Since quiescent cells appear to be ‘asleep’ it is easy to think of quiescence as a passive state of inactivity. However, as Dr. Alison Greenlaw, former graduate student in the Tsukiyama lab and author of a recently published paper in Nucleic Acids Research on RNA regulation in quiescence, explained, this is, in fact, “an active process that needs to be actively maintained”. While gene expression enters into a generally silenced state, there does appear to be quiescent-specific genomic transcription and regulation. Much of what is known about cellular activity during quiescence has focused on aspects of chromatin and genomic modifications, as well as some profiling of steady-state RNA in quiescent cells, but there have been limited studies on nascent (newly transcribed) RNA profiles or post-transcriptional regulation, which was the focus of Dr. Greenlaw’s recent publication.
It so happens that studying quiescence in yeast cells is uniquely ideal, as these single-celled eukaryotic cells can be easily pushed into quiescence or separated from non-quiescent cells. Using yeast as a model, the research team wanted to characterize the nascent RNA profiles in quiescent cells compared to a different cellular state called G1, immediately after cells have finished a round of division but before they begin the next round of DNA replication. By assessing nascent and steady-state RNA profiles, they hoped to recognize unique features of transcriptional regulation that define quiescence, including potential post-transcriptional modifications. Because quiescence is a more repressed state for cells, the team originally hypothesized that transcription would produce fewer RNA transcripts overall and with a lower proportion of non-coding (nc)RNAs, indicating a potentially more efficient state of transcription as a response to limited resources. When they assessed this in their model, however, what they observed was an increase in the variety of transcripts found, as well as an increase in the proportion of ncRNAs compared to mRNA, a result that seemed “unintuitive” noted Dr. Greenlaw, and which began to suggest that quiescent transcription was not as silenced as they had expected.
They also observed considerable differences between steady state and nascent transcriptomes, suggesting some contribution of post-transcriptional regulation. To investigate this further, they performed principal component analyses (PCA) on the mRNA and ncRNA transcripts, which allows for a 2-dimensional representation of highly complex data. This revealed that there were considerable global differences between G1 and quiescent cells (indicated by being farther apart on the PCA plot), consistent with their previous observations, but also between steady state and nascent profiles, particularly for ncRNA in quiescent cells. When comparing features of mRNAs contributing to PC1 and PC2, one particular finding was that Gene Ontology (GO) terms associated with nascent RNA included pathways consistent with post-transcriptional regulation. In addition to this, they also performed analyses of abundance of transcripts between steady state and nascent transcriptomes, and observed distributions which indicated an increase in degraded transcripts in quiescent cells. Together, this data pointed them towards post-transcriptional regulation as an important contributor to gene regulation in quiescent cells.
To further understand the mechanisms of post-transcriptional regulation in quiescent cells, they asked whether the nuclear exosome played a role. You may be more familiar with the term exosome referring to the small extracellular vesicles that have gained notoriety in the last few years, but in this case the nuclear exosome refers to a large protein complex with known roles in ncRNA degradation. By knocking out one key component of the nuclear exosome, RRP6, they found that mRNA accumulated more than ncRNA in quiescent cells compared to G1 cells, suggesting that mRNA degradation by the nuclear exosome is a major mechanism of post-transcriptional regulation in quiescence. They also observed that loss of RRP6 resulted in delayed entry into quiescence when the yeast cells were placed into nutrient-deficient conditions. The research team next targeted another part of this pathway, Nab3 (a part of the Nrd1-Nab3-Sen1 (NNS) complex, upstream of RRP6), through a depletion strategy. In this scenario, they observed increased transcription of both ncRNA and mRNA and increased abundance of transcripts in steady state, suggesting this complex contributed to decreased gene transcription during quiescence. When comparing the transcripts found to be regulated by both RRP6 and Nab3, the research team found a high degree of correlation, supporting their hypothesis that this pathway works together to regulate RNA abundance during quiescence. Interestingly, they also noted that following Nab3 depletion, there was an increase in antisense ncRNA, a subcategory of ncRNA which are complementary to mRNA and can act to block mRNA translation and control abundance. This observation seemed promising to Dr. Greenlaw, as she noted that they expected to be able to discuss functional ncRNA in the context of quiescence. However, she lamented that “there were [essentially] no functional antisense [ncRNAs]” in their analyses. When they further tried to assess specific features of Nab3-targeted RNAs, they found small associations with short mRNAs, lowly transcribed RNAs, and some GO terms indicative of cell cycle processes and metabolism. But future work will likely be needed to fully understand the specifics of how the NNS complex and nuclear exosome regulate RNA in quiescence.
Reading this article, it is clear that the impressive amount of data generated provides a novel look into the nascent transcriptomes and the importance of post-transcriptional regulation in quiescence. Dr. Greenlaw reflected that this study provided its own challenges as they were “figuring out all of our assumptions were incorrect”, and that the results presented more follow up questions than answers. She hopes that her data helps “get people thinking; it’s not just chromatin” as she conveyed that much of the focus on quiescence so far has been on genomic regulation and organization. “I think there’s a lot to be learned on the post transcriptional side of things, and even translation and post-translation areas”, she emphasized. And while it may seem easy to disregard quiescence if you work in other areas of research, there are broader implications for other organisms, such as humans, which rely on quiescence to maintain their stem cell niche and who have a large number of conserved genes with yeast. So next time you come across a mention of a quiescence, remember to consider how much may actually be going on in those seemingly sleepy cells.
The spotlighted research was funded by the National Institutes of Health and the National Science Foundation.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Toshio Tsukiyama contributed to this work.
Alison C Greenlaw, Kris G Alavattam, Toshio Tsukiyama, Post-transcriptional regulation shapes the transcriptome of quiescent budding yeast, Nucleic Acids Research, 2023;, gkad1147