Pharma Focus America

Pbp1 Associates With Puf3 and Promotes Translation of Its Target mRNAs Involved in Mitochondrial Biogenesis

Floortje van de Poll, Benjamin M. Sutter, Michelle Grace Acoba, Daniel Caballero, Samira Jahangiri, Yu-San Yang, Chien-Der Lee, Benjamin P. Tu

Abstract

Pbp1 (poly(A)-binding protein—binding protein 1) is a cytoplasmic stress granule marker that is capable of forming condensates that function in the negative regulation of TORC1 signaling under respiratory conditions. Polyglutamine expansions in its mammalian ortholog ataxin-2 lead to spinocerebellar dysfunction due to toxic protein aggregation. Here, we show that loss of Pbp1 in S. cerevisiae leads to decreased amounts of mRNAs and mitochondrial proteins which are targets of Puf3, a member of the PUF (Pumilio and FBF) family of RNA-binding proteins. We found that Pbp1 supports the translation of Puf3-target mRNAs in respiratory conditions, such as those involved in the assembly of cytochrome c oxidase and subunits of mitochondrial ribosomes. We further show that Pbp1 and Puf3 interact through their respective low complexity domains, which is required for Puf3-target mRNA translation. Our findings reveal a key role for Pbp1-containing assemblies in enabling the translation of mRNAs critical for mitochondrial biogenesis and respiration. They may further explain prior associations of Pbp1/ataxin-2 with RNA, stress granule biology, mitochondrial function, and neuronal health.

Introduction

Yeast cells are capable of rapidly adapting their metabolism to changes in environmental conditions. When grown in glucose media, yeast cells use glycolysis for energy production and suppress mitochondrial biogenesis. However, in the presence of a non-fermentable carbon source, such as lactate, yeast cells adapt by inducing mitochondrial biogenesis to increase ATP production by oxidative phosphorylation (OXPHOS). The majority of the protein components of the electron transport chain are nuclear-encoded and need to be imported into the mitochondria. This essential process also requires cytosolic protein participants and is tightly regulated according to the cell’s metabolic needs [1].

Materials and method

Yeast strains, growth, and media

The prototrophic Saccharomyces cerevisiae CEN.PK strain [41] was used in all experiments. All strains used in this study are listed in S2 Table. Gene deletions were performed using standard PCR-based strategies to amplify resistance cassettes with appropriate flanking sequences and replace the target gene through homologous recombination [42]. C-terminal tags were similarly made using PCR to amplify resistance cassettes with flanking sequences. Pbp1 and Puf3 mutants with various domain deletions or point mutations were first made using PCR and then integrated into the PBP1 or PUF3 locus in a pbp1Δ or puf3Δ strain with different selection markers. Yeast strains were grown in YPD (1% yeast extract (Bio Basic), 2% peptone (BD Biosciences) and 2% glucose) or YPL (0.5% yeast extract, 2% peptone and 2% lactate (Sigma L1375)). Cells from overnight cultures were inoculated into fresh YPD to 0.3 optical density (OD600)/ml and grown for at least two generations to log phase. Cells were then spun down, washed with YPL, and resuspended in the same volume of YPL. Samples were collected at indicated time points. For cells treated with rapamycin, 200 ng/ml rapamycin (Sigma) was added to cells grown in YPL and incubated for 30 min before harvesting.

Results

To interrogate a possible role for Pbp1 in mitochondrial function, we assayed mitochondrial protein abundance in response to switching from glycolytic (YPD) to respiratory (YPL) media, which are conditions that demand mitochondrial biogenesis, in wild type (WT) and pbp1Δ cells. We assessed Por1 (mitochondrial porin) and Cox2 (subunit II of cytochrome c oxidase) protein levels using readily available antibodies by immunoblot in YPD and at several time points after switching to YPL (Fig 1A). Por1 protein levels increased over time during growth in respiratory conditions in both WT and pbp1Δ cells. Strikingly, Cox2 protein levels were severely decreased in pbp1Δ compared to WT cells at all time points. Moreover, pbp1Δ cells showed a significantly reduced growth rate in YPL compared to WT cells (Fig 1B), but their growth rate in YPD was similar to WT. These observations are consistent with pbp1Δ cells having compromised mitochondrial function due to reduced amounts of proteins required for respiration, such as Cox2.

Discussion

In this study, we show that Pbp1 supports mitochondrial function by promoting the translation of Puf3-target mRNAs that are involved in mitochondrial biogenesis. Both Pbp1 and Puf3 harbor low complexity domains, and their association is required for normal Puf3 function. We speculate that Pbp1 self-associates under respiratory conditions to recruit Puf3 to the vicinity of mitochondria, where Puf3 promotes the translation of mRNAs central for mitochondrial biogenesis. Consistent with this hypothesis, we observed that Pbp1’s capacity to self-assemble correlated with its interaction with Puf3 and its ability to boost Cox2 protein amounts. Interestingly, Pbp1 has been suggested to function as a redox sensor [17]. Reactive oxygen species can accumulate during mitochondrial dysfunction, underlining the potential for a key sensor role for Pbp1 in modulating Puf3 functions in response to mitochondrial dysfunction or biogenesis.

Acknowledgments

We thank the Tu Lab for helpful discussions. We thank Dr. S.M. Claypool at Johns Hopkins University for the Atp2 and Cox4 antibodies.
Citation: van de Poll F, Sutter BM, Acoba MG, Caballero D, Jahangiri S, Yang Y-S, et al. (2023) Pbp1 associates with Puf3 and promotes translation of its target mRNAs involved in mitochondrial biogenesis. PLoS Genet 19(5): e1010774. https://doi.org/10.1371/journal.pgen.1010774

Editor: Anita K. Hopper, Ohio State University, UNITED STATES

Received: April 4, 2023; Accepted: May 7, 2023; Published: May 22, 2023

Copyright: © 2023 van de Poll et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Raw sequencing data have been deposited at Gene Expression Omnibus with accession number: GSE227356

Funding: This work was supported by the Howard Hughes Medical Institute (HHMI) to BPT; the HHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS): R01NS115546 to BPT and the HHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS): 3R01NS115546-04S1 to DC. The funders had no role in study 
design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors declare that they have no conflict of interest.

Thermo Fisher Scientific - mRNA ServicesFuture Labs Live USA 2024World Vaccine Congress Europe 2024World Orphan Drug Congress 2024Advanced Therapies USA 2024World Orphan Drug Congress Europe 2024