Exonuclease Xrn1 regulates TORC1 signaling in response to SAM availability

Reviewer #1 (Public Review):

Summary: The paper by McGinnis et al. uses a combination of genetic and biochemical approaches to understand how the conserved 5'-3' RNA exonuclease Xrn1 affects autophagy in response to methionine starvation in S. cerevisiae. The authors present evidence Xrn1 affects autophagy primarily via its effect on regulating TORC1 signaling. They present some evidence that Xrn1's effect on TORC1 singnaling is via its physical interaction with the SEACIT complex.

Strengths: The experiments in general for this paper are clear and have proper controls.

Weaknesses:
The authors seem to try and fit the data to a simplistic model rather than embrace the complexity of the data. I will give some examples below.

1. Figure 1 clearly shows that xrn1d results in loss of tight repression of autophagy. Specifically, the 0 timepoint has increased autophagy in both the idh-GFP and ALP assays. However, it is incorrect to say that it is related in any way to methionine deprivation. The same basic pattern of regulation occurs in WT and xrn1d strains. The only difference is the "leakiness" of repression at t=0.

2. Figure 2 shows that catalytically inactive Xrn1 has the same autophagy phenotype as a deletion, indicating that Xrn1 enzymatic activity is important for function. However, it is also clear that xrn1-deletion cells expressing wt Xm1-flag do not repress autophagy as well as XRN+ cells, even though the amount of expressed protein seems similar. Does this imply the flag-tag may be a less active version of the protein? This should be discussed.

3. Figure 3 shows Xrn1-loss effects TORC signaling and that npr2-deletion inhibits autophagy. The surprising result is that a xrn1d/npr2d behaves like WT with regards to autophagy. This needs to be discussed. To me, this seems to strongly suggest that methionine repression of autophagy is occurring downstream of both xrn1 and npr2. Measuring p-S6 in the double mutant may be informative.

4. Figure 4 appears to show that even in the absence of GTR1, autophagy is repressed in rich media, active in YPL-SL, but still responds to methionine repression. This does not seem consistent with the model presented in Figure 5. Shouldn't loss of GTR1 result in repressed Torc1? The GTP and GDP-lock mutants are either all on, or all off. Why is deletion different? This needs to be explained and discussed. Also, the Figure legend does not match figures (problem after Fig4b).

5. Figure 5B shows GTR1 IP with Xrn1-FLAG. However, there are no negative controls in this experiment, so the result could be background. RNAaseA and RNA addition experiments are convincing.

6. Line 254-255. The lead sentence is simply not supported by the data. There is no evidence that Xrn1 actually affects the regulation of Gtr1/2 binding states.

7. Line 259-260. This is again overstated. Just because a mutant can be rescued by Gtr1-GTP-locked, does not say anything about RNA decay. In fact, the double mutant has extra high levels of some ATG RNA's, so I have no idea how the Gtr1 rescues.

8. Line 268-281. Your model here ignores the fact that methionine regulation takes place in the absence of both xrn1 and npr2. Therefore the model, as proposed, can't be correct.

9. Line 290-300. The slow growth rate of Xrn1 mutants may be affecting the metabolite levels. I felt that this entire paragraph was overly speculative.

Reviewer #2 (Public Review):

Summary:
McGinnis and colleagues conducted a study to elucidate the precise mechanism through which SEACIT detects amino acids and regulates TORC1 signaling in yeast. In their research, the authors made a noteworthy discovery by identifying the conserved 5'-3' RNA exonuclease Xrn1 as a novel regulator of TORC1 activity, particularly in response to stress-induced autophagy. The study revealed that the impact of Xrn1 on TORC1 is contingent on its catalytic activity rather than the degradation of any specific category of mRNAs. Instead, Xrn1 plays a pivotal role in modulating the nucleotide-binding state of the Gtr1/2 complex. This modulation is crucial for the complex's interaction with and subsequent activation of TORC1.

Strengths:
This study holds considerable potential as it illuminates an intriguing function of Xrn1 in nutrient sensing and growth control, expanding beyond its conventional role in RNA degradation. Furthermore, the research suggests a novel pathway through which RNA metabolism can influence methionine signaling to activate TORC1.

