Translationskontrolle durch eIF2D: Von Ziel-mRNAs zu zell-spezifische Funktionen
Zellbiologie
Zusammenfassung der Projektergebnisse
Many eukaryotic mRNAs contain regulatory elements in their 5’ leader sequences that dynamically interact with the ribosome and initiation factors to regulate protein synthesis. Examples include upstream Open Reading Frames (uORFs) which are translated by ribosomes as they scan from the 5’ cap towards the main Open Reading Frame (mORF). We previously discovered that the DENR•MCTS1 complex promotes "translation reinitiation” initiation and it had been suggested that the related factor, eIF2D would function in a similar manner. However, as eIF2D had only been studied in highly reduced biochemical systems, its cellular mRNA targets and mechanism of regulation in vivo were not defined. At the start of our project, essentially nothing was known about eIF2D function and targets in vivo. The questions that motivated our study were: 1) What cellular processes depend on eIF2D in vivo and why? Work in cell-free translation systems had shown that eIF2D (aka ligatin) had interesting functions that partly overlapped with those of the DENR•MCTS1 in recycling of post-termination ribosomes, and can help recruit initiator tRNA in an eIF2-independent manner on a particular class of viral IRESs in vitro, How eIF2D’s molecular activities might affect translation of cellular mRNAs was not explored. 2) Which cellular mRNAs are regulated by eIF2D and how does this relate to the underlying phenotypes? 3) How does eIF2D regulate its mRNA targets? Does it use the same mechanism as the DENR•MCTS1 complex or function in a fundamentally different way? Hence the objectives of this project were to determine the in vivo processes requiring eIF2D, identify specific mRNA targets regulated by eIF2D, and ideally be able to connect the mRNA targets with the cellular processes affected. We also wanted to gain mechanistic insight into how eIF2D controls translation of the identified targets and to understand whether this was related to DENR•MCTS1 complex function. We discovered that the majority of mRNA targets and mode of regulation by eIF2D largely differs from that of the DENR•MCTS1 complex. We found that the crucial role for eIF2D in the nervous system to support locomotion behavior and glutamatergic synaptic transmission involves regulation of specific mRNA targets. Genome-wide characterization of eIF2D translational targets via polysome profiling identified a number of regulated mRNAs. By dissecting regulation using cellular reporter assays, we found that eIF2D regulates a largely non-overlapping set of mRNAs vs. and the unexpected result that eIF2D functions primarily as a translational repressor via 5’UTRs in a manner that is not dependent on uORFs. We also successfully set up a new generally applicable method to study translational control within a specific cell population contained in a complex mixture. The method combines sucrose density gradient fractionation, with cell-specific ribosome isolation via an affinity tag ("Gradient-TRAP’). This combines the strengths of each individual method and overcomes their inherent limitations of each method when applied individually. Importantly, the method can capture translational regulation of specific mRNA isoforms and can be applied in either directed or genome-wide manner. The potential applications of the Gradient-TRAP method are manifold and we hope further development enables broad adoption and refinement. Collectively, our findings provide a better understanding of how eIF2D functions both in an organismal context and on a mechanistic level to regulate its specific mRNA targets. They also establish the key conditions for a new, generally applicable method for identifying specific mRNA targets for translational control within a subset of cells in a complex mixture (e.g. the CNS).
Projektbezogene Publikationen (Auswahl)
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TDP-43 enhances translation of specific mRNAs linked to neurodegenerative disease. Nucleic Acids Research, 47(1), 341-361.
Neelagandan, Nagammal; Gonnella, Giorgio; Dang, Stefan; Janiesch, Philipp C.; Miller, Katharine K.; Küchler, Katrin; Marques, Rita F.; Indenbirken, Daniela; Alawi, Malik; Grundhoff, Adam; Kurtz, Stefan & Duncan, Kent E.
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Motor neuron translatome reveals deregulation of SYNGR4 and PLEKHB1 in mutant TDP-43 amyotrophic lateral sclerosis models. Human Molecular Genetics, 29(16), 2647-2661.
Marques, Rita F.; Engler, Jan B.; Küchler, Katrin; Jones, Ross A.; Lingner, Thomas; Salinas, Gabriela; Gillingwater, Thomas H.; Friese, Manuel A. & Duncan, Kent E.
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Pum2 and TDP-43 refine area-specific cytoarchitecture post-mitotically and modulate translation of Sox5, Bcl11b, and Rorb mRNAs in developing mouse neocortex. eLife, 11 (2022, 3, 9).
Harb, Kawssar; Richter, Melanie; Neelagandan, Nagammal; Magrinelli, Elia; Harfoush, Hend; Kuechler, Katrin; Henis, Melad; Hermanns-Borgmeyer, Irm; Calderon, de Anda Froylan & Duncan, Kent
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SYNGR4 and PLEKHB1 deregulation in motor neurons of amyotrophic lateral sclerosis models: potential contributions to pathobiology. Neural Regeneration Research, 17(2), 266.
Duncan, KentE & Marques, RitaF
