Esteban-Serna, S. | bioRxiv Science

bioRxiv2025

A simple, reversible and non-toxic anchor-away system for effective nuclear depletion of proteins

The anchor-away (AA) technique enables rapid depletion of nuclear proteins by tethering them to cytoplasmic anchors through rapamycin-induced heterodimerisation. Albeit powerful, in Saccharomyces cerevisiae, this system is restricted to rapamycin-resistant strains, as the drug inhibits TOR signalling and hinders the heat-shock response, limiting its application to stress-related studies. Moreover, this AA method is not fully reversible, limiting studies of dynamic cellular processes that require transient perturbation and functional restoration. To overcome these constraints, we developed an alternative AA system that uses the plant hormone abscisic acid (ABA) to induce conditional association of the target to its cytoplasmic anchor. The ABA-AA system enables efficient and fully reversible depletion of highly abundant nuclear proteins. Unlike rapamycin, ABA does not cause major gene expression changes and is suitable for diverse genetic backgrounds. The ABA-AA system provides a fully reversible, non-toxic, and broadly applicable alternative for nuclear protein depletion across eukaryotic systems.

molecular biology↗

bioRxiv2024

A co-transcriptional mechanism for tightly controlling RNA homeostasis in yeast

Transcription termination by the Nrd1-Nab3-Sen1 (NNS) complex is key in repressing pervasive transcription in Saccharomyces cerevisiae. Counterintuitively, during starvation, multiple mRNAs that are upregulated are also increasingly bound and prematurely terminated and degraded via NNS. Here we demonstrate that this NNS-mediated attenuation is important for controlling the expression and protein concentration of an evolutionarily conserved mitochondrial transporter, Pic2. Strikingly, we find that even a modest increase in Pic2 protein levels caused by defective NNS regulation has major phenotypical consequences, increasing cell volume and intracellular stress, prolonging cell cycle and decreasing growth rate. Disrupting Nab3 binding to PIC2 globally redistributed Nrd1 binding, changing the levels of other NNS-regulated transcripts. We propose that imbalances in the availability of the subunits constituting the NNS complex underlie the cell volume and cycle anomalies. Collectively our results illustrate that even subtle changes in how RNA-binding proteins interact with a single RNA substrate can cause global defects and they emphasise the crucial role of the NNS complex in preserving microbial fitness during stress. HighlightsO_LINNS regulates the expression and protein concentration of a stress-response protein-coding gene (PIC2), improving cell fitness and adaptability to environmental challenges. C_LIO_LICreating an imbalance in RNA binding of Nab3 and Nrd1 for PIC2 mRNA disturbs the homeostasis of co-regulated transcripts. C_LIO_LIEven a modest defect in NNS regulation of PIC2 elicits severe defects in cell growth, increases cell size and intracellular stress, and prolongs the cell cycle. C_LI

molecular biology↗