Weaknesses/General comments:

1. Previous work has shown that SAMTOR, upstream of mTORC1 in mammalian cells, senses methionine abundance through SAM levels (PMID: 29123071). However, this study suggests that Xrn1 senses and signals methionine to regulate mTORC1 signaling independently of SAM abundance. This finding appears to contradict the mentioned previous work. The authors should address this discrepancy. Moreover, the title of this manuscript does not seem to fit with actual findings. In the title, the authors mention that Xrn1 regulates TORC1 in response to SAM availability, but SAM levels do not seem to matter for Xrn1-dependent regulation of autophagy and TORC1.
2. This group has previously shown that the addition of methionine stimulates the synthesis of S-adenosylmethionine (SAM), which inhibits autophagy and promotes growth through the action of the methyltransferase Ppm1p, which modifies the catalytic subunit of PP2A in tune with SAM levels (PMID: 23870128). Since Xrn1 controls autophagy in a methionine-dependent manner, the authors should assess the effects of Xrn1 on SAM-dependent methylation of PP2A?
3. The authors should measure the effects of Xrn1 and TORC1 regulation on the methionine-SAM cycle activity through an isotope tracing approach, possibly by using U-13C-methionine.
4. The authors use mainly the GFP cleavage assay from Idh1-GFP to assess mitochondria degradation (or mitophagy) and generalize that autophagy is induced. Other assays should be employed more notably to assess globally non-mitochondrial specific degradation. For example, the authors could employ the Pho8∆60 assay.
5. In several blots (Panel 3D, 4D, 4B, 4F), the authors assess autophagy through GFP cleavage from Idh1-GFP but do not assess TORC1 activity in the same conditions. Showing autophagy induction and TORC1 activity on the same panels would be preferable.

Specific comments:

1. Panel 5E: As a control, the authors should use DNA instead of NMPs.
2. Panel S3B: Contrary to what is indicated in the text, this panel does not display ATG mRNA levels.
3. Panel S5K is not cited in the text. The rationale behind measuring the steady-state levels of GTP and GDP is not explained.
   Several panels are not subjected to statistical analysis. It is important for the authors to ensure that appropriate statistical methods are applied.

Reviewer #3 (Public Review):

Summary
This study investigated the role of the exonuclease Xrn1 in regulating autophagy in response to methionine deprivation in the budding yeast (S. cerevisiae). As a model system, wild-type and xrn1-deletion cells are switched from a nutrient-rich, lactate-based media (YPL) to a synthetic, minimal, lactate media with or without re-addition of methionine. Autophagy is measured by a previously reported Idh-GFP cleavage assay, and in some cases by quantification of alkaline phosphatase activity. The authors conclude that Xrn1 suppresses autophagy in response to methionine depletion based on the results of the Idh-GFP assay. However, the alkaline phosphatase assay could potentially suggest the opposite conclusion, with xnr1 deletion blocking the induction of autophagy relative to baseline in those cells, an interpretation which is complicated by higher basal autophagy induction upon xnr1 deletion. To address the mechanism of Xrn1 regulation of autophagy, a model is presented in which Xrn1 activates Target of Rapamycin Complex 1 (TORC1), which suppresses autophagy. This regulation is proposed to occur through physical association of Xrn1 with known upstream regulators of TORC1 activity, the SEACIT/GATOR1 and Gtr/Rag complexes. However, TORC1 activity is not measured under many key experimental conditions, making it difficult to determine the accuracy of this model. If the model ultimately proves correct, this would be an important finding that establishes a new player in the critical TORC1 pathway that controls cell growth and metabolism in response to changes in nutrient availability.

Strengths
Clear and highly reproducible results using the Idh-GFP cleavage assay to measure apoptosis.

Detailed characterization of the metabolic and transcriptomic effects of Xrn1 deletion through metabolomics and RNA-seq.

Use of a catalytically inactive Xrn1 mutant to demonstrate that its effects on autophagy require its catalytic activity.

Weaknesses
Predominant use of a single autophagy assay (Idh-GFP cleavage), with potentially conflicting results in another assay (alkaline phosphatase activity).

TORC1 activity is not measured under many key experimental conditions.

Protein-protein interactions are studied by overexpression of tagged proteins. While this may be essential for detection, the level of overexpression relative to endogenous protein is unclear, as well as whether this recapitulates the endogenous interactions and regulation.

Results from some experiments have several possible interpretations.