The Pat1-Lsm complex prevents 3′ to 5′ degradation of a specific subset of ATG mRNAs during nitrogen starvation-induced autophagy

ABSTRACT

Deregulation of macroautophagy/autophagy, a conserved catabolic recycling pathway, has been implicated in the onset and development of several diseases. While post-translational regulation of auto-phagy-related (Atg) proteins has been an important research focus leading to significant breakthroughs in understanding autophagy regulation, less is known about the post-transcriptional regulation of ATG transcripts. In a recent study we showed that, during nitrogen starvation, the RNA-binding complex Pat1-Lsm is involved in binding and preventing the 3ʹ to 5ʹ exosome-mediated degradation of a specific subset of ATG mRNAs. Dephosphorylation of Pat1 at residues S456 and S457 facilitates ATG mRNA binding, resulting in ATG mRNA accumulation, Atg protein synthesis and robust autophagy induction. In addition, we present evidence that these processes are conserved in human cells. These results further elucidate our understanding of the post-transcriptional mechanism necessary for efficient induction of autophagy during stress conditions.

KEYWORDS:

• 3ʹ-5ʹ degradation
• 5ʹ-3ʹ degradation
• exosome
• Lsm1
• mRNA decay
• Xrn1

Autophagy is an important regulatory mechanism for maintaining cellular homeostasis. This process must be tightly regulated to prevent excessive degradation of necessary cellular components. Under nutrient-rich conditions autophagy is kept at low levels; however, upon nutrient starvation auto-phagy is highly upregulated in part due to increased ATG gene transcription and protein synthesis. Previous studies have shown that ATG mRNA levels are dramatically increased in nutrient-starvation conditions. While this result is logical because autophagy is required during starvation conditions, it carries further significance because most gene transcription is either shut down or kept at minimum levels when essential nutrients are not available. Thus, there must be specific regulatory mechanisms by which ATG mRNAs are stabilized in order to increase Atg protein synthesis when autophagy is required.

The exoribonuclease Xrn1 has been recently proposed as a key regulator of ATG mRNA levels. Xrn1 catalyzes one of the two major mRNA decay pathways, targeting deadenylated transcripts whose 5ʹ cap has been removed. During nutrient-rich conditions Xrn1 catalyzes the 5ʹ to 3ʹ degradation of several ATG mRNAs, which is facilitated by the decapping enzyme Dcp2, thus maintaining basal autophagy levels during non-inducing conditions. Conversely, nitrogen starvation inhibits Dcp2 activity preventing Xrn1-mediated ATG mRNA degradation and leading to ATG transcript stabilization, increased Atg protein synthesis and autophagy induction. This research provided significant insight into the post-transcriptional regulation of autophagy and opened the possibility that other mRNA decay pathways could also have a role in the regulation of ATG transcripts.

The second major pathway for degrading transcripts is 3ʹ to 5ʹ mRNA decay, which is catalyzed by the exosome. The core exosome complex is composed of six proteins in a ring structure along with three associated proteins. Efficient transcript degradation by the exosome requires interaction with the Ski complex formed by Ski2 (an RNA helicase), Ski3 and Ski8, which, along with the Ski7 adaptor protein, is thought to modulate the cytoplasmic functions of the exosome. A third complex, formed by the mRNA binding protein Pat1 and a heptameric ring-shaped complex formed by Lsm1 through Lsm7, has been implicated in both 5ʹ to 3ʹ and 3ʹ to 5ʹ mRNA decay. Pat1-Lsm binds preferentially to the 3ʹ UTR of mRNA which has been reported to enhance mRNA decapping leading to Xrn1-mediated degradation. However, Pat1-Lsm mRNA binding has also been implicated in protecting mRNA from 3ʹ to 5ʹ degradation by the exosome.

Our study in S. cerevisiae provided evidence that the Pat1-Lsm complex is required for robust autophagy induction after nitrogen starvation [1]. The decrease in autophagy activity in PAT1 and LSM1 deletion strains compared to the wild-type after nitrogen starvation correlates with decreased protein and ATG mRNA levels of the essential ATG genes ATG1, ATG2, ATG7 and ATG9. Preventing efficient exosome-mediated 3ʹ to 5ʹ mRNA degradation by deleting SKI3, restores autophagy activity in the PAT1 deletion strain, as well as ATG1, ATG2, ATG7 and ATG9 protein and mRNA levels to those observed in a wild-type strain. Notably, measuring the mRNA levels of all known yeast ATG genes in a pat1Δ strain, revealed that the Pat1-Lsm complex affects only a subset of ATG mRNAs during nitrogen starvation; the levels of several other ATG mRNA transcripts, such as ATG17 and ATG18, remain unaffected by PAT1 deletion.

Because Pat1 is rapidly dephosphorylated at serine residues S456 and S457 under conditions of glucose starvation, we wondered if Pat1 was also dephosphorylated during nitrogen starvation, and if this dephosphorylation could be relevant to autophagy regulation. Using an SDS-PAGE phosphate-binding tag we determined that, indeed, Pat1 is dephosphorylated at the S456 and S457 residues during nitrogen starvation. Furthermore, a phosphomimetic Pat1 mutant (Pat1^S456,457E) displays a similar decrease in autophagy activity, as well as ATG1, ATG2, ATG7 and ATG9 protein and mRNA levels as that seen in a PAT1 deletion strain. Conversely, when ATG17 and ATG18 mRNA levels are measured in the Pat1^S456,457E strain, they fail to show any difference compared to a strain expressing wild-type Pat1. This difference between ATG mRNAs appears to be due to the ability of Pat1 to bind specific transcripts, as observed when binding is measured through mRNA immunoprecipitation as well as a protein-RNA bimolecular fluorescence complementation assay. Once again, preventing efficient 3ʹ to 5ʹ mRNA degradation by deleting SKI3 restores the autophagy activity, as well as ATG1, ATG2, ATG7 and ATG9 protein and mRNA levels in the Pat1^S456E,S457E strain to those observed in a wild-type strain.

Because the Pat1-Lsm complex binds preferentially to the 3ʹ UTR of mRNA, we decided to switch the 3ʹ UTR of ATG1 mRNA, which shows a decrease when PAT1 is deleted, with the 3ʹ UTR of ATG17 and ATG18 mRNA, which remain unaffected by PAT1 deletion. Switching the ATG1 mRNA 3ʹ UTR with that of either gene prevents the PAT1 deletion-mediated decrease of ATG1 mRNA and protein levels after nitrogen starvation. Finally, we determined if the Pat1-Lsm complex effect on ATG mRNA was conserved in humans. The siRNA-mediated knockdown of the PAT1 ortholog PATL1 leads to a decrease in ATG2 and ATG9A mRNA levels. Consistent with our yeast data, a PATL1 phosphomimetic mutant shows decreased ATG2 and ATG9A mRNA levels under autophagy-inducing conditions, whereas a PATL1 nonphosphorylatable mutant displays increased autophagy activity.

Our study further elucidates how autophagy is regulated at the post-transcriptional level by different mRNA binding proteins. However, important questions remain about which other mRNA-binding proteins are involved in autophagy regulation, as well as understanding the specific mechanisms by which these mRNA-binding proteins recognize their target mRNAs.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases [AI001123-01];Division of Intramural Research, National Institute of Allergy and Infectious Diseases [AI001124-01];National Institute of General Medical Sciences [GM053396].