The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent

Drosophila Boi limits Hedgehog levels to suppress follicle stem cell proliferation

The Boi receptor regulates stem cell function by sequestering the diffusible hedgehog ligand

STONEWALL PREVENTS EXPRESSION OF TESTIS-ENRICHED GENES AND BINDS TO INSULATOR ELEMENTS IN D. MELANOGASTER

Germline stem cells (GSCs) are the progenitor cells for the entire population of an organism’s germline. In Drosophila, these cells reside in a well-defined cellular niche that is required for both their maintenance (self-renewal) and differentiation (asymmetric division resulting in a daughter cell that differs from the GSC). The stem-cell maintenance factor Stonewall (Stwl) has undergone adaptive evolution across the Drosophila phylogeny and has been implicated in heterochromatin maintenance. In the hopes of identifying potential drivers of selection at Stwl, we investigated the molecular functions of Stwl. We performed RNA-Seq on stwl mutant ovaries and testes to assay the transcript abundance of transposable elements in the absence of functional Stwl. We found that stwl mutant ovaries (but not testes) show significant de-repression of many transposon families, but that heterochromatic genes are not preferentially misregulated relative to euchromatic genes. We also found that testis-enriched genes, including the differentiation factor bgcn and a large cluster on chromosome 2, are upregulated in stwl mutant ovaries. This abnormal masculinization of the ovary was accompanied by ectopic expression of a number of testis- and somatic-enriched genes. Surprisingly, we also found that RNAi knockdown of stwl in S2 cells results in ectopic expression of these gene classes. In order to understand how Stwl regulates transcription, we developed antibodies against a Stwl epitope and thoroughly validated it prior to performing parallel ChIP-Seq and RNA-Seq experiments in S2 cells. Analysis of Stwl binding sites shows that Stwl binds upstream of transcription start sites and localizes to heterochromatic sequences. We also show that Stwl is enriched at repetitive sequences associated with telomeres. Finally, we identify binding motifs that are shared with known insulator binding proteins. We propose that Stwl affects gene regulation by binding insulators and establishing chromatin boundaries

Sequential events during the quiescence to proliferation transition establish patterns of follicle cell differentiation in the Drosophila ovary

Stem cells cycle between periods of quiescence and proliferation to promote tissue health. In Drosophila ovaries, quiescence to proliferation transitions of follicle stem cells (FSCs) are exquisitely feeding-dependent. Here, we demonstrate feeding-dependent induction of follicle cell differentiation markers, eyes absent (Eya) and castor (Cas) in FSCs, a patterning process that does not depend on proliferation induction. Instead, FSCs extend micron-scale cytoplasmic projections that dictate Eya-Cas patterning. We identify still life and sickie as necessary and sufficient for FSC projection growth and Eya-Cas induction. Our results suggest that sequential, interdependent events establish long-term differentiation patterns in follicle cell precursors, independently of FSC proliferation induction

Inhibition of Eukaryotic Translation by the Antitumor Natural Product Agelastatin A

International audienceProtein synthesis plays an essential role in cell proliferation, differentiation, and survival. Inhibitors of eukaryotic translation have entered the clinic, establishing the translation machinery as a promising target for chemotherapy. A recently discovered, structurally unique marine sponge-derived brominated alkaloid, (-)-agelastatin A (AglA), possesses potent antitumor activity. Its underlying mechanism of action, however, has remained unknown. Using a systematic top-down approach, we show that AglA selectively inhibits protein synthesis. Using a high-throughput chemical footprinting method, we mapped the AglA-binding site to the ribosomal A site. A 3.5 Å crystal structure of the 80S eukaryotic ribosome from S. cerevisiae in complex with AglA was obtained, revealing multiple conformational changes of the nucleotide bases in the ribosome accompanying the binding of AglA. Together, these results have unraveled the mechanism of inhibition of eukaryotic translation by AglA at atomic level, paving the way for future structural modifications to develop AglA analogs into novel anticancer agents

Synonymous codons, ribosome speed, and eukaryotic gene expression regulation

Quantitative control of gene expression occurs at multiple levels, including the level of translation. Within the overall process of translation, most identified regulatory processes impinge on the initiation phase. However, recent studies have revealed that the elongation phase can also regulate translation if elongation and initiation occur with specific, not mutually compatible rate parameters. Translation elongation then limits the overall amount of protein that can be made from an mRNA. Several recently discovered control mechanisms of biological pathways are based on such elongation control. Here, we review the molecular mechanisms that determine ribosome speed in eukaryotic organisms, and discuss under which conditions ribosome speed can become the controlling parameter of gene expression levels

Non-coding RNA networks in cancer

Thousands of unique non-coding RNA (ncRNA) sequences exist within cells. Work from the past decade has altered our perception of ncRNAs from 'junk' transcriptional products to functional regulatory molecules that mediate cellular processes including chromatin remodelling, transcription, post-transcriptional modifications and signal transduction. The networks in which ncRNAs engage can influence numerous molecular targets to drive specific cell biological responses and fates. Consequently, ncRNAs act as key regulators of physiological programmes in developmental and disease contexts. Particularly relevant in cancer, ncRNAs have been identified as oncogenic drivers and tumour suppressors in every major cancer type. Thus, a deeper understanding of the complex networks of interactions that ncRNAs coordinate would provide a unique opportunity to design better therapeutic interventions