of post-translational modifications (PTMs). Furthermore, we also want to investigate what effect on the enzyme activity such an accumulation would have. We will particularly focus on glycation, which is a PTM through non-enzymatic reactions between the carbonyl groups (e.g., of methylglyoxal (MGO) or glyoxal (GO)) with amino groups of proteins or other biomolecules. It is already known that the levels of both MGO and GO increase with age. For our investigations, we express each domain of the GNE separately, treat them with one of the glycation agents, and determine their activity. We demonstrate that the enzymatic activity of the N-acetylmannosamine kinase (GNE-kinase domain) decreases dramatically after glycation with MGO or GO-with a remaining activity of 13% ± 5% (5 mM MGO) and 22% ± 4% (5 mM GO). Whereas the activity of the UDP-N-acetylglucosamine 2-epimerase (GNE-epimerase domain) is only slightly reduced after glycation-with a remaining activity of 60% ± 8% (5 mM MGO) and 63% ± 5% (5 mM GO).

Post-Transcriptional Expression Control in Platelet Biogenesis and Function.

Platelets are highly abundant cell fragments of the peripheral blood that originate from megakaryocytes. Beside their well-known role in wound healing and hemostasis, they are emerging mediators of the immune response and implicated in a variety of pathophysiological conditions including cancer. Despite their anucleate nature, they harbor a diverse set of RNAs, which are subject to an active sorting mechanism from megakaryocytes into proplatelets and affect platelet biogenesis and function. However, sorting mechanisms are poorly understood, but RNA-binding proteins (RBPs) have been suggested to play a crucial role. Moreover, RBPs may regulate RNA translation and decay following platelet activation. In concert with other regulators, including microRNAs, long non-coding and circular RNAs, RBPs control multiple steps of the platelet life cycle. In this review, we will highlight the different RNA species within platelets and their impact on megakaryopoiesis, platelet biogenesis and platelet function. Additionally, we will focus on the currently known concepts of post-transcriptional control mechanisms important for RNA fate within platelets with a special emphasis on RBPs.

LINC00261 Is Differentially Expressed in Pancreatic Cancer Subtypes and Regulates a Pro-Epithelial Cell Identity.

Pancreatic adenocarcinoma (PDAC) is one of the major causes of cancer-associated deaths worldwide, with a dismal prognosis that has not significantly changed over the last decades. Transcriptional analysis has provided valuable insights into pancreatic tumorigenesis. Specifically, pancreatic cancer subtypes were identified, characterized by specific mutations and gene expression changes associated with differences in patient survival. In addition to differentially regulated mRNAs, non-coding RNAs, including long non-coding RNAs (lncRNAs), were shown to have subtype-specific expression patterns. Hence, we aimed to characterize prognostic lncRNAs with deregulated expression in the squamous subtype of PDAC, which has the worst prognosis. Extensive in silico analyses followed by in vitro experiments identified long intergenic non-coding RNA 261 (LINC00261) as a downregulated lncRNA in the squamous subtype of PDAC, which is generally associated with transforming growth factor ß (TGFß) signaling in human cancer cells. Its genomic neighbor, the transcription factor forkhead box protein A2 (FOXA2), regulated LINC00261 expression by direct binding of the LINC00261 promoter. CRISPR-mediated knockdown and promoter knockout validated the importance of LINC00261 in TGFß-mediated epithelial-mesenchymal transition (EMT) and established the epithelial marker E-cadherin, an important cell adhesion protein, as a downstream target of LINC00261. Consequently, depletion of LINC00261 enhanced motility and invasiveness of PANC-1 cells in vitro. Altogether, our data suggest that LINC00261 is an important tumor-suppressive lncRNA in PDAC that is involved in maintaining a pro-epithelial state associated with favorable disease outcome.