Study of the RNA splicing kinetics via in vivo 5-ethynyl uridine labelling.

Splicing, a process of intron removal from eukaryotic RNA transcripts, is an important step of gene expression in all eukaryotes. Splice sites might be used with different efficiency giving rise to alternative splicing products. At the same time, splice sites might be utilised at a variable rate. We used 5-ethynyl uridine labelling to sequence a nascent transcriptome of HeLa cells and deduce the rate of splicing for each donor and acceptor splice site. The following correlation analysis allowed us to assess a correspondence of primary transcript features with the rate of splicing. Some dependencies we revealed were anticipated, such as splicing rate decrease with a decreased complementarity of donor splice site to U1 and acceptor sites to U2 snRNAs, or an acceleration of donor site usage if an upstream acceptor site is located at a shorter distance. Other dependencies were more surprising, like a negative influence of a distance to the 5' end on the rate of acceptor splicing site utilization, or the differences in splicing rate between long, short and RBM17-dependent introns. We also observed a deceleration of last intron splicing with an increase of the distance to the polyA site, which might be explained by a cooperativity of the splicing and polyadenylation. In addition, we performed the analysis of splicing kinetics of SF3B4 knockdown cells which suggested the impairment of U2 snRNA recognition step. As a result, we deconvoluted the effects of several examined features on the splicing rate into a single regression model. The data obtained here are useful for further studies in the field as it provides general splicing rate dependencies as well as helps justify the existence of slowly removed splice sites, e.g. to ensure alternative splicing.

An easy tool to monitor the elemental steps of in vitro translation via gel electrophoresis of fluorescently labeled small peptides.

Several methods are available to visualize and assess the kinetics and efficiency of elemental steps of protein biosynthesis. However, each of these methods has its own limitations. Here, we present a novel, simple and convenient tool for monitoring stepwise in vitro translation initiated by BODIPY-Met-tRNA. Synthesis and release of very short, 1-7 amino acids, BODIPY-labeled peptides, can be monitored using urea-polyacrylamide gel electrophoresis. Very short BODIPY-labeled oligopeptides might be resolved this way, in contrast to widely used Tris-tricine gel electrophoresis, which is suitable to separate peptides larger than 1 kDa. The method described in this manuscript allows one to monitor the steps of translation initiation, peptide transfer, translocation, and termination as well as their inhibition at an unprecedented single amino acid resolution.

Gausemycin Antibiotic Family Acts via Ca2+-Dependent Membrane Targeting.

We report the molecular mechanism of action of gausemycins and the isolation of new members of the family, gausemycins C (1c), D (1d), E (1e), and F (1f), the minor components of the mixture. To elucidate the mechanism of action of gausemycins, we investigated the antimicrobial activity of the most active compounds, gausemycins A and B, in the presence of Ca2+, other metal ions, and phosphate. Gausemycins require a significantly higher Ca2+ concentration for maximum activity than daptomycin but lower than that required for malacidine and cadasides. Species-specific antimicrobial activity was found upon testing against a wide panel of Gram-positive bacteria. Membranoactivity of gausemycins was demonstrated upon their interactions with model lipid bilayers and micelles. The pore-forming ability was found to be dramatically dependent on the Ca2+ concentration and the membrane lipid composition. An NMR study of gausemycin B in zwitterionic and anionic micelles suggested the putative structure of the gausemycin/membrane complex and revealed the binding of Ca2+ by the macrocyclic domain of the antibiotic.

Synthetic Activators of Autophagy.

Autophagy is a central process for degradation of intracellular components that do not operate correctly. Molecular mechanisms underlying this process are extremely difficult to study, since they involve a large number of participants. The main task of autophagy is redistribution of cellular resources in response to environmental changes, such as starvation. Recent studies show that autophagy regulation could be the key to achieve healthy longevity, as well as to create therapeutic agents for treatment of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Thus, development of autophagy activators with established detailed mechanism of action is a really important area of research. Several commercial companies are at various stages of development of such molecules, and some of them have already begun to introduce autophagy activators to the market.

Natural Activators of Autophagy.

Autophagy is the process by which cell contents, such as aggregated proteins, dysfunctional organelles, and cell structures are sequestered by autophagosome and delivered to lysosomes for degradation. As a process that allows the cell to get rid of non-functional components that tend to accumulate with age, autophagy has been associated with many human diseases. In this regard, the search for autophagy activators and the study of their mechanism of action is an important task for treatment of many diseases, as well as for increasing healthy life expectancy. Plants are rich sources of autophagy activators, containing large amounts of polyphenolic compounds in their composition, which can be autophagy activators in their original form, or can be metabolized by the intestinal microbiota to active compounds. This review is devoted to the plant-based autophagy activators with emphasis on the sources of their production, mechanism of action, and application in various diseases. The review also describes companies commercializing natural autophagy activators.

