Efficient protein depletion by genetically controlled deprotection of a dormant N‐degron

Methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. Here, we describe a method that allows the control of protein abundance by a genetically encoded regulatory system. We developed a dormant N‐degron that can be attached to the N‐terminus of a protein of interest. Upon expression of a site‐specific protease, the dormant N‐degron becomes deprotected. The N‐degron then targets itself and the attached protein for rapid proteasomal degradation through the N‐end rule pathway. We use an optimized tobacco etch virus (TEV) protease variant combined with selective target binding to achieve complete and rapid deprotection of the N‐degron‐tagged proteins. This method, termed TEV protease induced protein inactivation (TIPI) of TIPI‐degron (TDeg) modified target proteins is fast, reversible, and applicable to a broad range of proteins. TIPI of yeast proteins essential for vegetative growth causes phenotypes that are close to deletion mutants. The features of the TIPI system make it a versatile tool to study protein function in eukaryotes and to create new modules for synthetic or systems biology.     

Toyocamycin specifically inhibits auxin signaling mediated by SCF pathway

The auxins, plant hormones, play a crucial role in many aspects of plant development by regulating cell division, elongation and differentiation. Toyocamycin, a nucleoside-type antibiotic, was identified as auxin signaling inhibitor in a screen of microbial extracts for inhibition of the auxin-inducible reporter gene assay. Toyocamycin specifically inhibited auxin-responsive gene expression, but did not affect other hormone-inducible gene expression. Toyocamycin also blocked auxin-enhanced degradation of the Aux/IAA repressor modulated by the SCF(TIR1) ubiquitin-proteasome pathway without inhibiting proteolytic activity of proteasome. Furthermore, toyocamycin inhibited auxin-induced lateral root formation and epinastic growth of cotyledon in the Arabidopsis thaliana plant. This evidence suggested that toyocamycin would act on the ubiquitination process regulated by SCF(TIR1) machineries. To address the structural requirements for the specific activity of toyocamycin on auxin signaling, the structure-activity relationships of nine toyocamycin-related compounds, including sangivamycin and tubercidin, were investigated.     

A mitochondrial mutator system in maize

Terfestatin A (TrfA), terphenyl-beta-glucoside, was isolated from Streptomyces sp. F40 in a forward screen for compounds that inhibit the expression of auxin-inducible genes in Arabidopsis (Arabidopsis thaliana). TrfA specifically and competitively inhibited the expression of primary auxin-inducible genes in Arabidopsis roots, but did not affect the expression of genes regulated by other plant hormones such as abscisic acid and cytokinin. TrfA also blocked the auxin-enhanced degradation of auxin/indole-3-acetic acid (Aux/IAA) repressor proteins without affecting the auxin-stimulated interaction between Aux/IAAs and the F-box protein TIR1. TrfA treatment antagonized auxin responses in roots, including primary root inhibition, lateral root initiation, root hair promotion, and root gravitropism, but had only limited effects on shoot auxin responses. Taken together, these results indicate that TrfA acts as a modulator of Aux/IAA stability and thus provides a new tool for dissecting auxin signaling.     

Silencing of Toll pathway components by direct injection of double-stranded RNA into Drosophila adult flies

Double-stranded RNA (dsRNA) gene interference is an efficient method to silence gene expression in a sequence-specific manner. Here we show that the direct injection of dsRNA can be used in adult Drosophila flies to disrupt function of endogenous genes in vivo. As a proof of principle, we have used this method to silence components of a major signaling cascade, the Toll pathway, which controls fruit fly resistance to fungal and Gram-positive bacterial infections. We demonstrate that the knockout is efficient only if dsRNA is injected in 4- or more day-old flies and that it lasts for at least 1 week. Furthermore, we report dsRNA-based epistatic gene analysis via injection of a mixture of two dsRNAs and propose that injection of dsRNA represents a powerful method for rapid functional analysis of genes in Drosophila melanogaster adults, particularly of those whose mutations are lethal during development.     

A simplified miRNA-based gene silencing method for Drosophila melanogaster

MicroRNA-based RNA interference is commonly used to produce loss-of-function phenotypes in mammalian systems, but is used only sparingly in invertebrates such as Caenorhabditis elegans and Drosophila melanogaster. Here, we evaluate this method in transgenic strains of D. melanogaster and cultured S2 cells. High throughput-ready expression vectors were developed that permit rapid cloning of synthetic hairpin RNAs. As proof of concept, this method was used for the efficient silencing of dpp gene activity in the adult wing, and the analysis of the general RNA Polymerase II (Pol II) elongation factor, Nelf-E.     

RNA Interference and Development

RNA interference consists in specific mRNA degradation in response to introduction of a double-stranded RNA, homologous in nucleotide sequence. RNA interference was found in eukaryotes and is used in genomics as a powerful method to determine the functions of genes with known nucleotide sequences. RNA interference is considered as a tool of protection against viruses and harmful consequences of mobile elements' transposals. The involvement of the components of RNA interference is considered in spermatogenesis of Drosophila melanogaster and regulation of the expression of genes in Caenorhabditis elegans responsible for temporal patterns of development. The role of RNA interference in stem cell formation and functioning is also considered.     

