Co-transcriptional mechanism for tightly controlling RNA homeostasis in yeast

Abstract

Transcription termination by the Nrd1-Nab3-Sen1 (NNS) complex plays a pivotal role in repressing pervasive transcription in Saccharomyces cerevisiae. Intriguingly, many upregulated protein-coding RNAs are also increasingly bound by the NNS complex during starvation. This implies that a subset of mRNAs encoding stressresponsive proteins are targeted for degradation shortly after transcription initiation. However, the biological significance of this observation has hitherto remained unclear. Premature termination of stress-responsive mRNAs has been proposed as a putative beneficial cellular mechanism to keep the expression of such genes low and tightly regulated during nutrient deprivation. To test this, I focused on the effect that NNS regulation exerted on one of its stress-specific targets, PIC2, which encodes a mitochondrial phosphate and copper importer.

Using strains lacking Nab3 and Nrd1 RNA-binding sites in PIC2 RNA, I have demonstrated that this NNS-mediated attenuation is important for fine-tuning the expression of an evolutionarily conserved mitochondrial transporter, Pic2, when cells rely on respiration to produce ATP. Remarkably, single-cell microfluidic analyses showed that NNS regulation of PIC2 not only reduced Pic2 protein levels but also decreased cell-to-cell variability in Pic2 expression, revealing a novel role for NNS as a transcriptional noise suppressor. Using GFP reporters, I show that this attenuation mechanism is generally applicable.

To investigate whether impairing NNS regulation of PIC2 affected cellular physiology, I characterised the mutants and compared their phenotype to that of the parental strain. My results prove that specifically disrupting Nab3 binding to PIC2 disturbs energy homeostasis, decreases cell fitness and leads to severe cell size increases and cell cycle delays. To determine whether these phenotypes solely emerged from the increase in activity of Pic2, I generated and inspected a PIC2 overexpression mutant, which only exhibited defective growth and energy homeostasis. Despite proving that maintaining an optimal expression of PIC2 is critical to enhancing microbial fitness during adaptation, this evidence also illustrated that larger levels of Pic2 did not underlie all the observed anomalies.

Combining multi-omics profiling and transcriptome-wide NNS-RNA binding footprinting, I demonstrate that disrupting Nab3 binding to PIC2 leads to redistribution of Nrd1 among its targets and changes levels of many other NNS-regulated transcripts. Given that depleting Nab3 from the nucleus causes an enlargement of cell size and a prolongation of the cell cycle, I posit that alterations in Nrd1 transcriptome occupancy underlie the cell volume and cycle anomalies observed upon abrogation of Nab3 binding to PIC2. Collectively, my findings illustrate that even subtle changes in how RNA-binding proteins interact with their RNA substrates can drive significant systemwide defects and emphasise the crucial role of the NNS complex in preserving microbial fitness during stress.