Search

Deep-UV resonance Raman spectroscopy has been shown to offer great potential for probing the in situ stability of mRNA vaccines. In this study, a vaccine model was subjected to controlled degradation using RNase A or through aging at room temperature. The degradation of mRNA was confirmed by using a cell transfection test and by gel electrophoresis. Under both settings, DUVRR spectroscopy successfully revealed the mRNA degradation signs of the vaccine model.

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., (© 2023. The Author(s).)

The exosome complex plays a central and essential role in RNA metabolism. However, comprehensive studies of exosome substrates and functional analyses of its subunits are lacking. Here, we demonstrate that as opposed to yeast and metazoans the plant exosome core possesses an unanticipated functional plasticity and present a genome-wide atlas of Arabidopsis exosome targets. Additionally, our study provides evidence for widespread polyadenylation- and exosome-mediated RNA quality control in plants, reveals unexpected aspects of stable structural RNA metabolism, and uncovers numerous novel exosome substrates. These include a select subset of mRNAs, miRNA processing intermediates, and hundreds of noncoding RNAs, the vast majority of which have not been previously described and belong to a layer of the transcriptome that can only be visualized upon inhibition of exosome activity. These first genome-wide maps of exosome substrates will aid in illuminating new fundamental components and regulatory mechanisms of eukaryotic transcriptomes.

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.

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.

Previously characterized by us barley chloroplast psbA gene, which encodes one of the main Photosystem II components--D1 protein, has been inserted in a set of special plasmid vectors and its expression in vitro and in vivo has been investigated. Experiments on the in vitro expression in the rabbit reticulocyte lysate system revealed a major product with a molecular weight ca. 33.5 kD, which corresponds to the unprocessed D1 barley protein. A lower molecular weight protein (about 29 kD) was also observed. These results are in agreement with the existence of two potential translation start sites in the psbA gene in the same reading frame, the second one starting from Met37 residue. The results fully correlate with the earlier data on the in vitro expression of psbA genes of maize, pea, and tobacco. Experiments on the in vivo expression of psbA gene in E. coli cells with the above constructions also revealed proteins with m. w. about 33.5 and 29 kD. The yield of the target recombinant protein in some cases was about 25-30% of the total E. coli cellular protein. The correspondence of the bands to the desired products was proved by the immunoenzyme analysis with the use of polyclonal antibodies. The data obtained show for the first time the construction of E. coli strains producing recombinant D1 protein of cereals in a high level.

Mutagenesis of two previously cloned plant genes, maize storage protein cZ22B1 gene and barley Photosystem II protein D1 gene (psbA), was carried out. To improve the nutritional quality of zein, the DNA region corresponding to the protein sixth alpha-helix rod was substituted by a synthetic segment bearing three codon changes for Lys. Additional stabilization of this helix was achieved by three more codon changes for Glu. By means of oligonucleotide directed mutagenesis five different copies of psbA gene were obtained, bearing single codon change of Ser264 (wild type) for Gly, Ala, Cys, Asn, and Thr, respectively. These constructs can be used for studying functional topography of protein D1 and core region.

In order to determine structures of the barley photosystem II subunits, the following genes have been cloned: psbB, encoding 47 kDa chlorophyll-binding subunit; psbH, encoding 7.7 kDa phosphoprotein; psbE and psbF, encoding 9.3 and 4.4 kDa subunits of the cytochrome b559 apoprotein, respectively; and a fragment of psbC gene, encoding the 43 kDa chlorophyll-binding subunit. The nucleotide sequences of these genes and the deduced amino acid sequences of their products are highly homologous to the corresponding sequences for other plant species.

A useful and efficient approach to the synthesis of DNA duplexes of practically unlimited length has been developed. The proposed methodology is based on the use of temporary restriction sites for subcloning and assembling the segments of the desired DNA. It allows the utilization of chemically synthesized oligonucleotides of various length (from 10- to 100-mers) for the duplex construction. The application of this approach to the synthesis of a gene for the functionally active bacteriorhodopsin fragment is described.

The psbD gene for the membrane polypeptide D2 has been isolated from barley chloroplast DNA and its sequence, along with the flanking regions, has been determined. The 3'-end of the psbD gene is overlapped with a 50 bp stretch of a second open reading frame which belongs to the psbC gene encoding the P6 protein of photosystem II.

A modified phosphotriester method has been employed for the efficient chemical synthesis of long-chain deoxyribooligonucleotides. During the course of this work, a general and rapid procedure was developed for the preparation of 24-62-mers in solution. Preparative reversed phase column chromatography on silanized silica gel was used to purify triester intermediates starting from 10-mers. The rapid synthesis of 32-mer and 42-mer on glass and silica gel supports using suitably protected 2-8-mer blocks as coupling units has been also accomplished. In particular, a convenient procedure for the solid-phase synthesis of oligonucleotide blocks bearing 3'-terminal phosphodiester groups is described.

A rapid and convenient method for the synthesis of deoxyribooligonucleotides has been developed using the phosphotriester approach. The advantage of this methodology for work in solution was successfully demonstrated in synthesis of a number of DNA fragments up to 32-long. Adaptation of the presented method to solid-phase synthesis allows a pentadecamer to be assembled in 4-5 hours using dinucleotides as coupling units.

A chemical-enzymatic synthesis of 271- and 286-bp DNA duplexes, each of which contains the entire sequence coding for human proinsulin has been accomplished. In addition to the coding sequence, the 271-bp fragment carries translation initiation and termination signals plus EcoRI-HindIII restriction enzyme sites for insertion into an appropriate plasmid vector. The 286-bp fragment also contains a Shine-Dalgarno (SD) sequence preceding an ATG codon. Employing the 286-bp polynucleotide, the 568-bp tandem proinsulin gene has been obtained. The synthesis of these DNA fragments involved preparation of 42 oligonucleotides by a rapid N-methylimidazolide phosphotriester method and enzymatic conversion of the oligonucleotides into the gene subfragments, which were cloned separately and fused to yield the desired DNAs coding for proinsulin. The proinsulin gene fragments were cloned in Escherichia coli and shown to have the correct sequences.