Exonuclease Xrn1 regulates TORC1 signaling in response to SAM availability

Autophagy is a conserved process of cellular self-digestion that promotes survival during nutrient stress. In yeast, methionine starvation is sufficient to induce autophagy. One pathway of autophagy induction is governed by the SEACIT complex, which regulates TORC1 activity in response to amino acids through the Rag GTPases Gtr1 and Gtr2. However, the precise mechanism by which SEACIT senses amino acids and regulates TORC1 signaling remains incompletely understood. Here, we identify the conserved 5’−3’ RNA exonuclease Xrn1 as a surprising and novel regulator of TORC1 activity in response to methionine starvation. This role of Xrn1 is dependent on its catalytic activity, but not on degradation of any specific class of mRNAs. Instead, Xrn1 modulates the nucleotide-binding state of the Gtr1/2 complex, which is critical for its interaction with and activation of TORC1. This work identifies a critical role for Xrn1 in nutrient sensing and growth control that extends beyond its canonical housekeeping function in RNA degradation and indicates an avenue for RNA metabolism to function in amino acid signaling into TORC1.


Fig. S1 | Loss of Xrn1 causes elevated levels of SAM and other sulfur-containing metabolites.
(A), (B) Xrn1 protein abundance is not altered under different metabolic conditions.Anti-flag Western blot assessing protein amounts of Xrn1 under the indicated conditions.(C) Methionine restores growth of WT but not xrn1∆ cells.Growth curve measuring OD600 of WT or xrn1∆ cells in the indicated media.OD600 was measured every 30 min.Fold change is plotted.The data are represented as mean ± SD (n=3).(D) Schematic of sulfur-containing metabolites in yeast produced from methionine and transsulfuration.(E) Many sulfur-containing metabolites are elevated in cells lacking Xrn1.WT and xrn1∆ cells were grown in the indicated conditions.Metabolite samples were collected and analyzed by LC-MS/MS.Data are represented as mean ± SD (n=2).The metabolite data are also presented in Table S1.(A) Autophagy under nitrogen starvation conditions is induced more rapidly in xrn1∆ cells.WT and xrn1∆ cells harboring a centromeric plasmid expressing GFP-Atg8 were grown to mid-log phase in YPD then starved for nitrogen (SD-N) for the indicated times.Free GFP, indicative of autophagy induction, was detected by Western blot.(B) Autophagy under nitrogen starvation conditions in xrn1∆ cells as monitored by ALP assay.Cells were grown to mid-log phase in YPD then starved of nitrogen (SD-N) for the indicated times.ALP activity was measured and normalized to the WT cells in rich media.Mean±SD, n=3, statistical analysis performed using student's t-test.Fold change in OD600 is plotted.(C) Cells lacking both Xrn1 and Gtr2 exhibit enhanced autophagy.The indicated strains were assayed for autophagy as in (A).(D) Schematic depicting how the nucleotide binding states of Gtr1 and Gtr2 are controlled by GAPs and GEFs.(E) Growth curves of cells lacking Xrn1 and either Lst4 or Lst7 reveals a synthetic growth defect.The indicated strains were grown in YPL and OD600 was measured every 30 min.Fold change in OD600 is plotted.(F) Loss of the Gtr2 GAPs Lst4 and Lst7 phenocopy the methionine-insensitive autophagy phenotype of xrn1∆ mutants.Autophagy was assayed as in (A).(G) Cells lacking Xrn1 in combination with Lst4 or Lst7 exhibit significantly enhanced autophagy.Autophagy was assayed as described as in (A).(H) Loss of Vam6 phenocopies npr2∆ cells in regulation of autophagy.Vam6 is annotated as a GEF for Gtr1.The indicated strains were assayed for autophagy as in (A).(I) Loss of Ait1 and Ivy1 do not play a role in regulation of autophagy in response to methionine deprivation.The indicated strains were assayed for autophagy as in (A).(J) Abundance of Kog1 protein is not altered in xrn1∆ compared to WT.Either WT or xrn1∆ with HA-tagged Kog1 were grown in the indicated conditions, and Kog1 protein abundance was assayed by Western blotting.(K) Targeted metabolomics reveals slightly reduced levels of both GDP and GTP in cells lacking Xrn1.WT and xrn1∆ cells were grown in the indicated conditions.Metabolites were extracted at the indicated times and analyzed by targeted LC-MS/MS.

Fig. S2 |
Fig. S2 | Xrn1 is a negative regulator of nitrogen starvation-induced autophagy.(A)Autophagy under nitrogen starvation conditions is induced more rapidly in xrn1∆ cells.WT and xrn1∆ cells harboring a centromeric plasmid expressing GFP-Atg8 were grown to mid-log phase in YPD then starved for nitrogen (SD-N) for the indicated times.Free GFP, indicative of autophagy induction, was detected by Western blot.(B) Autophagy under nitrogen starvation conditions in xrn1∆ cells as monitored by ALP assay.Cells were grown to mid-log phase in YPD then starved of nitrogen (SD-N) for the indicated times.ALP activity was measured and normalized to the WT cells in rich media.Mean±SD, n=3, statistical analysis performed using student's t-test.

Fig. S3 |
Fig. S3 | Amounts of select autophagy mRNAs and proteins in cells lacking Xrn1.(A) Key autophagy protein Atg13 exhibits reduced abundance in cells lacking Xrn1.HA-tagged Atg13 protein abundance was assayed in WT and xrn1∆ cells following methionine deprivation for the indicated times by Western blotting.(B) Gene ontology enrichment from RNA-seq identifies gene groups that are altered in xrn1∆ cells.

Fig
Fig. S4 | Xrn1 interacts with Rag GTPase Gtr1.(A)Xrn1 and Gtr1 interact independent of methionine availability.Cells expressing flag-tagged Xrn1 and HA-tagged Gtr1 were grown in YPL and switched to SL for the indicated times.Interaction between these proteins was assessed by co-IP followed by Western blot.(B) GTPlocked mutation of Gtr1 restores TORC1 activity in cells lacking xrn1∆, assayed by Western blot for phosphorylated S6 ribosomal protein.(C) Xrn1 preferentially interacts with the GDP-locked form of Gtr1 by co-IP.Cells expressing flag-tagged Gtr1 constructs and HA-tagged Xrn1 were grown in the indicated conditions.Interaction between these proteins was assessed by co-IP followed by Western blot.Note the GDP-locked (S20L) mutation destabilizes the Gtr1 protein compared to WT or GTP-locked (Q65L) mutation.

Fig. S5 |
Fig. S5 | Xrn1 does not act through known Gtr1/Gtr2 regulatory proteins.(A)GTP-and GDP-locked mutations in Gtr2 synergize with loss of Xrn1 in regulating autophagy following methionine deprivation.The indicated strains expressing point mutations of Gtr2 to lock it in its GTP-binding (Q66L) or GDP-binding (S23L) state were grown in YPL and then switched to SL for 6 h, in the absence or presence of 1 mM methionine.Autophagy was measured by the GFP cleavage assay.(B) GTP-and GDP-locked mutations in Gtr2 exacerbate the growth defect of xrn1∆ cells.The indicated strains were grown in YPL and OD600 was measured every 30 min.

Table S3 : Yeast strains used in this study. 741
All strains are in the prototrophic CEN.PK background.742