Cells exposed to heat stress experience an increase in the amount of misfolded and aggregated proteins. Cells respond to this threat through coordinated and finely tuned ad- justments in gene expression. When the ambient temperature increases, cells activate the heat stress response (HSR), a process in which the transcription of mRNAs encoding heat shock proteins (Hsps) is upregulated. During severe heat stress, cells also downregulate the synthesis of misfolding-prone housekeeping proteins while the synthesis of Hsps takes precedence. Consequently, the amount of misfolded and aggregated proteins is reduced by Hsps. While the transcriptional HSR has been studied in depth over the last 50 years, our understanding of protein translation regulation during heat stress remains limited.
Biomolecular condensates have been proposed as a new way to regulate cellular functions. In budding yeast exposed to severe heat stress, the repression in the synthesis of housekeep- ing proteins coincides with the formation of condensates called heat stress granules (HSGs). HSGs are enriched for translation factors and translationally-repressed mRNAs and they have been implicated in translation regulation. However, if and how HSGs regulate translation during severe heat stress has remained elusive. Using in vitro reconstitution assays, I demonstrate that the heat-induced condensation of translation factors together with mRNA is an adaptive mechanism to regulate protein synthesis during severe heat stress.
My thesis work focused on the translation initiation factor complex eIF4F from Saccharomyces cerevisiae. eIF4F was previously shown to promote global translation of capped mRNAs. One subunit of eIF4F is eIF4G, an RNA-binding and scaffold protein that interacts with numerous translation initiation factors. Two other subunits of eIF4F are the RNA helicase eIF4A and the mRNA cap-binding protein eIF4E. eIF4G also interacts with the poly(A) binding protein (Pab1p) and the RNA helicase Ded1p, which like eIF4F, are crucial in translation initiation. Importantly, eIF4G, eIF4E, Pab1p and Ded1p condense into HSGs in yeast upon severe heat stress, while eIF4A remains soluble in the cytosol. To investigate the function of these translation factors in regulating translation, I purified eIF4F, Pab1p and Ded1p. Using purified eIF4F, nanoluciferase-encoding reporter mRNAsand an in vitro translation assay, I showed that eIF4F enhances general protein synthesis.
Together with Pab1p and Ded1p, eIF4F enhances the translation of reporter mRNAs with 5’ UTRs of housekeeping transcripts to a greater extent than reporter mRNAs with 5’ UTRs of Hsp-encoding genes. These findings suggest important differences in translation regulation at physiological temperatures and that efficient translation of housekeeping mRNAs requires synergy between eIF4F, Pab1p and Ded1p. Next, I reconstituted eIF4G condensates in vitro using biochemical approaches. I found that eIF4G forms condensates with mRNA. The condensation of eIF4G-mRNA is promoted by heat-induced structural rearrangements and interaction valences between eIF4G RNAbinding domains (RBDs). eIF4G has three RBDs, where the removal of either RBDs did not affect the RNA binding affinity but repressed condensation. Thus, eIF4G-mRNA condensation requires cooperativity between the three RBDs. Critically, I found that the mechanism of heat-induced condensation is conserved and adapted in eIF4G orthologues from yeast species that thrive in colder or warmer temperatures. Using multi-component in vitro assays, I found that heated eIF4G-mRNA condensates recruit eIF4E and Pab1p. In agreement with the fact that eIF4A does not assemble into HSGs in cells, eIF4A did not partition into eIF4G-mRNA condensates, which is likely due to a heat-induced weakening of interactions with eIF4G. I next characterized eIF4G variants with targeted mutations in the eIF4E- and Pab1-binding sites of eIF4G. This allowed me to demonstrate that the recruitment of eIF4E into eIF4G-mRNA condensates is driven by protein-mediated interactions. Furthermore, I found that heterotypic interactions between eIF4G, Pab1 and the poly(A) tail of mRNA promote the solidification of heated condensates. This is consistent with previous observations reporting solid-like properties of HSGs. Finally, I investigated the translation activity of heated translation factor condensates in yeast cell-free extracts. Solid-like eIF4F-mRNA condensates with Pab1p or Ded1p resulted in a pronounced repression of translation. This coincided with the recruitment of reporter mRNAs into condensates. Based on these findings, I thus propose that the repression in translation of housekeeping mRNAs during severe heat stress in yeast is a consequence of the formation of solid-like translation factor and mRNA condensates. Further analyses revealed that mRNA outside of condensates are translated in an eIF4A-dependent manner. This is because eIF4A is not recruited to the condensates and remains active upon heating. In summary, I propose that heat stress promotes the condensation of mRNA with eIF4G, eIF4E, Pab1p and Ded1p into solid-like condensates. In vitro assays suggest that translation factors inside of condensates are inactive while the mRNA is translationally repressed. This model highlights a mechanism for the downregulation in the synthesis of housekeeping proteins during severe heat stress in yeast. My findings also suggest that the preferential translation of mRNAs encoding Hsps occurs independently of the condensate-forming translation factors and may be mediated by eIF4A, which does not localise into HSGs. I thus conclude that translation regulation during severe heat stress is achieved by specific translation initiation factors that form inactive and solid-like condensates with mRNA.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:90321 |
| Date | 05 March 2024 |
| Creators | Desroches Altamirano, Christine |
| Contributors | Alberti, Simon, Franzmann, Titus, Grill, Stephan Wolfgang, Stoecklin, Georg, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | info:eu-repo/grantAgreement/Human Frontier Science Program/Research grant/RGP0034/2017//Elucidating the molecular logic of membrane-free compartment function and assembly, info:eu-repo/grantAgreement/European Research Council/Horizon 2020/725836//PhaseAge - the chemistry and physics of RNP granules: how they form, age and cause disease/Phase Age |
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