Essential gene

Abstract: N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site, and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia.

Abstract: To enhance the research capabilities of investigators interested in Staphylococcus aureus, the Nebraska Center for Staphylococcal Research (CSR) has generated a sequence-defined transposon mutant library consisting of 1,952 strains, each containing a single mutation within a nonessential gene of the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) isolate USA300. To demonstrate the utility of this library for large-scale screening of phenotypic alterations, we spotted the library on indicator plates to assess hemolytic potential, protease production, pigmentation, and mannitol utilization. As expected, we identified many genes known to function in these processes, thus validating the utility of this approach. Importantly, we also identified genes not previously associated with these phenotypes. In total, 71 mutants displayed differential hemolysis activities, the majority of which were not previously known to influence hemolysin production. Furthermore, 62 mutants were defective in protease activity, with only 14 previously demonstrated to be involved in the production of extracellular proteases. In addition, 38 mutations affected pigment formation, while only 7 influenced mannitol fermentation, underscoring the sensitivity of this approach to identify rare phenotypes. Finally, 579 open reading frames were not interrupted by a transposon, thus providing potentially new essential gene targets for subsequent antibacterial discovery. Overall, the Nebraska Transposon Mutant Library represents a valuable new resource for the research community that should greatly enhance investigations of this important human pathogen. IMPORTANCE Infections caused by Staphylococcus aureus cause significant morbidity and mortality in both community and hospital environments. Specific-allelic-replacement mutants are required to study the biology of this organism; however, this process is costly and time-consuming. We describe the construction and validation of a sequence-defined transposon mutant library available for use by the scientific community through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) strain repository. In addition, complementary resources, including a website (http://app1.unmc.edu/fgx/) and genetic tools that expedite the allelic replacement of the transposon in the mutants with useful selectable markers and fluorescent reporter fusions, have been generated. Overall, this library and associated tools will have a significant impact on studies investigating S. aureus pathogenesis and biology and serve as a useful paradigm for the study of other bacterial systems.

Abstract: We completed a large insertional mutagenesis screen in zebrafish to identify genes essential for embryonic and early larval development. We isolated 525 mutants, representing lesions in ≈390 different genes, and we cloned the majority of these. Here we describe 315 mutants and the corresponding genes. Our data suggest that there are roughly 1,400 embryonic-essential genes in the fish. Thus, we have mutations in ≈25% of these genes and have cloned ≈22% of them. Re-screens of our collection to identify mutants with specific developmental defects suggest that ≈50 genes are essential for the development of some individual organs or cell types. Seventy-two percent of the embryonic-essential fish genes have homologues in yeast, 93% have homologues in invertebrates (fly or worm), and 99% have homologues in human. Yeast and worm orthologues of genes that are essential for early zebrafish development have a strong tendency to be essential for viability in yeast and for embryonic development in the worm. Thus, the trait of being a genetically essential gene is conserved in evolution. This mutant collection should be a valuable resource for diverse studies of cell and developmental biology.