Elucidation of novel biosynthetic pathways and metabolite flux patterns by retrobiosynthetic NMR analysis

The labelling patterns of metabolites from experiments with stable isotope-labelled precursors can be determined by NMR spectroscopy. Complex isotopomer mixtures are found when general metabolites such as glucose are used as stable isotope-labelled precursors which are diverted to all branches of intermediary metabolism. The complex results can be interpreted by a pattern recognition approach based on comparison between the labelling patterns of secondary metabolites and primary metabolites such as amino acids and ribonucleosides. The isotope labelling patterns of intermediates in central metabolic pools such as carbohydrate phosphates, dicarboxylic acids, and acetyl CoA can be obtained by biosynthetic retroanalysis. Biosynthetic pathways as well as metabolite flux patterns can be determined from these data. The method is illustrated using the classical mevalonate pathway and the more recently discovered deoxyxylulose pathway of terpenoid biosynthesis as examples. Applications of the retrobiosynthetic method of the biosynthesis of molybdopterin and of riboflavin are also discussed. Stable isotope experiments monitored by NMR spectroscopy have also been shown to be a powerful tool for the elucidation of metabolic flux in microorganisms with unusual lifestyles and in fermentation processe

Organization of chromatin and histone modifications at a transcription site

According to the transcription factory model, localized transcription sites composed of immobilized polymerase molecules transcribe chromatin by reeling it through the transcription site and extruding it to form a surrounding domain of recently transcribed decondensed chromatin. Although transcription sites have been identified in various cells, surrounding domains of recently transcribed decondensed chromatin have not. We report evidence that transcription sites associated with a tandem gene array in mouse cells are indeed surrounded by or adjacent to a domain of decondensed chromatin composed of sequences from the gene array. Formation of this decondensed domain requires transcription and topoisomerase IIα activity. The decondensed domain is enriched for the trimethyl H3K36 mark that is associated with recently transcribed chromatin in yeast and several mammalian systems. Consistent with this, chromatin immunoprecipitation demonstrates a comparable enrichment of this mark in transcribed sequences at the tandem gene array. These results provide new support for the pol II factory model, in which an immobilized polymerase molecule extrudes decondensed, transcribed sequences into its surroundings

Driver mutations in <i>GNAQ</i> and <i>GNA11</i> genes as potential targets for precision immunotherapy in uveal melanoma patients

Uveal melanoma (UM) is the most common ocular malignancy in adults. Nearly 95% of UM patients carry the mutually exclusive mutations in the homologous genes GNAQ (amino acid change Q209L/Q209P) and GNA11 (aminoacid change Q209L). UM is located in an immunosuppressed organ and does not suffer immunoediting. Therefore, we hypothesize that driver mutations in GNAQ/11 genes could be recognized by the immune system. Genomic and transcriptomic data from primary uveal tumors were collected from the TCGA-UM dataset (n = 80) and used to assess the immunogenic potential for GNAQ/GNA11 Q209L/Q209P mutations using a variety of tools and HLA type information. All prediction tools showed stronger GNAQ/11 Q209L binding to HLA than GNAQ/11 Q209P. The immunogenicity analysis revealed that Q209L is likely to be presented by more than 73% of individuals in 1000 G databases whereas Q209P is only predicted to be presented in 24% of individuals. GNAQ/11 Q209L showed a higher likelihood to be presented by HLA-I molecules than almost all driver mutations analyzed. Finally, samples carrying Q209L had a higher immune-reactive phenotype. Regarding cancer risk, seven HLA genotypes with low Q209L affinity show higher frequency in uveal melanoma patients than in the general population. However, no clear association was found between any HLA genotype and survival. Results suggest a high potential immunogenicity of the GNAQ/11 Q209L variant that could allow the generation of novel therapeutic tools to treat UM like neoantigen vaccinations

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Methylated cytosines in total poly(A) ESC RNA. (XLSX 761 kb

Arxes: retrotransposed genes required for adipogenesis

Retrotransposed sequences arise from messenger RNAs (mRNAs) that have been reinserted into genomic DNA by reverse transcription. Usually, these sequences are embedded in dormant regions, collect missense mutations over time and constitute processed, nonfunctional pseudogenes. There are thousands of processed pseudogenes in the mouse and human genome. Here, we report evidence for two paralog genes (termed Arxes1 and Arxes2), which arose by retrotransposition of the signal peptidase Spcs3 followed by a segmental duplication event. They gained a functional promoter that we show to be transactivated by adipogenic transcription factors. We further show that the Arxes mRNAs are highly expressed in adipose tissue and strongly upregulated during adipogenesis in different cell models. Additionally, their expression is elevated by an anti-diabetic agent in vitro and in vivo. Importantly, we provide evidence that the Arxes genes are translated and that the proteins are located in the endoplasmic reticulum. Although the sequence similarity and subcellular location are reminiscent of their parental gene, our data suggest that the Arxes have developed a different function, since their expression is required for adipogenesis, whereas Spcs3 is dispensable. In summary, we report retrotransposed-duplicated genes that evolved from a parental gene to function in a tissue and adipogenesis-specific context

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.</p

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.</p