Xrn1 is a negative regulator of autophagy induced by methionine deprivation.

(A) Scheme depicting the media switch from rich lactate medium (YPL) to minimal, synthetic lactate media (SL). Cells are supplemented with either 1 mM methionine (met) or non-sulfur amino (non-S AA) acid mix containing 1 mM of all amino acids except methionine, cysteine, and tyrosine. (B) Cells lacking Xrn1 exhibit increased autophagy. Autophagy was measured by the Idh1-GFP cleavage assay34. The accumulation of free GFP following switch to SL medium indicates autophagy. Note xrn1Δ cells induce autophagy even in rich YPL media. (C) Cells lacking Xrn1 exhibit increased autophagy, which cannot be inhibited by methionine. Autophagy was assayed quantitatively by the alkaline phosphatase (ALP) activity assay. WT and xrn1Δ cells were switched from YPL to SL for the indicated times, in the absence or presence of methionine supplementation. Note autophagy is inhibited by the addition of methionine to WT but not xrn1Δ cells. Mean±SD, n=6, statistical analysis performed using unpaired student’s t-test. (D) Methionine is unable to repress autophagy in cells lacking Xrn1. As shown by the GFP cleavage assay, autophagy is inhibited by addition of methionine but not by a mix of non-sulfur amino acids in WT cells, but not xrn1Δ cells.

The regulation of autophagy by Xrn1 is dependent on its catalytic activity, but not dysregulation of autophagy-related mRNA transcripts.

(A) Catalytic activity of Xrn1 is required for methionine-responsive autophagy regulation. Xrn1 was knocked out and WT or catalytically dead (D208A) flag-tagged Xrn1 was expressed ectopically from a plasmid using the endogenous Xrn1 promoter. WT Xrn1, but not Xrn1 D208A, was able to rescue the methionine-sensitive autophagy phenotype of xrn1Δ cells. (B) Cells lacking Xrn1 exonuclease activity have a severe growth defect. The indicated strains were grown in YPL media and growth rate was monitored by automated measurement of OD600 every 30 min. (C) Differentially expressed genes in WT and xrn1Δ cells before and after the switch to SL for 1 h. Known differentially expressed gene groups are highlighted to demonstrate their muted response in xrn1Δ compared to WT cells. (D) Differentially expressed genes between xrn1Δ and WT cells in either YPL or SL media. Autophagy (ATG) genes are highlighted. RNA-seq data are also available in Table S2.

Xrn1 regulates autophagy through modulation of TORC1 signaling.

(A) Cells lacking Xrn1 exhibit diminished TORC1 activity. WT and xrn1Δ cells were grown in YPL and switched to SL media, or SL containing methionine, for the indicated times. Western blot analysis was used to assess phosphorylation of the TORC1-dependent substrate, ribosomal protein S6. (B) Npr2 and the SEACIT complex regulate TORC1 activity through the Rag GTPases Gtr1 and Gtr2. The nucleotide binding state of this heterodimer determines whether the complex activates or inhibits TORC1. (C) GFP cleavage autophagy assay in cells lacking Xrn1 and/or Npr2. The indicated strains were grown in YPL and then switched to SL for 6 h, in the absence or presence of 1 mM methionine. (D) Growth curves of cells lacking Xrn1, Npr2, or both. The indicated strains were grown in YPL and OD600 was measured every 30 min. Fold change in OD600 is plotted. (E) Xrn1 interacts with Npr2. Cells expressing Flag-tagged Xrn1 and HA-tagged Npr2 were grown in YPL and then switched to SL for the indicated times. Cells were harvested and the interaction between Xrn1 and Npr2 was determined by co-immunoprecipitation followed by Western blotting.

Xrn1 regulates autophagy through the Rag GTPase proteins Gtr1/2.