Nitric oxide influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor

Previous studies have demonstrated that auxin (indole-3-acetic acid) and nitric oxide (NO) are plant growth regulators that coordinate several plant physiological responses determining root architecture. Nonetheless, the way in which these factors interact to affect these growth and developmental processes is not well understood. The Arabidopsis thaliana F-box proteins TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) are auxin receptors that mediate degradation of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to induce auxin-regulated responses. A broad spectrum of NO-mediated protein modifications are known in eukaryotic cells. Here, we provide evidence that NO donors increase auxin-dependent gene expression while NO depletion blocks Aux/IAA protein degradation. NO also enhances TIR1-Aux/IAA interaction as evidenced by pull-down and two-hybrid assays. In addition, we provide evidence for NO-mediated modulation of auxin signaling through S-nitrosylation of the TIR1 auxin receptor. S-nitrosylation of cysteine is a redox-based post-translational modification that contributes to the complexity of the cellular proteome. We show that TIR1 C140 is a critical residue for TIR1-Aux/IAA interaction and TIR1 function. These results suggest that TIR1 S-nitrosylation enhances TIR1-Aux/IAA interaction, facilitating Aux/IAA degradation and subsequently promoting activation of gene expression. Our findings underline the importance of NO in phytohormone signaling pathways.     

Function of the ubiquitin–proteosome pathway in auxin response

Proteolysis of important regulatory proteins by the ubiquitin–proteosome pathway is a key aspect of cellular regulation in eukaryotes. Genetic studies in Arabidopsis indicate that response to auxin depends on the function of proteins in this pathway. The auxin transport inhibitor resistant 1 (TIR1) protein is part of a ubiquitin–protein–ligase complex (E3), known as SKP1 CDC53 F-box^TIR1 (SCF^TIR1), that possibly directs ubiquitin-modification of protein regulators of the auxin response. In yeast, a similar E3 complex, SCF^CDC4, is regulated by conjugation of the ubiquitin-related protein Rub1 to the Cdc53 protein. In Arabidopsis, the auxin-resistant1 (AXR1) gene encodes a subunit of the RUB1-activating enzyme, the first enzyme in the RUB-conjugation pathway. Loss of AXR1 results in loss of auxin response. These results suggest a model in which RUB1 modification regulates the activity of SCF^TIR1, thereby directing the degradation of the repressors of the auxin response.     

A mitochondrial carrier gene, <Emphasis Type="Italic">CG32103</Emphasis>, is highly expressed in the corpora allata in the fruit fly <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> (Diptera: Drosophilidae)

Here we describe a novel gene that is highly expressed in the corpora allata, an endocrine organ responsible for synthesizing juvenile hormones (JHs), in the fruit fly, Drosophila melanogaster Meigen. We isolated an enhancer-trap line in which the transgene was inserted at the locus CG32103, which encodes a mitochondrial carrier family protein with calcium-binding motifs. RNA in situ hybridization revealed that CG32103 is predominantly expressed in the corpora allata in D. melanogaster larvae. Putative orthologs of CG32103 are conserved in many insect species. Mitochondrial carriers are responsible for transporting metabolites across the inner mitochondrial membrane. Given that both mitochondrial membrane transport and cytoplasmic calcium signaling are important for JH biosynthesis regulation, we speculated that CG32103 represents a new member of the family of JH biosynthesis regulators in insects.     

2A and the Auxin-Based Degron System Facilitate Control of Protein Levels in Plasmodium falciparum

Analysis of gene function in Plasmodium falciparum, the most important human malaria parasite, is restricted by the lack of robust and simple reverse genetic tools. Approaches to manipulate protein levels post-translationally are powerful tools to study protein-off effects especially in the haploid malaria parasite where genetic knockouts of essential genes are lethal. We investigated if the auxin-inducible degron system is functional in P. falciparum and found that degron-tagged yellow fluorescent protein levels were efficiently reduced upon addition of auxin which otherwise had no effect on parasite viability. The genetic components required in this conditional approach were co-expressed in P. falciparum by applying the small peptide 2A. 2A is a self-processing peptide from Foot-And-Mouth Disease virus that allows the whole conditional system to be accommodated on a single plasmid vector and ensures stoichiometric expression levels.     

An expanded auxin-inducible degron toolkit for Caenorhabditis elegans

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.     

Data mining for protein–protein interactions in invertebrate model organisms

Well-annotated genome databases are available for many invertebrate species, notably the fruitfly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. An adequate interpretation of this information at the biological level requires the exploration of the interactions between the gene products. Knowledge of protein interactions and the components of cell signalling pathways in the fly and worm are particularly valuable as hypotheses can be rapidly tested using the powerful genetic toolkits available. Invertebrates offer additional experimental advantages when attempting to characterise protein–protein interactions (PPIs). Their relatively small genome size compared to mammals helps to reduce missed interactions due to redundancy, and their function can be addressed using forward (mutants) and reverse (RNA interference) genetics. However, the researcher looking for evidence of PPIs for a protein of interest is faced with the challenge of extracting interaction data from sources that are highly varied, such as the results of microarray experiments in the unstructured text of research papers. This challenge is greatly reduced by a range of public databases of curated information, as well as publicly available, enhanced search engines, which can provide either direct experimental evidence for a PPI, or valuable clues for generating new hypotheses.