CUL3BPM E3 ubiquitin ligases regulate MYC2, MYC3, and MYC4 stability and JA responses

The jasmonate (JA)-pathway regulators MYC2, MYC3, and MYC4 are central nodes in plant signaling networks integrating environmental and developmental signals to fine-tune JA defenses and plant growth. Continuous activation of MYC activity is potentially lethal. Hence, MYCs need to be tightly regulated in order to optimize plant fitness. Among the increasing number of mechanisms regulating MYC activity, protein stability is arising as a major player. However, how the levels of MYC proteins are modulated is still poorly understood. Here, we report that MYC2, MYC3, and MYC4 are targets of BPM (BTB/POZ-MATH) proteins, which act as substrate adaptors of CUL3-based E3 ubiquitin ligases. Reduction of function of CUL3BPM in amiR-bpm lines, bpm235 triple mutants, and cul3ab double mutants enhances MYC2 and MYC3 stability and accumulation and potentiates plant responses to JA such as root-growth inhibition and MYC-regulated gene expression. Moreover, MYC3 polyubiquitination levels are reduced in amiR-bpm lines. BPM3 protein is stabilized by JA, suggesting a negative feedback regulatory mechanism to control MYC activity, avoiding harmful runaway responses. Our results uncover a layer for JA-pathway regulation by CUL3BPM-mediated degradation of MYC transcription factors.This work was funded by Spanish Ministry for Science and Innovation Grants BIO2016-77216-R (Ministerio de Economia [MINECO]/Fondos Europeos de Desarrollo Regional [FEDER]) (to R.S.) and BIO2016-80551-R (MINECO/FEDER) (to V.R.). E.C. was the recipient of a Formación de Personal Investigador grant from MINECO (Reference BES-2017-081147). The mass spectrometry instrumentation was funded by the University of Strasbourg (IdEx “Equipement mi-Lourd” 2015) and by “Laboratoires d’Excellence” Grant ANR-10-LABX-0036 (NETRNA)

Pain exacerbates chronic mild stress-induced changes in noradrenergic transmission in rats

Depression can influence pain and vice versa, yet the biological mechanisms underlying how one influences the pathophysiology of the other remains unclear. Dysregulation of locus coeruleus-noradrenergic transmission is implicated in both conditions, although it is not known whether this effect is exacerbated in cases of co-morbid depression and chronic pain. We studied locus coeruleus activity using immunofluorescence and electrophysiological approaches in rats subjected to unpredictable chronic mild stress (CMS, an experimental model of depression) and/or chronic constriction injury (CCI, a model of chronic neuropathic pain) for 2 weeks. CCI alone had no effect on any of the locus coeruleus parameters studied, while CMS led to a slight reduction in the electrophysiological activity of the locus coeruleus. Furthermore, CMS was associated with an increase in the number of tyrosine hydroxylase-positive cells in the locus coeruleus, although they were smaller in size. Interestingly, these effects of CMS were exacerbated when combined with CCI, even though no changes in the α2-adrenoreceptors or the noradrenaline transporter were observed in any group. Together, these findings suggest that CMS triggers several modifications in locus coeruleus-noradrenergic transmission that are exacerbated by co-morbid chronic pain

Regulation and Role of Arabidopsis CUL4-DDB1A-DDB2 in Maintaining Genome Integrity upon UV Stress

Plants use the energy in sunlight for photosynthesis, but as a consequence are exposed to the toxic effect of UV radiation especially on DNA. The UV-induced lesions on DNA affect both transcription and replication and can also have mutagenic consequences. Here we investigated the regulation and the function of the recently described CUL4-DDB1-DDB2 E3 ligase in the maintenance of genome integrity upon UV-stress using the model plant Arabidopsis. Physiological, biochemical, and genetic evidences indicate that this protein complex is involved in global genome repair (GGR) of UV-induced DNA lesions. Moreover, we provide evidences for crosstalks between GGR, the plant-specific photo reactivation pathway and the RAD1-RAD10 endonucleases upon UV exposure. Finally, we report that DDB2 degradation upon UV stress depends not only on CUL4, but also on the checkpoint protein kinase Ataxia telangiectasia and Rad3-related (ATR). Interestingly, we found that DDB1A shuttles from the cytoplasm to the nucleus in an ATR-dependent manner, highlighting an upstream level of control and a novel mechanism of regulation of this E3 ligase

Arabidopsis CULLIN3 Genes Regulate Primary Root Growth and Patterning by Ethylene-Dependent and -Independent Mechanisms

CULLIN3 (CUL3) together with BTB-domain proteins form a class of Cullin-RING ubiquitin ligases (called CRL3s) that control the rapid and selective degradation of important regulatory proteins in all eukaryotes. Here, we report that in the model plant Arabidopsis thaliana, CUL3 regulates plant growth and development, not only during embryogenesis but also at post-embryonic stages. First, we show that CUL3 modulates the emission of ethylene, a gaseous plant hormone that is an important growth regulator. A CUL3 hypomorphic mutant accumulates ACS5, the rate-limiting enzyme in ethylene biosynthesis and as a consequence exhibits a constitutive ethylene response. Second, we provide evidence that CUL3 regulates primary root growth by a novel ethylene-dependant pathway. In particular, we show that CUL3 knockdown inhibits primary root growth by reducing root meristem size and cell number. This phenotype is suppressed by ethylene-insensitive or resistant mutations. Finally, we identify a function of CUL3 in distal root patterning, by a mechanism that is independent of ethylene. Thus, our work highlights that CUL3 is essential for the normal division and organisation of the root stem cell niche and columella root cap cells

