UC Berkeley

The endoplasmic reticulum (ER) maintains a lumenal environment favorable for the folding of at least 30% of the cell’s protein complement. ER quality control mechanisms, including chaperone systems, ensure proper protein folding in this compartment. However, the burden of misfolded proteins within the ER lumen can exceed the capacity of the ER folding machinery. In such instances of ER stress, a conserved pathway called the unfolded protein response (UPRER) is induced. The budding yeast Saccharomyces cerevisiae harbor only the most conserved branch of this pathway, at the crux of which is the cytoplasmic splicing of a translationally repressed mRNA encoding the transcription factor Hac1. Splicing of the HAC1 mRNA is executed by a conserved ER-localized stress sensor and endonuclease, Ire1. Transcriptional targets of the UPRER in yeast include ER resident chaperones, ER associated decay (ERAD) machinery, and membrane biogenesis factors, which all serve to mitigate ER stress.

Despite conservation of this pathway, several key differences exist between UPRER signaling in budding yeast and metazoans. First, widespread gene repression is a component of the UPRER gene expression program in metazoans, but has previously not been appreciated to be an important aspect of UPRER signaling in budding yeast. Second, even though many of the mechanistic details of the pathway were first elucidated in budding yeast, physiological significance of the pathway has only been established in metazoans. This is in part because UPRER activation in budding yeast is generally achieved by external perturbation, such as via drug treatment. Furthermore, hac1∆ and ire1∆ cells do not exhibit severe phenotypes during vegetative yeast growth, suggesting that the UPRER may not play a crucial role under standard laboratory growth conditions. Based on our observations that budding yeast cells activate the UPRER during meiotic differentiation and recent findings that gene repression can occur through transcription of poorly translated alternate mRNA isoforms, I investigated whether and how pharmacological activation of the UPRER in budding yeast involves Hac1-dependent gene repression, and additionally sought to characterize the basis for and importance of meiotic physiological UPRER induction in yeast.

To capture a more nuanced view of the Hac1-dependent gene expression program during the UPRER, I generated deep, parallel gene expression datasets (measuring mRNA, translation, and protein levels). I found that a recently described mode of gene repression relying on the expression of long undecoded transcript isoforms (LUTIs) contributes to global proteome remodeling in response to ER stress. Namely, Hac1-dependent LUTI expression resulted in decreased protein levels of one set of targets, while Hac1-dependent transcription of canonical mRNAs resulted in increased protein levels of a separate set of targets. My findings elucidate how gene repression during UPRER can be achieved by the same transcription factor, Hac1, that has been previously associated with only gene activation. The same principle may be applicable to other transcriptionally driven stress response pathways.

To investigate the physiological UPRER induction seen in the absence of external perturbation during meiosis, I evaluated whether an intact UPRER is required for the meiotic program in budding yeast. I determined that having HAC1 prior to and/or during meiotic entry is necessary for meiotic progression. I showed that while many key meiotic events were dispensable for meiotic UPRER induction, expression of the meiotic transcription factor Ndt80 was required. I developed strategies to identify transcriptional targets of the UPRER in meiosis, and used them to generate rich datasets allowing for physiological target identification, which will enable a better understanding of the meiotic function of UPRER activation.

Whether gene repression via the LUTI mechanism and/or physiological induction as part of the meiotic program are conserved characteristics of the UPRER remains to be determined. However, my findings resolve two of the discrepancies between UPRER signaling in budding yeast and metazoans, elucidating a mechanism by which gene repression is achieved and characterizing a physiological context in which the pathway is activated in budding yeast.