Biology subjects

Powers, E. N.

Publications and source records attributed to Powers, E. N..

4 recordsLinked to original sources

bioRxiv2024

Dbp1 is a low performance paralog of RNA helicase Ded1 that drives impaired translation and heat stress response

Ded1 and Dbp1 are paralogous conserved RNA helicases that enable translation initiation in yeast. Ded1 has been heavily studied but the role of Dbp1 is poorly understood. We find that the expression of these two helicases is controlled in an inverse and condition-specific manner. In meiosis and other long-term starvation states, Dbp1 expression is upregulated and Ded1 is downregulated, whereas in mitotic cells, Dbp1 expression is extremely low. Inserting the DBP1 ORF in place of the DED1 ORF cannot replace the function of Ded1 in supporting translation, partly due to inefficient mitotic translation of the DBP1 mRNA, dependent on features of its ORF sequence but independent of codon optimality. Global measurements of translation rates and 5 leader translation, activity of mRNA-tethered helicases, ribosome association, and low temperature growth assays show that--even at matched protein levels--Ded1 is more effective than Dbp1 at activating translation, especially for mRNAs with structured 5 leaders. Ded1 supports halting of translation and cell growth in response to heat stress, but Dbp1 lacks this function, as well. These functional differences in the ability to efficiently mediate translation activation and braking can be ascribed to the divergent, disordered N- and C-terminal regions of these two helicases. Altogether, our data show that Dbp1 is a "low performance" version of Ded1 that cells employ in place of Ded1 under long-term conditions of nutrient deficiency.

genomics↗

bioRxiv2022

Functional promiscuity of small multidrug resistance transporters from Staphylococcus aureus, Pseudomonas aeruginosa, and Francisella tularensis

Small multidrug resistance (SMR) transporters efflux toxic substrates from bacterial cells and were recently divided into two subfamilies: specific toxic metabolite transporters and promiscuous drug exporters. These drug exporters are thought to function similarly to EmrE, the model system for this subfamily of SMR transporters. Studies of EmrE homologs indicate that they are able to confer resistance to EmrE substrates in E. coli and in their native organisms. Recent work from our lab showed that functional EmrE can confer resistance or susceptibility in vivo depending on the drug substrate. Here, we test whether this functional promiscuity of EmrE extends to SMR transporters from three additional human or animal pathogens: SAsmr from Staphylococcus aureus, PAsmr from Pseudomonas aeruginosa, and FTsmr from Francisella tularensis. We find that these SMR homologs can confer either resistance or susceptibility to different toxic substrates in E. coli. This demonstrates that the ability of a single transporter to lead to opposite biological outcomes when transporting different substrates is a general property of the promiscuous multidrug transporters in the SMR family. It also suggests the potential for novel antibiotic development targeting these transporters with small molecules that trigger susceptibility. Such a strategy does not require that the target be the primary mode for antibiotic resistance because the goal is not simple inhibition of activity, but rather activation of an alternative transport function that is detrimental to bacteria.

microbiology↗

bioRxiv2022

Use of a ubiquitous gene-editing tool in budding yeast causes off-target repression of neighboring gene protein synthesis

Precision genome-editing approaches have long been available in budding yeast, enabling introduction of gene deletions, epitope tag fusions, and promoter swaps through a selection-based strategy. Such approaches allow loci to be modified without disruption of coding or regulatory sequences of neighboring genes. Use of this approach to delete DBP1 however, led to silencing of expression and the resultant loss of function for the neighboring gene MRP51. We found that insertion of a resistance cassette to delete DBP1, drove a 5 extended alternative transcript for MRP51 which dampened Mrp51 protein synthesis. Misregulation of MRP51 occurred through an integrated transcriptional and translational repressive long undecoded transcript isoform (LUTI)-based mechanism that was recently shown to naturally regulate gene expression in yeast and other organisms. Cassette-induced MRP51 repression drove all mutant phenotypes we detected in cells deleted for DBP1. Selection cassette-mediated aberrant transcription events are not specific to this locus or a unique cassette but can be prevented by insertion of transcription insulators flanking the cassette. Our study suggests the existence of confounding off-target mutant phenotypes resulting from misregulated neighboring loci following genome edits in yeast. Furthermore, features of LUTI-based regulation are broadly conserved to eukaryotic organisms which indicates the potential that similar misregulation could be unnoticed in other edited organisms as well.

genetics↗

bioRxiv2019

Aurora B-dependent Ndc80 Degradation Regulates Kinetochore Composition in Meiosis

The kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We have previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule-kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N-terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments.

genetics↗