Hormone-Mediated Gene-Specific Translation Regulation

The central role of translation regulation in the control of critical cellular processes has long been recognized. Yet the systematic exploration of quantitative changes in translation at a genome-wide scale in response to specific stimuli has only recently become technically feasible. Using a genetic approach, we have identified new Arabidopsis weak-ethylene insensitive mutants that also display defects in translation, which suggested the existence of a previously unknown molecular module involved in ethylene-mediated translation regulation of components of this signaling pathway. To explore this link in detail, we implemented for Arabidopsis the ribosome-footprinting technology, which enables the study of translation at a whole-genome level at single codon resolution[1]. Using ribosome-footprinting we examined the effects of short exposure to ethylene on the Arabidopsis translatome looking for ethylene-triggered changes in translation rates that could not be explained by changes in transcript levels. The results of this research, in combination with the characterization of a subset of the aforementioned weak-ethylene insensitive mutants that are defective in the UPF genes (core-components of the nonsense-mediated mRNA decay machinery), uncovered a translation-based branch of the ethylene signaling pathway[2]. In the presence of ethylene, translation of a negative regulator of ethylene signaling EBF2 is repressed, despite induced transcription of this gene. These translational effects of ethylene require the long 3´UTR of EBF2 (3´EBF2), which is recognized by the C-terminal end of the key ethylene-signaling protein EIN2 (EIN2C) in the cytoplasm once EIN2C is released from the ER-membrane by proteolytic cleavage. EIN2C binds the 3´EBF2, recruits the UPF proteins and moves to P-bodies, where the translation of EBF2 in inhibited despite its mRNA accumulation. Once the ethylene signal is withdrawn, the translation of the stored EBF2 mRNAs is resumed, thus rapidly dampening the ethylene response. These findings represent a mechanistic paradigm of gene-specific regulation of translation in response to a key growth regulator. Translation regulatory elements can be located in both 3′ and 5′ UTRs. We are now focusing on the ead1 and ead2 mutants, another set of ethylene-signaling mutants defective in translational regulation. Ribosome-footprinting on the ead1 mutant revealed an accumulation of translating ribosomes in the 5´UTRs of uORF-containing genes and reduction in the levels of ribosomes in the main ORF. The mutant is also impaired in the translation of GFP when this reporter is fused to WT 5´UTR of potential EAD1 targets but not when GFP is fused to the uORF-less versions of the same 5´UTRs. Our hypothesis is that EAD1/2 work as a complex that is required for the efficient translation of mRNAs that have common structural (complex 5´UTR with uORFs) and functional (regulation of key cellular processes) features. We are working towards the identification of the conditions where the EAD1 regulation of translation is required. [1] Ingolia, N. et al. (2009) Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling. Science, 324; 218-222 [2] Merchante, C. et al. (2015) Gene-Specific Translation Regulation Mediated by the Hormone-Signaling Molecule EIN2. Cell, 163(3): 684-697Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Translation regulation of uORFs-containing genes in Arabidopsis

Translational regulation has long been recognized as a vital process in the cell responses to the environment and in the execution of developmental programs, yet still little is known about the selective translation of specific mRNAs and its regulation. By the implementation of the Ribo-Seq technology in Arabidopsis [1], we uncovered a translation regulation module in response to ethylene that involves the key-signalling gene EIN2, the 3´UTRs of EBF1 and 2 and the NMD machinery [2]. We now focus on the ead1,2 mutants, which present altered responses to ethylene and auxin. The two corresponding genes represent the likely orthologs of translation factors from yeast and humans, and this, together with their inferred roles in multiple response pathways, offer an excellent opportunity to investigate the role of signal integration at the translational level. EAD1 and 2 interact and Ribo-seq on ead1 revealed an accumulation of translating ribosomes in the 5´UTRs of uORF-containing genes and reduction in the levels of ribosomes in the main ORF. ead1 is also impaired in the translation of GFP when fused to WT 5´UTR of potential EAD1 targets but not when fused to uORF-less versions of the same 5´UTRs. Our hypothesis is that, under certain hormonal and/or environmental conditions, EAD1/2 work as a complex required for the efficient translation of mRNAs that have common structural and functional features. Our progress towards the identification of the conditions where the EAD1 regulation of translation is required will be presented. [1] Merchante C et al. Bio-Protocol, 6(21), 1–34. [2] Merchante C et al. (2015) Cell, 163(3): 684-697Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Gene-Specific Translation Regulation Mediated by the Hormone-Signaling Molecule EIN2

SummaryThe central role of translation in modulating gene activity has long been recognized, yet the systematic exploration of quantitative changes in translation at a genome-wide scale in response to a specific stimulus has only recently become technically feasible. Using the well-characterized signaling pathway of the phytohormone ethylene and plant-optimized genome-wide ribosome footprinting, we have uncovered a molecular mechanism linking this hormone’s perception to the activation of a gene-specific translational control mechanism. Characterization of one of the targets of this translation regulatory machinery, the ethylene signaling component EBF2, indicates that the signaling molecule EIN2 and the nonsense-mediated decay proteins UPFs play a central role in this ethylene-induced translational response. Furthermore, the 3′UTR of EBF2 is sufficient to confer translational regulation and required for the proper activation of ethylene responses. These findings represent a mechanistic paradigm of gene-specific regulation of translation in response to a key growth regulator

Systems analysis of auxin transport in the Arabidopsis root apex

Auxin is a key regulator of plant growth and development. Within the root tip, auxin distribution plays a crucial role specifying developmental zones and coordinating tropic responses. Determining how the organ-scale auxin pattern is regulated at the cellular scale is essential to understanding how these processes are controlled. In this study, we developed an auxin transport model based on actual root cell geometries and carrier subcellular localizations. We tested model predictions using the DII-VENUS auxin sensor in conjunction with state-of-the-art segmentation tools. Our study revealed that auxin efflux carriers alone cannot create the pattern of auxin distribution at the root tip and that AUX1/LAX influx carriers are also required. We observed that AUX1 in lateral root cap (LRC) and elongating epidermal cells greatly enhance auxin’s shootward flux, with this flux being predominantly through the LRC, entering the epidermal cells only as they enter the elongation zone. We conclude that the nonpolar AUX1/LAX influx carriers control which tissues have high auxin levels, whereas the polar PIN carriers control the direction of auxin transport within these tissues

Multilevel Interactions between Ethylene and Auxin in Arabidopsis Roots[W]

Hormones play a central role in the coordination of internal developmental processes with environmental signals. Herein, a combination of physiological, genetic, cellular, and whole-genome expression profiling approaches has been employed to investigate the mechanisms of interaction between two key plant hormones: ethylene and auxin. Quantification of the morphological effects of ethylene and auxin in a variety of mutant backgrounds indicates that auxin biosynthesis, transport, signaling, and response are required for the ethylene-induced growth inhibition in roots but not in hypocotyls of dark-grown seedlings. Analysis of the activation of early auxin and ethylene responses at the cellular level, as well as of global changes in gene expression in the wild type versus auxin and ethylene mutants, suggests a simple mechanistic model for the interaction between these two hormones in roots, according to which ethylene and auxin can reciprocally regulate each other's biosyntheses, influence each other's response pathways, and/or act independently on the same target genes. This model not only implies existence of several levels of interaction but also provides a likely explanation for the strong ethylene response defects observed in auxin mutants

Author Correction: A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate

The original version of this Article omitted the following from the Acknowledgements: ‘We also thank DBT-CREST BT/HRD/03/01/2002.’ This has been corrected in both the PDF and HTML versions of the Article