Identification and functional analysis of an autophagy-related gene TdAtg8 in wild emmer wheat under biotic (fusarium culmorum) and abiotic (drought) stress conditions

Autophagy, literally self eating, is an evolutionary conserved catalytic process for vacuolar degradation of intracellular components, previously examined in yeast, mammals and plants. Abiotic stress factors, including nutrient starvation, oxidative stress, salt stress and osmotic stress have been previously reported to induce autophagy in plants. In this study, for the first time, Atg8 gene was cloned from wild emmer wheat (TdAtg8) and the role of autophagy under biotic and abiotic stress conditions was investigated. Examination of TdAtg8 expression patterns indicates that Atg8 expression was immensely upregulated under drought stress, especially in the roots. Monodansylcadaverine (MDC) and Lysotracker Red markers utilized to observe autophagosomes revealed that autophagy is constitutively active in T. dicoccoides. Moreover, autophagy was determined to be more active in plants exposed to drought stress when compared to plants grown under normal conditions. TdAtg8 gene was demonstrated to complement Atg8 yeast mutants grown under starvation conditions in a drop test assay. For further functional analysis, ATG8 protein from T. dicoccoides were expressed in yeast and analyzed with western blotting. TdAtg8 was also silenced in wild emmer wheat by virus-induced gene silencing and its role was investigated in the presence of a plant pathogen, Fusarium culmorum. This response, for the first time, showed that fungi sporulation decreased in Atg8 silenced plants in comparison to controls. Based on the data obtained, we conclude that the plants become more resistant against the plant pathogen when the autophagy was inhibited

The Caenorhabditis elegans GW182 protein AIN-1 interacts with PAB-1 and subunits of the PAN2-PAN3 and CCR4-NOT deadenylase complexes

GW182 family proteins are essential for miRNA-mediated gene silencing in animal cells. They are recruited to miRNA targets via interactions with Argonaute proteins and then promote translational repression and degradation of the miRNA targets. The human and Drosophila melanogaster GW182 proteins share a similar domain organization and interact with PABPC1 as well as with subunits of the PAN2-PAN3 and CCR4-NOT deadenylase complexes. The homologous proteins in Caenorhabditis elegans, AIN-1 and AIN-2, lack most of the domains present in the vertebrate and insect proteins, raising the question as to how AIN-1 and AIN-2 contribute to silencing. Here, we show that both AIN-1 and AIN-2 interact with Argonaute proteins through GW repeats in the middle region of the AIN proteins. However, only AIN-1 interacts with C. elegans and D. melanogaster PABPC1, PAN3, NOT1 and NOT2, suggesting that AIN-1 and AIN-2 are functionally distinct. Our findings reveal a surprising evolutionary plasticity of the GW182 protein interaction network and demonstrate that binding to PABPC1 and deadenylase complexes has been maintained throughout evolution, highlighting the significance of these interactions for silencing

HELZ directly interacts with CCR4-NOT and causes decay of bound mRNAs

Eukaryotic superfamily (SF) 1 helicases have been implicated in various aspects of RNA metabolism, including transcription, processing, translation, and degradation. Nevertheless, until now, most human SF1 helicases remain poorly understood. Here, we have functionally and biochemically characterized the role of a putative SF1 helicase termed "helicase with zinc-finger," or HELZ. We discovered that HELZ associates with various mRNA decay factors, including components of the carbon catabolite repressor 4-negative on TATA box (CCR4-NOT) deadenylase complex in human and Drosophila melanogaster cells. The interaction between HELZ and the CCR4-NOT complex is direct and mediated by extended low-complexity regions in the C-terminal part of the protein. We further reveal that HELZ requires the deadenylase complex to mediate translational repression and decapping-dependent mRNA decay. Finally, transcriptome-wide analysis of Helz-null cells suggests that HELZ has a role in the regulation of the expression of genes associated with the development of the nervous system

A DDX6-CNOT1 Complex and W-Binding Pockets in CNOT9 Reveal Direct Links between miRNA Target Recognition and Silencing

CCR4-NOT is a major effector complex in miRNA-mediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)-containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. Here, we elucidate the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. We show that the conserved CNOT9 subunit attaches to a domain of unknown function (DUF3819) in the CNOT1 scaffold. The resulting complex provides binding sites for TNRC6/GW182, and its crystal structure reveals tandem W-binding pockets located in CNOT9. We further show that the CNOT1 MIF4G domain interacts with the C-terminal RecA domain of DDX6, a translational repressor and decapping activator. The crystal structure of this complex demonstrates striking similarity to the eIF4G-eIF4A complex. Together, our data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping

Structure and assembly of the NOT module of the human CCR4–NOT complex

The CCR4-NOT deadenylase complex is a master regulator of translation and mRNA stability. Its NOT module orchestrates recruitment of the catalytic subunits to target mRNAs. We report the crystal structure of the human NOT module formed by the CNOT1, CNOT2 and CNOT3 C-terminal (-C) regions. CNOT1-C provides a rigid scaffold consisting of two perpendicular stacks of HEAT-like repeats. CNOT2-C and CNOT3-C heterodimerize through their SH3-like NOT-box domains. The heterodimer is stabilized and tightly anchored to the surface of CNOT1 through an unexpected intertwined arrangement of peptide regions lacking defined secondary structure. These assembly peptides mold onto their respective binding surfaces and form extensive interfaces. Mutagenesis of individual interfaces and perturbation of endogenous protein ratios cause defects in complex assembly and mRNA decay. Our studies provide a structural framework for understanding the recruitment of the CCR4-NOT complex to mRNA targets