The fail-safe mechanism of post-transcriptional silencing of unspliced HAC1 mRNA

Abstract

HAC1 encodes a transcription factor that is the central effector of the unfolded protein response (UPR) in budding yeast. When the UPR is inactive, HAC1 mRNA is stored as an unspliced isoform in the cytoplasm and no Hac1 protein is detectable. Intron removal is both necessary and sufficient to relieve the post-transcriptional silencing of HAC1 mRNA, yet the precise mechanism by which the intron prevents Hac1 protein accumulation has remained elusive. Here, we show that a combination of inhibited translation initiation and accelerated protein degradation—both dependent on the intron—prevents the accumulation of Hac1 protein when the UPR is inactive. Functionally, both components of this fail-safe silencing mechanism are required to prevent ectopic production of Hac1 protein and concomitant activation of the UPR. Our results provide a mechanistic understanding of HAC1 regulation and reveal a novel strategy for complete post-transcriptional silencing of a cytoplasmic mRNA.Molecular machines called ribosomes read the genetic instructions in an mRNA molecule and then translate them to make proteins. However, cells do not translate all of the template mRNAs that they have available into proteins; instead they have a number of ways to block the process to control when certain proteins are made.In budding yeast, the mRNA that codes for a protein called Hac1 is always present in the cell but the protein is normally not detected. The Hac1 protein is responsible for helping the cell deal with certain types of stress, so it only accumulates when the cell is experiencing such stresses. The mRNA that encodes Hac1 (referred to as HAC1 mRNA) contains a sequence called an intron. These sequences are normally cut out of mRNAs before they are read by the ribosome. However, the intron in the HAC1 mRNA is unusual, because it is only removed when cells are subjected to stress. The rest of the time, this intron serves to block the production of Hac1 through a poorly understood mechanism.Now, Di Santo et al. show the HAC1 mRNA uses two strategies to keep itself fully repressed—both of which involve its intron. One strategy relies on a structure formed in the HAC1 mRNA that prevents ribosomes from starting translation in the first place. However, this block is occasionally bypassed, causing some Hac1 protein to be produced when it should not be. To deal with this, the Hac1 protein that is produced contains a short protein sequence, encoded by the intron, that targets this unneeded protein for degradation. These two strategies together comprise a “fail-safe” mechanism to completely repress the HAC1 mRNA.Following on from these findings, it will be important to determine whether other mRNAs – both in budding yeast and in other species including humans – use a similar fail-safe strategy to block proteins from being made when they should not be.