A lncRNA-regulated gene expression system with rapid induction kinetics in the fission yeast Schizosaccharomyces pombe

Abstract

The fission yeast Schizosaccharomyces pombe is an excellent model organism for the study of eukaryotic cellular physiology. The organism is genetically tractable and several tools to study the functions of individual genes are available. One such tool is regulatable gene expression and overproduction of proteins. Limitations of currently available overexpression systems include delay in expression after induction, narrow dynamic range, and system-wide changes due to induction conditions. Here I describe a new long noncoding RNA (lncRNA)-regulated, thiamine-inducible expression system that integrates lncRNA-based transcriptional interference at the fission yeast tgp1 promoter with the fast repression kinetics of the thiamine-repressible nmt1 promoter. This hybrid system has rapid induction kinetics, broad dynamic range, and tunable expression via thiamine concentration. The lncRNA-regulated thiamine-inducible system will be advantageous for the study of individual genes and for potential applications in the production of heterologous proteins in fission yeast.

Activities and Structure-Function Analysis of Fission Yeast Inositol Pyrophosphate (IPP) Kinase-Pyrophosphatase Asp1 and Its Impact on Regulation of pho1 Gene Expression

“…To gauge the impact of Asp1 kinase mutations in vivo , we established a Pho1 activity-based reporter system in which pTIN plasmids expressing wild-type or mutated versions of Asp1-(1-385) were introduced into asp1 Δ cells. The pTIN expression vector ( 24 ) places the Asp1-(1-385) open reading frame under the transcriptional control of the tgp1 promoter, which is situated adjacent to the transcription unit specifying the nc-tgp1 lncRNA driven by the thiamine-repressible nmt1 promoter. In the absence of thiamine, lncRNA transcription interferes with firing of the tgp1 promoter.…”

“…In the absence of thiamine, lncRNA transcription interferes with firing of the tgp1 promoter. In the presence of thiamine, lncRNA synthesis is turned off and expression of the downstream mRNA—encoding Asp1-(1-385) in this case—is turned on ( 24 ).…”

“…pTIN plasmids ( 24 ) encoding the Asp1 kinase domain (aa 1–385) or alanine mutants thereof were transfected by the lithium acetate method into S. pombe asp1 Δ cells. Control transfection was performed with the empty pTIN vector.…”

An improved tetracycline-inducible expression system for fission yeast

“…Over the past 35 years, a large number of regulatable promoter systems have been developed to enable the manipulation of gene expression in the fission yeast Schizosaccharomyces pombe . These systems include glucose-repressible promoters (Hoffman and Winston, 1989; Iacovoni et al, 1999), hormone-inducible promoters (Ohira et al, 2017; Picard et al, 1990), thiamine-repressible promoters and their derivatives (Basi et al, 1993; Garg, 2020; Kjaerulff and Nielsen, 2015; Kumar and Singh, 2006; Maundrell, 1993, 1990; Moreno et al, 2000), tetracycline-inducible promoters (Erler et al, 2006; Faryar and Gatz, 1992; Patterson et al, 2019; Zilio et al, 2012), a copper-repressible promoter (Bellemare et al, 2001), a heat shock-inducible promoter (Fujita et al, 2006), a uracil- inducible promoter (Watson et al, 2011; Watt et al, 2008), an ethanol-inducible promoter (Matsuzawa et al, 2013), and pheromone-inducible promoters (Hennig et al, 2018).…”