Sergei V. Reverdatto

Deadenylation of mRNA is often the first and rate-limiting step in mRNA decay. PARN, a poly(A)-specific 3' --> 5' ribonuclease which is conserved in many eukaryotes, has been proposed to be primarily responsible for such a reaction, yet the importance of the PARN function at the whole-organism level has not been demonstrated in any species. Here, we show that mRNA deadenylation by PARN is essential for viability in higher plants (Arabidopsis thaliana). Yet, this essential requirement for the PARN function is not universal across the phylogenetic spectrum, because PARN is dispensable in Fungi (Schizosaccharomyces pombe), and can be at least severely downregulated without any obvious consequences in Metazoa (Caenorhabditis elegans). Development of the Arabidopsis embryos lacking PARN (AtPARN), as well as of those expressing an enzymatically inactive protein, was markedly retarded, and ultimately culminated in an arrest at the bent-cotyledon stage. Importantly, only some, rather than all, embryo-specific transcripts were hyperadenylated in the mutant embryos, suggesting that preferential deadenylation of a specific select subset of mRNAs, rather than a general deadenylation of the whole mRNA population, by AtPARN is indispensable for embryogenesis in Arabidopsis. These findings indicate a unique, nonredundant role of AtPARN among the multiple plant deadenylases.

A multisubunit form of acetyl coenzyme A (CoA) carboxylase (ACCase) from soybean (Glycine max) was characterized. The enzyme catalyzes the formation of malonyl CoA from acetyl CoA, a rate-limiting step in fatty acid biosynthesis. The four known components that constitute plastid ACCase are biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and the alpha- and beta-subunits of carboxyltransferase (alpha- and beta-CT). At least three different cDNAs were isolated from germinating soybean seeds that encode BC, two that encode BCCP, and four that encode alpha-CT. Whereas BC, BCCP, and alpha-CT are products of nuclear genes, the DNA that encodes soybean beta-CT is located in chloroplasts. Translation products from cDNAs for BC, BCCP, and alpha-CT were imported into isolated pea (Pisum sativum) chloroplasts and became integrated into ACCase. Edman microsequence analysis of the subunits after import permitted the identification of the amino-terminal sequence of the mature protein after removal of the transit sequences. Antibodies specific for each of the chloroplast ACCase subunits were generated against products from the cDNAs expressed in bacteria. The antibodies permitted components of ACCase to be followed during fractionation of the chloroplast stroma. Even in the presence of 0.5 M KCl, a complex that contained BC plus BCCP emerged from Sephacryl 400 with an apparent molecular mass greater than about 800 kD. A second complex, which contained alpha- and beta-CT, was also recovered from the column, and it had an apparent molecular mass of greater than about 600 kD. By mixing the two complexes together at appropriate ratios, ACCase enzymatic activity was restored. Even higher ACCase activities were recovered by mixing complexes from pea and soybean. The results demonstrate that the active form of ACCase can be reassembled and that it could form a high-molecular-mass complex.

Inter-organelle contact and communication between mitochondria and sarco/endoplasmic reticulum (SR/ER) maintain cellular homeostasis and are profoundly disturbed during tissue ischemia. We tested the hypothesis that the formin Diaphanous-1 (DIAPH1), which regulates actin dynamics, signal transduction and metabolic functions, contributes to these processes. We demonstrate that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to shorten mitochondria-SR/ER distance, thereby enhancing mitochondria-ER contact in cells including cardiomyocytes, endothelial cells and macrophages. Solution structure studies affirm the interaction between the Diaphanous Inhibitory Domain and the cytosolic GTPase domain of MFN2. In male rodent and human cardiomyocytes, DIAPH1-MFN2 interaction regulates mitochondrial turnover, mitophagy, and oxidative stress. Introduction of synthetic linker construct, which shorten the mitochondria-SR/ER distance, mitigated the molecular and functional benefits of DIAPH1 silencing in ischemia. This work establishes fundamental roles for DIAPH1-MFN2 interaction in the regulation of mitochondria-SR/ER contact networks. We propose that targeting pathways that regulate DIAPH1-MFN2 interactions may facilitate recovery from tissue ischemia.

Two subtilisin-like proteases (SLP) were identified in soybean (Glycine max [L.] Merr.). The first, SLP-1, was localized in seed coats early in seed development, but became undetectable with anti-SLP-1 antibodies as seed fill progressed. A partial purification of SLP-1 was achieved using a two step chromatographic procedure. NH2-terminal sequence analysis of the partially purified enzyme permitted primers to be designed that were used to amplify cDNA encoding SLP-1. A genomic clone encoding SLP-1 was also obtained. Characterization of the cDNA and partially purified SLP-1 revealed the initial translation product was an 82 694 MW precursor. After removal of a signal peptide, the mature protein was formed by removal of an NH2-terminal propeptide. A COOH-terminal peptide also appeared to be removed from some of the protease molecules. DNA blot analysis suggested that at least one additional SLP gene was present in soybean. The second gene, SLP-2, was subsequently cloned and characterized. Although the coding regions for SLP-1 and SLP-2 were homologous, their promoters were quite divergent. RT-PCR revealed that SLP-2 message was found in the mature plant and in cotyledons of germinating seeds. Although SLP-2 mRNA could be identified in developing seeds, the message was at least an order of magnitude less abundant than that for SLP-1, and it was mis-spliced such that a chain termination event would preclude obtaining a product. As with SLPs from other organisms, the functions of the soybean proteases are unknown. However, SLP-1 is one of only a few proteins from soybean seed coats that have been described.