Multiple decay events target HAC1 mRNA during splicing to regulate the unfolded protein response

Essential revisions:

1) The authors should explicitly do an experiment along the lines of Figure 4F to determine in tpt1Δ hac1Δ cells if the 5' extended 3' exon or the 3' shortened 5' exon are produced when HAC1-u or HAC1-s are expressed. This experiment would unequivocally examine the origin of these fragments.

We generated and verified cells with the ​tpt1​∆ hac1​∆ (10​ x tRNA​) genotype and transformed them with HAC1​ and ​HAC1​ expression plasmids. We analyzed HAC1 by northern blot and present the new result in a new panel Figure 4G. We describe the result with the additional sentence: “Furthermore, expression of ​HAC1​^s in a ​tpt1​∆ background also fails to produce additional fragments (Figure 4G), whereas expression of ​HAC1​ is sufficient in the tpt1​∆ background to produce the double-band secondary cleavage molecular phenotype.”

2) The authors should do a northern as part of Figure 6 to explicitly show that HAC1-s is substantially increased in tpt1Δ xrn1Δ and in tpt1Δ ski2Δ cells, relative to that in tpt1Δ cells. It is a major finding, discussed in the Abstract, that this decay is occurring, and the direct comparison by Northern is easy to do.

We analyzed both the time 0 and 120 minute samples from the relevant genotypes and blot for HAC1​ 3′-exon, quantifying the splicing percentages in each lane below (Figure 6E).

3) In both examples of the kinase mediated decay pathway (Figure 2 for decay of the 3' exon, and Figure 3 for the decay of the excised intron), the authors have provided convincing evidence that the kinase activity of Trl1 is required for the accumulation of 3' exon and the intron. However, the authors should also show results from expressing a ligase-dead mutant to demonstrate that ligase activity is not required.

We generated and confirmed yeast expression plasmids for the Trl1 alleles trl1​ ​-K114A (ligase-dead/adenylylation-dead) (Sawaya et al., 2003) and double-mutant trl1​ ​-K114A-D425N. We expressed those in a ​trl1​∆ (10x tRNA) background and performed intron and 3′-exon northern blots on these samples (new Figure 2C and Figure 3I).

In doing so, we add the sentences:

A) “We also tested whether the ligase activity of Trill affected HAC1​ 3′-exon abundance using an adenylyl-transferase/ligase defective allele (Trl1-K114A) ​(Sawaya et al., 2003) and found that additional 3′-exon accumulates compared to wild-type (​Figure 2C​), indicating that ligation also contributes to processing of free 3′-exon.”

And

B) “Interestingly, expression of the adenylyl-transferase-dead/ligase-dead allele, trl1​ ​-K114A, also led to accumulation of some free HAC1​ intron (Figure ​ 3I​), potentially indicating a role for ligation in the processing of liberated HAC1​ ​ intron.”

4) In Figure 3, it is not clear to this reader why trl1Δ mutants have less accumulated intron than xrn1Δ cells, if intron decay is occurring by kinase mediated decay. Would the authors speculate?

In fulfilling essential revision 3, we added a blot using trl1-K114A, a adenylylation-dead/ligase-dead allele of Trl1. This blot showed that both the kinase and adenylylation domains contribute to clearance of liberated ​HAC1​ intron from the cells. Sentence (2) from above addresses this point.