Structure and Composition of Spermatozoa Fibrous Sheath in Diverse Groups of Metazoa.

The proper functioning and assembly of the sperm flagella structures contribute significantly to spermatozoa motility and overall male fertility. However, the fine mechanisms of assembly steps are poorly studied due to the high diversity of cell types, low solubility of the corresponding protein structures, and high tissue and cell specificity. One of the open questions for investigation is the attachment of longitudinal columns to the doublets 3 and 8 of axonemal microtubules through the outer dense fibers. A number of mutations affecting the assembly of flagella in model organisms are known. Additionally, evolutionary genomics data and comparative analysis of flagella morphology are available for a set of non-model species. This review is devoted to the analysis of diverse ultrastructures of sperm flagellum of Metazoa combined with an overview of the evolutionary distribution and function of the mammalian fibrous sheath proteins.

Ribosomal protein S18 acetyltransferase RimI is responsible for the acetylation of elongation factor Tu.

N-terminal acetylation is widespread in the eukaryotic proteome but in bacteria is restricted to a small number of proteins mainly involved in translation. It was long known that elongation factor Tu (EF-Tu) is N-terminally acetylated, whereas the enzyme responsible for this process was unclear. Here, we report that RimI acetyltransferase, known to modify ribosomal protein S18, is likewise responsible for N-acetylation of the EF-Tu. With the help of inducible tufA expression plasmid, we demonstrated that the acetylation does not alter the stability of EF-Tu. Binding of aminoacyl tRNA to the recombinant EF-Tu in vitro was found to be unaffected by the acetylation. At the same time, with the help of fast kinetics methods, we demonstrate that an acetylated variant of EF-Tu more efficiently accelerates A-site occupation by aminoacyl-tRNA, thus increasing the efficiency of in vitro translation. Finally, we show that a strain devoid of RimI has a reduced growth rate, expanded to an evolutionary timescale, and might potentially promote conservation of the acetylation mechanism of S18 and EF-Tu. This study increased our understanding of the modification of bacterial translation apparatus.

Nybomycin inhibits both types of E. coli DNA gyrase - fluoroquinolone-sensitive and fluoroquinolone-resistant.

Bacterial type II topoisomerases, DNA gyrase and topoisomerase IV, are targets of many antibiotics including fluoroquinolones (FQs). Unfortunately, a number of bacterial species easily acquire resistance to FQs by mutations in either DNA gyrase or topoisomerase IV genes. The emergence of resistant pathogenic strains is a global problem in healthcare, therefore, identifying alternative pathways to thwart their persistence is the current frontier in drug discovery. An attractive class of compounds is nybomycins, reported to be "reverse antibiotics" that selectively inhibit growth of some Gram-positive FQ-resistant bacteria by targeting the mutant form of DNA gyrase, while being inactive against wild-type strains with FQ-sensitive gyrases. The strong "reverse" effect was demonstrated only for a few Gram-positive organisms resistant to FQs due to the S83L/I mutation in GyrA subunit of DNA gyrase. However, the activity of nybomycins has not been extensively explored among Gram-negative species. Here, we observed that in Gram-negative E. coli ΔtolC strain with enhanced permeability, wild-type gyrase and GyrA S83L mutant, resistant to fluoroquinolones, are both similarly sensitive to nybomycin.

Multiple competing RNA structures dynamically control alternative splicing in the human ATE1 gene.

The mammalian Ate1 gene encodes an arginyl transferase enzyme with tumor suppressor function that depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. We report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore RNA structure revert splicing to that of the wild type. In the endogenous Ate1 pre-mRNA, blocking the competing base pairings by LNA/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of R2R5 interaction changes the ratio of MXE. That is, Ate1 splicing is controlled by two independent, dynamically interacting, and functionally distinct RNA structure modules. Exon 7a becomes more included in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted, indicating that exon 7a/7b ratio depends on co-transcriptional RNA folding. In sum, these results demonstrate that splicing is coordinated both in time and in space over very long distances, and that the interaction of these components is mediated by RNA structure.

Post-Transcriptional and Post-Translational Modifications in Telomerase Biogenesis and Recruitment to Telomeres.

