FRIENDLY (FMT) is an RNA binding protein associated with cytosolic ribosomes at the mitochondrial surface.

The spatial organization of protein synthesis in the eukaryotic cell is essential for maintaining the integrity of the proteome and the functioning of the cell. Translation on free polysomes or on ribosomes associated with the endoplasmic reticulum has been studied for a long time. More recent data have revealed selective translation of mRNAs in other compartments, in particular at the surface of mitochondria. Although these processes have been described in many organisms, particularky in plants, the mRNA targeting and localized translation mechanisms remain poorly understood. Here, the Arabidopsis thaliana Friendly (FMT) protein is shown to be a cytosolic RNA binding protein that associates with cytosolic ribosomes at the surface of mitochondria. FMT knockout delays seedling development and causes mitochondrial clustering. The mutation also disrupts the mitochondrial proteome, as well as the localization of nuclear transcripts encoding mitochondrial proteins at the surface of mitochondria. These data indicate that FMT participates in the localization of mRNAs and their translation at the surface of mitochondria.

Transcriptional Regulation of Congocidine (Netropsin) Biosynthesis and Resistance.

The production of specialized metabolites by Streptomyces bacteria is usually temporally regulated. This regulation is complex and frequently involves both global and pathway-specific mechanisms. Streptomyces ambofaciens ATCC23877 produces several specialized metabolites, including spiramycins, stambomycins, kinamycins and congocidine. The production of the first three molecules has been shown to be controlled by one or several cluster-situated transcriptional regulators. However, nothing is known regarding the regulation of congocidine biosynthesis. Congocidine (netropsin) belongs to the family of pyrrolamide metabolites, which also includes distamycin and anthelvencins. Most pyrrolamides bind into the minor groove of DNA, specifically in A/T-rich regions, which gives them numerous biological activities, such as antimicrobial and antitumoral activities. We previously reported the characterization of the pyrrolamide biosynthetic gene clusters of congocidine (cgc) in S. ambofaciens ATCC23877, distamycin (dst) in Streptomyces netropsis DSM40846, and anthelvencins (ant) in Streptomyces venezuelae ATCC14583. The three gene clusters contain a gene encoding a putative transcriptional regulator, cgc1, dst1, and ant1, respectively. Cgc1, Dst1, and Ant1 present a high percentage of amino acid sequence similarity. We demonstrate here that Cgc1, an atypical orphan response regulator, activates the transcription of all cgc genes in the stationary phase of S. ambofaciens growth. We also show that the cgc cluster is constituted of eight main transcriptional units. Finally, we show that congocidine induces the expression of the transcriptional regulator Cgc1 and of the operon containing the resistance genes (cgc20 and cgc21, coding for an ABC transporter), and propose a model for the transcriptional regulation of the cgc gene cluster. IMPORTANCE Understanding the mechanisms of regulation of specialized metabolite production can have important implications both at the level of specialized metabolism study (expression of silent gene clusters) and at the biotechnological level (increase of the production of a metabolite of interest). We report here a study on the regulation of the biosynthesis of a metabolite from the pyrrolamide family, congocidine. We show that congocidine biosynthesis and resistance are controlled by Cgc1, a cluster-situated regulator. As the gene clusters directing the biosynthesis of the pyrrolamides distamycin and anthelvencin encode a homolog of Cgc1, our findings may be relevant for the biosynthesis of other pyrrolamides. In addition, our results reveal a new type of feed-forward induction mechanism, in which congocidine induces its own biosynthesis through the induction of the transcription of cgc1.

A genome-scale analysis of mRNAs targeting to plant mitochondria: upstream AUGs in 5' untranslated regions reduce mitochondrial association.

Intracellular sorting of mRNAs is an essential process for regulating gene expression and protein localization. Most mitochondrial proteins are nuclear-encoded and imported into the mitochondria through post-translational or co-translational processes. In the latter case, mRNAs are found to be enriched in the vicinity of mitochondria. A genome-scale analysis of mRNAs associated with mitochondria has been performed to determine plant cytosolic mRNAs targeted to the mitochondrial surface. Many messengers encoding mitochondrial proteins were found associated with mitochondria. These mRNAs correspond to particular functions and complexes, such as respiration or mitoribosomes, which indicates a coordinated control of mRNA localization within metabolic pathways. In addition, upstream AUGs in 5' untranslated regions (UTRs), which modulate the translation efficiency of downstream sequences, were found to negatively affect the association of mRNAs with mitochondria. A mutational approach coupled with in vivo mRNA visualization confirmed this observation. Moreover, this technique allowed the identification of 3'-UTRs as another essential element for mRNA localization at the mitochondrial surface. Therefore, this work offers new insights into the mechanism, function and regulation of the association of cytosolic mRNAs with plant mitochondria.

Removing nucleic acids from nucleoid-associated proteins purified by affinity column.

In bacteria, DNA is tightly compacted in a supercoiled organization, which is mediated in part by nucleoid-associated proteins (NAPs). NAPs are well characterized for their ability to bind nucleic acids and for their involvement in gene regulation. A method commonly used to study protein-nucleic acid interactions involves immunoprecipitation of the protein of interest which is subsequently incubated with nucleic acids. A common cause of artifact is due to nucleic acids that remains bound to the protein of interest during the whole purification process. We developed an optimized method for the purification of tagged NAPs on affinity columns. The combination of three known methods allows removal of most of the nucleic acids bound to proteins during the purification process. This protocol is designed to improve the quality and specificity of results of in vitro experiments involving nucleic acid binding tests on purified NAPs. It can be used for in vitro studies of other RNA/DNA binding proteins.

Natural combinatorial biosynthesis involving two clusters for the synthesis of three pyrrolamides in Streptomyces netropsis.

The pyrrolamides constitute a small family of secondary metabolites that are known for their ability to bind noncovalently to the DNA minor groove with some sequence specificity. To date, only a single pyrrolamide biosynthetic gene cluster has been reported, directing the synthesis of congocidine (netropsin) in Streptomyces ambofaciens. In this study, we improve our understanding of pyrrolamide biosynthesis through the identification and characterization of the gene cluster responsible for the production of distamycin in Streptomyces netropsis DSM40846. We discover that the strain produces two other pyrrolamides, the well-characterized congocidine and a congocidine/distamycin hybrid that we named disgocidine. S. netropsis DSM40846 genome analysis led to the identification of two distinct pyrrolamide-like biosynthetic gene clusters. We show here that these two clusters are reciprocally dependent for the production of the three pyrrolamide molecules. Furthermore, based on detailed functional analysis of these clusters, we propose a biosynthetic route to congocidine and distamycin and an updated model for pyrrolamide assembly. The synthesis of disgocidine, the distamycin/congocidine hybrid, appears to constitute the first example of "natural combinatorial biosynthesis" between two related biosynthetic pathways. Finally, we analyze the genomic context of the two biosynthetic gene clusters and suggest that the presently interdependent clusters result from the coevolution of two ancestral independent pyrrolamide gene clusters.