Alternative RNA splicing modulates ribosomal composition and determines the spatial phenotype of glioblastoma cells.

Glioblastoma (GBM) is characterized by exceptionally high intratumoral heterogeneity. However, the molecular mechanisms underlying the origin of different GBM cell populations remain unclear. Here, we found that the compositions of ribosomes of GBM cells in the tumour core and edge differ due to alternative RNA splicing. The acidic pH in the core switches before messenger RNA splicing of the ribosomal gene RPL22L1 towards the RPL22L1b isoform. This allows cells to survive acidosis, increases stemness and correlates with worse patient outcome. Mechanistically, RPL22L1b promotes RNA splicing by interacting with lncMALAT1 in the nucleus and inducing its degradation. Contrarily, in the tumour edge region, RPL22L1a interacts with ribosomes in the cytoplasm and upregulates the translation of multiple messenger RNAs including TP53. We found that the RPL22L1 isoform switch is regulated by SRSF4 and identified a compound that inhibits this process and decreases tumour growth. These findings demonstrate how distinct GBM cell populations arise during tumour growth. Targeting this mechanism may decrease GBM heterogeneity and facilitate therapy.

Flow-Seq Evaluation of Translation Driven by a Set of Natural Escherichia coli 5'-UTR of Variable Length.

Flow-seq is a method that combines fluorescently activated cell sorting and next-generation sequencing to deduce a large amount of data about translation efficiency from a single experiment. Here, we constructed a library of fluorescent protein-based reporters preceded by a set of 648 natural 5'-untranslated regions (5'-UTRs) of Escherichia coli genes. Usually, Flow-seq libraries are constructed using uniform-length sequence elements, in contrast to natural situations, where functional elements are of heterogenous lengths. Here, we demonstrated that a 5'-UTR library of variable length could be created and analyzed with Flow-seq. In line with previous Flow-seq experiments with randomized 5'-UTRs, we observed the influence of an RNA secondary structure and Shine-Dalgarno sequences on translation efficiency; however, the variability of these parameters for natural 5'-UTRs in our library was smaller in comparison with randomized libraries. In line with this, we only observed a 30-fold difference in translation efficiency between the best and worst bins sorted with this factor. The results correlated with those obtained with ribosome profiling.

Deep Functional Profiling of Wild Animal Microbiomes Reveals Probiotic Bacillus pumilus Strains with a Common Biosynthetic Fingerprint.

The biodiversity of microorganisms is maintained by intricate nets of interactions between competing species. Impaired functionality of human microbiomes correlates with their reduced biodiversity originating from aseptic environmental conditions and antibiotic use. Microbiomes of wild animals are free of these selective pressures. Microbiota provides a protecting shield from invasion by pathogens in the wild, outcompeting their growth in specific ecological niches. We applied ultrahigh-throughput microfluidic technologies for functional profiling of microbiomes of wild animals, including the skin beetle, Siberian lynx, common raccoon dog, and East Siberian brown bear. Single-cell screening of the most efficient killers of the common human pathogen Staphylococcus aureus resulted in repeated isolation of Bacillus pumilus strains. While isolated strains had different phenotypes, all of them displayed a similar set of biosynthetic gene clusters (BGCs) encoding antibiotic amicoumacin, siderophore bacillibactin, and putative analogs of antimicrobials including bacilysin, surfactin, desferrioxamine, and class IId cyclical bacteriocin. Amicoumacin A (Ami) was identified as a major antibacterial metabolite of these strains mediating their antagonistic activity. Genome mining indicates that Ami BGCs with this architecture subdivide into three distinct families, characteristic of the B. pumilus, B. subtilis, and Paenibacillus species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated B. pumilus strains efficiently inhibit the growth of both Gram-positive S. aureus and Gram-negative E. coli in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing B. pumilus can be attributed to the metabolomic profile predetermined by their biosynthetic fingerprint. Ultrahigh-throughput isolation of natural probiotic strains from wild animal microbiomes, as well as their metabolic reprogramming, opens up a new avenue for pathogen control and microbiome remodeling in the food industry, agriculture, and healthcare.

Little but Loud. The Diversity of Functions of Small Proteins and Peptides - Translational Products of Short Reading Frames.

Cell functioning is tightly regulated process. For many years, research in the fields of proteomics and functional genomics has been focused on the role of proteins in cell functioning. The advances in science have led to the uncovering that short open reading frames, previously considered non-functional, serve a variety of functions. Short reading frames in polycistronic mRNAs often regulate their stability and translational efficiency of the main reading frame. The improvement of proteomic analysis methods has made it possible to identify the products of translation of short open reading frames in quantities that suggest the existence of functional role of those peptides and short proteins. Studies demonstrating their role unravel a new level of the regulation of cell functioning and its adaptation to changing conditions. This review is devoted to the analysis of functions of recently discovered peptides and short proteins.

PARP1 Regulates the Biogenesis and Activity of Telomerase Complex Through Modification of H/ACA-Proteins.