Caractérisation moléculaire et génétique de différentes sous-unités du complexe SCF chez Arabidopsis thaliana

La protéolyse ubiquitine-dépendante est un mécanisme conservé permettant la dégradation ciblée de nombreuses protéines chez les eucaryotes. Dans ce système, l'ubiquitine sert d'étiquette pour adresser la protéine à la machinerie de dégradation, le complexe multiprotéolytique protéasome 26S. Les ubiquitine proteine-ligases (E3) sont les enzymes responsables de la spécifité de reconnaissance du substrat. Mon travail de thèse a porté principalement sur une classe de E3, le complexe SCF. Le SCF, initialement identifié chez la levure, est composé de 4 sous unités majeures. Skp1 et Culline1 forment la charpente du complexe, la protéine à F-box assure la reconnaisance du substrat et RBX1 est la sous-unité catalytique. L'implication du SCF dans différentes voies de signalisation a été montrée par l'analyse de mutants perte de fonction chez Arabidopsis: réponse à l'auxine, au jasmonate, le développement floral, rythme circadien, photomorphogénèse, sénescence?Les résultats obtenus et décrits dans ce mémoire ont permis d'identifier et de caractériser différentes sous-unités du complexe SCF chez Arabidopsis thaliana. Le travail de caractérisation du gène AtCulline1 a révélé le rôle critique du SCF au cours du développement embryonnaire.D'autre part, nous avons caractérisé la protéine AtRBX1 et nous avons démontré qu'elle joue un rôle actif au sein du complexe SCF chez Arabidopsis thaliana. L'étude de plantes transgéniques RNAi-RBX1 a montré qu'un niveau d'accumulation réduit de AtRBX1 provoque de fortes modifications au cours de la croissance de la plante mais aussi dans la voie de signalisation du jasmonate.Enfin la caractérisation moléculaire et génétique de deux protéines à F-box, EBF1 et EBF2 a mis en évidence l'implication de la protéolyse SCF dépendante dans la voie de signalisation de l'éthylène. Nous proposons un modèle dans lequel EIN3 est constitutivement ubiquitinée et degradée par les complexes SCFEBF1/EBF2 et ne peut s'accumuler qu'après un traitement à l'éthylène.The ubiquitin/proteasome pathway promotes the degradation of key regulatory proteins in eukaryotes. Ubiquitin, a small protein, is used as a tag that targets a substrate protein to a multiproteolytic complex, the 26S proteasome. E3 ubiquitin-ligases are responsible for the substrate recognition. I focused my work on one class of E3 ligases, the SCF complex. The SCF complex has been first characterized in yeast. It is a multiprotein complex composed of at least four subunits: Cullin1 and SKP1 that form the scaffold of the complex, the F-box protein that binds the substrate and RBX1, the catalytic subunit. In Arabidopsis thaliana each subunit is encoded by a multigene family. Analyses of several F-box mutants have already shown that SCF-dependent proteolysis is involved in many signalling and developmental pathways, for example in auxin and jasmonate perception, floral development, circadian rhythm, photomorphogenesis, senescence?.In this thesis, I describe the identification and functional characterization of different SCF subunits from Arabidopsis thaliana. By studying the AtCullin1, we showed that the SCF complex plays a critical role during embryogenesis.We also identified AtRBX1, and demonstrated that it is indeed a component of the SCF complex in Arabidopsis thaliana. The down-regulation of AtRBX1 by a RNAi strategy causes severe developmental defects. More over these plants are impaired in jasmonate response.The molecular end genetic characterisation of two F-box proteins EBF1 and EBF2 revealed a role for SCF-dependant proteolysis in ethylene signal transduction. We proposed a model in which the EIN3 transcription factor is constitutively degraded via the SCFEBF1/EBF2 and only transiently accumulate in the presence of ethylene.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

The emerging family of CULLIN3-RING ubiquitin ligases (CRL3s): cellular functions and disease implications

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The Exoribonuclease XRN4 Is a Component of the Ethylene Response Pathway in \u3cem\u3eArabidopsis\u3c/em\u3e

EXORIBONUCLEASE4 (XRN4), the Arabidopsis thaliana homolog of yeast XRN1, is involved in the degradation of several unstable mRNAs. Although a role for XRN4 in RNA silencing of certain transgenes has been reported, xrn4 mutant plants were found to lack any apparent visible phenotype. Here, we show that XRN4 is allelic to the unidentified components of the ethylene response pathway ETHYLENE-INSENSITIVE5/ACC-INSENSITIVE1 (EIN5/AIN1) and EIN7. xrn4 mutant seedlings are ethylene-insensitive as a consequence of the upregulation of EIN3 BINDING F-BOX PROTEIN1 (EBF1) and EBF2 mRNA levels, which encode related F-box proteins involved in the turnover of EIN3 protein, a crucial transcriptional regulator of the ethylene response pathway. Epistasis analysis placed XRN4/EIN5/AIN1 downstream of CTR1 and upstream of EBF1/2. XRN4 does not appear to regulate ethylene signaling via an RNA-INDUCED SILENCING COMPLEX–based RNA silencing mechanism but acts by independent means. The identification of XRN4 as an integral new component in ethylene signaling adds RNA degradation as another posttranscriptional process that modulates the perception of this plant hormone