Telomere length is associated with the proliferative potential of cells. Telomerase is an enzyme that elongates telomeres throughout the entire lifespan of an organism in stem cells, germ cells, and cells of constantly renewed tissues. It is activated during cellular division, including regeneration and immune responses. The biogenesis of telomerase components and their assembly and functional localization to the telomere is a complex system regulated at multiple levels, where each step must be tuned to the cellular requirements. Any defect in the function or localization of the components of the telomerase biogenesis and functional system will affect the maintenance of telomere length, which is critical to the processes of regeneration, immune response, embryonic development, and cancer progression. An understanding of the regulatory mechanisms of telomerase biogenesis and activity is necessary for the development of approaches toward manipulating telomerase to influence these processes. The present review focuses on the molecular mechanisms involved in the major steps of telomerase regulation and the role of post-transcriptional and post-translational modifications in telomerase biogenesis and function in yeast and vertebrates.

Mitoregulin Contributes to Creatine Shuttling and Cardiolipin Protection in Mice Muscle.

Small peptides compose a large share of the mitochondrial proteome. Mitoregulin (Mtln) is a mitochondrial peptide known to contribute to the respiratory complex I functioning and other processes in mitochondria. In our previous studies, we demonstrated that Mtln knockout mice develop obesity and accumulate triglycerides and other oxidation substrates in serum, concomitant with an exhaustion of tricarboxylic acids cycle intermediates. Here we examined the functional role of Mtln in skeletal muscles, one of the major energy consuming tissues. We observed reduced muscle strength for Mtln knockout mice. Decrease of the mitochondrial cardiolipin and concomitant increase in monolysocardiolipin concentration upon Mtln inactivation is likely to be a consequence of imbalance between oxidative damage and remodeling of cardiolipin. It is accompanied by the mitochondrial creatine kinase octamer dissociation and suboptimal respiratory chain performance in Mtln knockout mice.

Kidney-Related Function of Mitochondrial Protein Mitoregulin.

A small protein, Mitoregulin (Mtln), localizes in mitochondria and contributes to oxidative phosphorylation and fatty acid metabolism. Mtln knockout mice develop obesity on a high-fat diet, demonstrating elevated cardiolipin damage and suboptimal creatine kinase oligomerization in muscle tissue. Kidneys heavily depend on the oxidative phosphorylation in mitochondria. Here we report kidney-related phenotypes in aged Mtln knockout mice. Similar to Mtln knockout mice muscle mitochondria, those of the kidney demonstrate a decreased respiratory complex I activity and excessive cardiolipin damage. Aged male mice carrying Mtln knockout demonstrated an increased frequency of renal proximal tubules' degeneration. At the same time, a decreased glomerular filtration rate has been more frequently detected in aged female mice devoid of Mtln. An amount of Mtln partner protein, Cyb5r3, is drastically decreased in the kidneys of Mtln knockout mice.

Synthesis and Structures of Lead(II) Complexes with Substituted Derivatives of the Closo-Decaborate Anion with a Pendant N3 Group.

In this work, we studied lead(II) and cobalt(II) complexation of derivatives [2-B10H9O(CH2)2O(CH2)2N3]2- and [2-B10H9O(CH2)5N3]2- of the closo-decaborate anion containing pendant azido groups in the presence of 1,10-phenanthroline and 2,2'-bipyridyl. Mononuclear [PbL2{An}] and binuclear [Pb2L4(NO3)2{An}] lead complexes (where {An} is the N3-substituted boron cluster) were isolated and studied by IR spectroscopy and elemental analysis. The mononuclear lead(II) complex [Pb(phen)2[B10H9O(CH2)2O(CH2)2N3] and the binuclear lead(II) complex [Pb2(phen)4(NO3)2[B10H9O(CH2)5)N3] were determined by single-crystal X-ray diffraction. In complex [Pb2(phen)4(NO3)2[B10H9O(CH2)5)N3], the boron cluster is coordinated by the metal atom only via the 3c2e MHB bonds. In complex [Pb(phen)2[B10H9O(CH2)2O(CH2)2N3], the coordination environment of the metal includes BH groups of the boron cluster and the oxygen atom of the exo-polyhedral substituent. When the reaction was performed in a CH3CN/water mixture, the binuclear lead(II) complex [(Pb(bipy)NO3)(Pb(bipy)2NO3)(B10H9O(CH2)2O(CH2)2N3)]·CH3CN·H2O was isolated, where the boron cluster acts as a bridging ligand between lead atoms coordinated by the boron cage via the O atoms of the substituent and/or the BH groups. In the course of cobalt(II) complexation, the starting compound (Ph4P)2[B10H9O(CH2)5N3] was isolated and its structure was also determined by X-ray diffraction. Although a number of lead(II) complexes with coordinated N3 are known from the literature, no complexes with the boron cluster coordinated by the pendant N3 group involved in the metal coordination have been isolated.