(A) The loss of Xrn1 and Gtr1 results in autophagy induction regardless of methionine availability. The indicated strains were grown in YPL and then switched to SL for 6 h, in the absence or presence of 1 mM methionine. Autophagy was measured using the GFP-cleavage assay. (B) The nucleotide-binding state of Gtr1 regulates autophagy. Point mutations of Gtr1 to lock it in its GTP-binding (Q65L) or GDP-binding (S20L) state affect its role in regulating autophagy through TORC1. Autophagy was assayed as in (A). (C) GTP-and GDP-locked mutations in Gtr1 predominate the growth defect of xrn1Δ cells. The indicated strains were grown in YPL and OD600 was measured every 30 min. Fold change in OD600 is plotted. (D) (E) GTP-and GDP-locked mutations in Gtr1 are sufficient to bypass the role of Xrn1 in regulating autophagy following methionine deprivation. The indicated strains were grown in YPL and then switched to SL for 6 h, in the absence or presence of 1 mM methionine. Autophagy was measured as in (A). (F) GTP-locked mutation of Gtr1 is unable to restore WT levels of many autophagy gene transcripts despite restoration of TORC1 signaling, repression of autophagy, and enhanced growth. mRNA abundance was assayed by qPCR for the indicated genes. (G) Loss of Xrn1 results in decreased interaction between Gtr1 and TORC1 component Kog1. WT and xrn1Δ cells expressing Gtr1-Flag and Kog1-HA were grown in YPL and switched to SL for the indicated times. The interaction between Gtr1 and Kog1 was assessed by co-IP followed by Western blotting.

The interaction between Xrn1 and Npr2 is regulated by RNA availability.

(A), (B) Interaction between catalytically dead Xrn1 mutant and Npr2 or Gtr1. In xrn1Δ cells expressing either HA-tagged Npr2 (A) or Gtr1 (B), WT or catalytically dead (D208A = DA) flag-tagged Xrn1 was expressed ectopically from a plasmid using the endogenous Xrn1 promoter. Cells were grown in the indicated conditions, and the interaction between Xrn1 and Npr2 or Gtr1 was assessed by co-IP followed by Western blotting. (C), (D) The interaction of Xrn1 with Npr2 (C) or Gtr1 (D) is sensitive to RNase treatment. Cells with flag-tagged Xrn1 and either HA-tagged Npr2 or Gtr1 were grown in the indicated conditions. The interaction between Xrn1 and Npr2 or Gtr1 was assessed by co-IP followed by Western blotting. Addition of RNase A (25 ng/μL) to the co-IP lysate increases interaction of Xrn1 with both Npr2 and Gtr1. (E), (F) The interaction of Xrn1 with Npr2 (E) but not Gtr1 (F) is sensitive to RNA concentration. Cells with flag-tagged Xrn1 and either HA-tagged Npr2 or Gtr1 were grown in YPL. The interaction between Xrn1 and Npr2 or Gtr1 was assessed by co-IP with RNA or NMPs added to the lysate followed by Western blotting. The addition of total RNA (from exponentially growing WT cells in YPL media) at 125 μg/mL (+) or 313 μg/mL (++), but not free NMPs, is sufficient to disrupt the interaction between Xrn1 and Npr2, but not Xrn1 and Gtr1. (G), (H) Treatment with RNA was sufficient to disrupt the interaction of Xrn1 with Npr2 (G) but not Gtr1 (H). Cells with flag-tagged Xrn1 and either HA-tagged Npr2 or Gtr2 were grown in YPL. The interaction between Xrn1 and Npr2 or Gtr1 was assessed by co-IP, followed by addition of RNA to the wash buffer, then analyzed by Western blotting. (I) Model for regulation of TORC1 activity by Xrn1 through Gtr1/2.

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 xrn1Δ cells. Growth curve measuring OD600 of WT or xrn1Δ 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 xrn1Δ 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.

Xrn1 is a negative regulator of nitrogen starvation-induced autophagy.

(A) Autophagy under nitrogen starvation conditions is induced more rapidly in xrn1Δ cells. WT and xrn1Δ 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 xrn1Δ 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.

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 xrn1Δ 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 xrn1Δ cells.

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) GTP-locked mutation of Gtr1 restores TORC1 activity in cells lacking xrn1Δ, 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.

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 xrn1Δ cells. The indicated strains were grown in YPL and OD600 was measured every 30 min. 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 xrn1Δ 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 npr2Δ 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 xrn1Δ compared to WT. Either WT or xrn1Δ 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 xrn1Δ cells were grown in the indicated conditions. Metabolites were extracted at the indicated times and analyzed by targeted LC-MS/MS.

Yeast strains used in this study.

All strains are in the prototrophic CEN.PK background.