RESUMO
To better understand the gene regulatory mechanisms that program developmental processes, we carried out simultaneous genome-wide measurements of mRNA, translation, and protein through meiotic differentiation in budding yeast. Surprisingly, we observed that the levels of several hundred mRNAs are anti-correlated with their corresponding protein products. We show that rather than arising from canonical forms of gene regulatory control, the regulation of at least 380 such cases, or over 8% of all measured genes, involves temporally regulated switching between production of a canonical, translatable transcript and a 5' extended isoform that is not efficiently translated into protein. By this pervasive mechanism for the modulation of protein levels through a natural developmental program, a single transcription factor can coordinately activate and repress protein synthesis for distinct sets of genes. The distinction is not based on whether or not an mRNA is induced but rather on the type of transcript produced.
Assuntos
Meiose/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Modelos Biológicos , Anotação de Sequência Molecular , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismoRESUMO
While m6A modification of mRNAs is now known to be widespread, the cellular roles of this modification remain largely mysterious. In this issue of Molecular Cell, Zhou et al. (2018) show that m6A modification unexpectedly contributes to the established uORF- and eIF2α-â-dependent mechanism of ATF4 translational regulation in response to stress.
Assuntos
Fator de Iniciação 2 em Eucariotos , RNA MensageiroRESUMO
Targeted selection-based genome-editing approaches have enabled many fundamental discoveries and are used routinely with high precision. We found, however, that replacement of DBP1 with a common selection cassette in budding yeast led to reduced expression and function for the adjacent gene, MRP51, despite all MRP51 coding and regulatory sequences remaining intact. Cassette-induced repression of MRP51 drove all mutant phenotypes detected in cells deleted for DBP1. This behavior resembled the 'neighboring gene effect' (NGE), a phenomenon of unknown mechanism whereby cassette insertion at one locus reduces the expression of a neighboring gene. Here, we leveraged strong off-target mutant phenotypes resulting from cassette replacement of DBP1 to provide mechanistic insight into the NGE. We found that the inherent bidirectionality of promoters, including those in expression cassettes, drives a divergent transcript that represses MRP51 through combined transcriptional interference and translational repression mediated by production of a long undecoded transcript isoform (LUTI). Divergent transcript production driving this off-target effect is general to yeast expression cassettes and occurs ubiquitously with insertion. Despite this, off-target effects are often naturally prevented by local sequence features, such as those that terminate divergent transcripts between the site of cassette insertion and the neighboring gene. Thus, cassette-induced off-target effects can be eliminated by the insertion of transcription terminator sequences into the cassette, flanking the promoter. Because the driving features of this off-target effect are broadly conserved, our study suggests it should be considered in the design and interpretation of experiments using integrated expression cassettes in other eukaryotic systems, including human cells.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Helicases DEAD-box/metabolismoRESUMO
Ribosome profiling, first developed in 2009, is the gold standard for quantifying and qualifying changes to translation genome-wide (Ingolia et al., Science, 2009). Though first designed and optimized in vegetative budding yeast, it has since been modified and specialized for use in diverse cellular states in yeast, as well as in bacteria, plants, human cells, and many other organisms (Ingolia et al. Science, 2009, reviewed in (Ingolia et al., Cold Spring Harb Perspect Biol, 2019; Brar and Weissman, Nat Rev Mol Cell Biol, 2015)). Here we report the current ribosome profiling protocol used in our lab to study genome-wide changes to translation in budding yeast undergoing the developmental process of meiosis (Brar et al., Science, 2012; Cheng et al., Cell, 2018). We describe this protocol in detail, including the following steps: collection and flash freezing samples, cell lysis and extract preparation, sucrose gradient centrifugation and monosome collection, RNA extraction, library preparation, and library quality control. Almost every step presented here should be directly applicable to performing ribosome profiling in other eukaryotic cell types or cell states.