Insights into the improved macrolide inhibitory activity from the high-resolution cryo-EM structure of dirithromycin bound to the E. coli 70S ribosome.

Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the Thermus thermophilus 70S ribosome. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome, improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using high-resolution cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating Escherichia coli 70S ribosome, which suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain.

Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel.

The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.

Amicoumacin a inhibits translation by stabilizing mRNA interaction with the ribosome.

We demonstrate that the antibiotic amicoumacin A (AMI) is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G.

METTL15 interacts with the assembly intermediate of murine mitochondrial small ribosomal subunit to form m4C840 12S rRNA residue.

Mammalian mitochondrial ribosomes contain a set of modified nucleotides, which is distinct from that of the cytosolic ribosomes. Nucleotide m4C840 of the murine mitochondrial 12S rRNA is equivalent to the dimethylated m4Cm1402 residue of Escherichia coli 16S rRNA. Here we demonstrate that mouse METTL15 protein is responsible for the formation of m4C residue of the 12S rRNA. Inactivation of Mettl15 gene in murine cell line perturbs the composition of mitochondrial protein biosynthesis machinery. Identification of METTL15 interaction partners revealed that the likely substrate for this RNA methyltransferase is an assembly intermediate of the mitochondrial small ribosomal subunit containing an assembly factor RBFA.

Rewiring of growth-dependent transcription regulation by a point mutation in region 1.1 of the housekeeping σ factor.

In bacteria, rapid adaptation to changing environmental conditions depends on the interplay between housekeeping and alternative σ factors, responsible for transcription of specific regulons by RNA polymerase (RNAP). In comparison with alternative σ factors, primary σs contain poorly conserved region 1.1, whose functions in transcription are only partially understood. We found that a single mutation in region 1.1 in Escherichia coli σ70 rewires transcription regulation during cell growth resulting in profound phenotypic changes. Despite its destabilizing effect on promoter complexes, this mutation increases the activity of rRNA promoters and also decreases RNAP sensitivity to the major regulator of stringent response DksA. Using total RNA sequencing combined with single-cell analysis of gene expression we showed that changes in region 1.1 disrupt the balance between the "greed" and "fear" strategies thus making the cells more susceptible to environmental threats and antibiotics. Our results reveal an unexpected role of σ region 1.1 in growth-dependent transcription regulation and suggest that changes in this region may facilitate rapid switching of RNAP properties in evolving bacterial populations.

Translation at first sight: the influence of leading codons.

First triplets of mRNA coding region affect the yield of translation. We have applied the flowseq method to analyze >30 000 variants of the codons 2-11 of the fluorescent protein reporter to identify factors affecting the protein synthesis. While the negative influence of mRNA secondary structure on translation has been confirmed, a positive role of rare codons at the beginning of a coding sequence for gene expression has not been observed. The identity of triplets proximal to the start codon contributes more to the protein yield then more distant ones. Additional in-frame start codons enhance translation, while Shine-Dalgarno-like motifs downstream the initiation codon are inhibitory. The metabolic cost of amino acids affects the yield of protein in the poor medium. The most efficient translation was observed for variants with features resembling those of native Escherichia coli genes.

LINC00116 codes for a mitochondrial peptide linking respiration and lipid metabolism.

Genes coding for small peptides have been frequently misannotated as long noncoding RNA (lncRNA) genes. Here we have demonstrated that one such transcript is translated into a 56-amino-acid-long peptide conserved in chordates, corroborating the work published while this manuscript was under review. The Mtln peptide could be detected in mitochondria of mouse cell lines and tissues. In line with its mitochondrial localization, lack of the Mtln decreases the activity of mitochondrial respiratory chain complex I. Unlike the integral components and assembly factors of NADH:ubiquinone oxidoreductase, Mtln does not alter its enzymatic activity directly. Interaction of Mtln with NADH-dependent cytochrome b5 reductase stimulates complex I functioning most likely by providing a favorable lipid composition of the membrane. Study of Mtln illuminates the importance of small peptides, whose genes might frequently be misannotated as lncRNAs, for the control of vitally important cellular processes.