TRIBE editing reveals specific mRNA targets of eIF4E-BP in<i>Drosophila</i>and in mammals

Jin, Hua; Na, Daxiang; Rahman, Reazur; Xu, Wei; Fieldsend, Allegra; Song, Wei; Liu, Shaobo; Li, Chong; Rosbash, Michael

doi:10.1101/2020.02.24.962852

Cited by 8 publications

(14 citation statements)

References 40 publications

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“…We speculate that the cells benefit from an excess of eIF4E by ensuring homogeneous distribution throughout the cytoplasm to rapidly activate bursts of translation necessary to preserve cellular homeostasis in response to environmental changes leading to, for instance increased mitochondria activity 36,37 or cell proliferation 38,39 . Live imaging revealed additional insights into the regulatory dynamics of Despite being the major cap-binding protein, eIF4E activity is primarily rate-limiting in translating mRNAs characterized by a highly structured 5'UTR 37,42 , a model that has been recently challenged [43][44][45] . In living cells, mTOR inhibition triggered the release of eIF4E from the mRNA after 30 minutes without affecting global translation as previously reported (Suppl.…”

Section: Discussionmentioning

confidence: 99%

Cap-dependent translation initiation monitored in living cells

Gandin

English

Freeman

et al. 2021

Preprint

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Despite extensive biochemical, genetic, and structural studies, a complete understanding of mRNA translation initiation is still lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-molecule resolution are necessary to fully elucidate regulation of this paramount process. We fused tags suitable for live imaging to eIF4E, eIF4G1 and 4E-BP1 without affecting their function. We combined Fluorescence Correlation Spectroscopy (FCS) and Single-Particle Tracking (SPT) to interrogate the binding dynamics of initiation factors to the 5'cap. Both FCS and SPT were able to detect eIF4E:eIF4G1 binding to the mRNA in the cytoplasm of proliferating cells and neuronal processes. Upon inhibition of phosphorylation by mTOR, 4E-BP1:eIF4E complexes rapidly dissociated from the 5'cap followed by eIF4G1 dissociation. Imaging of the binding dynamics of individual translation factors in living cells revealed the temporal regulation of translation at unprecedented resolution.

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Section: Discussionmentioning

confidence: 99%

Cap-dependent translation initiation monitored in living cells

Gandin

English

Freeman

et al. 2021

Preprint

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“…However, Drosophila ADARcd shows low editing efficiency in human PC3 cells (25). To utilize HyperTRIBE in human cells, the catalytic domain of human ADAR2 was used to improve its editing efficiency.…”

Section: Development Of the Hypertribe Methods In Plantsmentioning

confidence: 99%

A new method to identify global targets of RNA-binding proteins in plants

Cheng

Hsieh

2021

Preprint

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Background: RNA-binding proteins (RBPs) play crucial roles in various aspects of post-transcriptional gene expression; their functions can vary between tissues, cell types, developmental stages, and environmental conditions. Identifying RBP target RNAs and investigating whether they are differentially bound by RBPs in different cell types, stages, or conditions could shed light on RBP functions. Although several strategies have been designed to identify RBP targets, they involve complicated biochemical steps and require large quantities of material, and only a few studies using these techniques have been performed in plants. The TRIBE (targets of RNA binding proteins identified by editing) method was recently developed to identify RBP targets using a RBP coupled to the catalytic domain of a Drosophila RNA editing enzyme and expressing this fusion protein in vivo. The resulting novel editing events can be identified by sequencing. This technique uses little material and does not require complex biochemical steps, however it is not yet adapted for use in plants. Results: We successfully applied an optimized genome-wide TRIBE method in plants. We selected the splicing regulator polypyrimidine tract-binding protein (PTB) as a model protein for testing the TRIBE system in the moss Physcomitrium patens. We demonstrated that 13.81% of protein-coding gene transcripts in P. patens are targets of PTB. Most potential PTB binding sites are located in coding sequences and 3 prime untranslated regions, suggesting that PTB performs multiple functions besides pre-mRNA splicing in this moss. In addition, TRIBE showed reproducible results compared to other methods. Conclusions: We have developed an alternative method based on the TRIBE system to identify RBP targets in plants globally, and we provide guidance here for its application in plants.

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“…Hence the adaptive modification of the translatome is responding to features within the mRNA 5’ TL. Torin1 induced translational stress targets mainly the availability of eIF4E1 via eIF4E1-4EBP1 (Heberle et al, 2015; Jin et al, 2020). Our KO studies reveal that it is specifically as a response to this stress that eIF4E3 drives the formation of the adaptive translatome.…”

Section: Discussionmentioning

confidence: 99%

“…What is happening at the molecular level with regards to how the eIF4F S selects these short TLs is unclear. Recent studies position the eIF4E1-4EBP on the 5’ cap (Jin et al, 2020; Ptushkina et al, 1999). The eIF4E1 has higher cap affinity than eIF4E3 and would presumably be positioned on the most accessible 5’ ends, i.e.…”

Section: Discussionmentioning

confidence: 99%

eIF4E3 Forms an Active eIF4F Complex during Stresses (eIF4F^S) Targeting mTOR and Re-Programs the Translatome

Weiss

Allen

Kloehn

et al. 2021

Preprint

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The eIF4E are a family of initiation factors that bind the mRNA 5’ cap, regulating the proteome and the cellular phenotype. eIF4E1 mediates global translation and its activity is controlled via the PI3K/AKT/mTOR pathway. mTOR down-regulation results in eIF4E1 sequestration into an inactive complex with the 4E binding proteins (4EBPs). The second member, eIF4E2, regulates the translatome during hypoxia. However, the exact function of the third member, eIF4E3, has remained elusive. We have dissected its function using a range of techniques. Starting from the observation that it does not interact with 4EBP1, we demonstrate that eIF4E3 recruitment into an eIF4F complex occurs when Torin1 inhibits the mTOR pathway. Ribo-seq studies demonstrate that this complex (eIF4FS) is translationally active only during stress and that it selects specific mRNA populations based on 5’ TL (UTR) length. The interactome reveals that it associates with cellular proteins beyond the cognate initiation factors, suggesting that it may have “moon-lighting” functions. Finally, we provide evidence that cellular metabolism is altered in an eIF4E3 KO background but only upon Torin1 treatment. We propose that eIF4E3 acts as a second branch of the integrated stress response, re-programming the translatome to promote “stress resistance” and adaptation.

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TRIBE editing reveals specific mRNA targets of eIF4E-BP inDrosophilaand in mammals

Cited by 8 publications

References 40 publications

Cap-dependent translation initiation monitored in living cells

Cap-dependent translation initiation monitored in living cells

A new method to identify global targets of RNA-binding proteins in plants

eIF4E3 Forms an Active eIF4F Complex during Stresses (eIF4F^S) Targeting mTOR and Re-Programs the Translatome

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TRIBE editing reveals specific mRNA targets of eIF4E-BP inDrosophilaand in mammals

Cited by 8 publications

References 40 publications

Cap-dependent translation initiation monitored in living cells

Cap-dependent translation initiation monitored in living cells

A new method to identify global targets of RNA-binding proteins in plants

eIF4E3 Forms an Active eIF4F Complex during Stresses (eIF4FS) Targeting mTOR and Re-Programs the Translatome

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eIF4E3 Forms an Active eIF4F Complex during Stresses (eIF4F^S) Targeting mTOR and Re-Programs the Translatome