Poly(ADP-ribose) polymerase 1 (PARP1) is established as a key regulator of the cellular DNA damage response and apoptosis. In addition, PARP1 participates in the global regulation of DNA repair, transcription, telomere maintenance, and inflammation response by modulating various DNA-protein and protein-protein interactions. Recently, it was reported that PARP1 also influences splicing and ribosomal RNA biogenesis. The H/ACA ribonucleoprotein complex is involved in a variety of cellular processes such as RNA maturation. It contains non-coding RNAs with specific H/ACA domains and four proteins: dyskerin (DKC1), GAR1, NHP2, and NOP10. Two of these proteins, DKC1 and GAR1, are targets of poly(ADP-ribosyl)ation catalyzed by PARP1. The H/ACA RNA-binding proteins are involved in the regulation of maturation and activity of the telomerase complex, which maintains telomere length. In this study, we demonstrated that of poly(ADP-ribosyl)ation influences on RNA-binding properties of DKC1 and GAR1 and telomerase assembly and activity. Our data provide the evidence that poly(ADP-ribosyl)ation regulates telomerase complex assembly and activity, in turn regulating telomere length that may be useful for design and development of anticancer therapeutic approaches that are based on the inhibition of PARP1 and telomerase activities.

Panel of potential lncRNA biomarkers can distinguish various types of liver malignant and benign tumors.

PURPOSE: Liver cancers are among the deadliest malignancies due to a limited efficacy of early diagnostics, the lack of appropriate biomarkers and insufficient discrimination of different types of tumors by classic and molecular methods. In this study, we searched for novel long non-coding RNA (lncRNA) as well as validated several known candidates suitable as probable biomarkers for primary liver tumors of various etiology. METHODS: We described a novel lncRNA HELIS (aka "HEalthy LIver Specific") and estimated its expression by RT-qPCR in 82 paired tissue samples from patients with hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), combined HCC-CCA, pediatric hepatoblastoma (HBL) and non-malignant hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH). Additionally, we examined expression of cancer-associated lncRNAs HULC, MALAT1, UCA1, CYTOR, LINC01093 and H19, which were previously studied mainly in HCC. RESULTS: We demonstrated that down-regulation of HELIS strongly correlates with carcinogenesis; whereas in tumors with non-hepatocyte origin (HBL, CCA) or in a number of poorly differentiated HCC, this lncRNA is not expressed. We showed that recently discovered LINC01093 is dramatically down-regulated in all malignant liver cancers; while in benign tumors LINC01093 expression is just twice decreased in comparison to adjacent samples. CONCLUSION: Our study revealed that among all measured biomarkers only down-regulated HELIS and LINC01093, up-regulated CYTOR and dysregulated HULC are perspective for differential diagnostics of liver cancers; whereas others demonstrated discordant results and cannot be considered as potential universal biomarkers for this purpose.

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.

Influence of the spacer region between the Shine-Dalgarno box and the start codon for fine-tuning of the translation efficiency in Escherichia coli.

Translation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine-Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next-generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine-Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100-fold range.

Oligoglutamylation of E. coli ribosomal protein S6 is under growth phase control.

Ribosomal protein S6 in Escherichia coli is modified by ATP-dependent glutamate ligase RimK. Up to four glutamate residues are added to the C-terminus of S6 protein. In this work we demonstrated that unlike the majority of ribosome modifications in E. coli, oligoglutamylation of S6 protein is regulated and happens only in the stationary phase of bacterial culture. Only S6 protein incorporated into assembled small ribosomal subunits, but not newly made free S6 protein is a substrate for RimK protein. Overexpression of the rimK gene leads to the modification of S6 protein even in the exponential phase of bacterial culture. Thus, it is unlikely that any stationary phase specific factor is needed for the modification. We propose a model that S6 modification is regulated solely via the rate of ribosome biosynthesis at limiting concentration of RimK enzyme.

Autocrine-based selection of ligands for personalized CAR-T therapy of lymphoma.

We report the development of a novel platform to enhance the efficacy and safety of follicular lymphoma (FL) treatment. Since lymphoma is a clonal malignancy of a diversity system, every tumor has a different antibody on its cell surface. Combinatorial autocrine-based selection is used to rapidly identify specific ligands for these B cell receptors on the surface of FL tumor cells. The selected ligands are used in a chimeric antigen receptor T cell (CAR-T) format for redirection of human cytotoxic T lymphocytes. Essentially, the format is the inverse of the usual CAR-T protocol. Instead of being a guide molecule, the antibody itself is the target. Thus, these studies raise the possibility of personalized treatment of lymphomas using a private antibody binding ligand that can be obtained in a few weeks.

Ultrahigh-throughput functional profiling of microbiota communities.

Microbiome spectra serve as critical clues to elucidate the evolutionary biology pathways, potential pathologies, and even behavioral patterns of the host organisms. Furthermore, exotic sources of microbiota represent an unexplored niche to discover microbial secondary metabolites. However, establishing the bacterial functionality is complicated by an intricate web of interactions inside the microbiome. Here we apply an ultrahigh-throughput (uHT) microfluidic droplet platform for activity profiling of the entire oral microbial community of the Siberian bear to isolate Bacillus strains demonstrating antimicrobial activity against Staphylococcus aureus Genome mining allowed us to identify antibiotic amicoumacin A (Ami) as responsible for inhibiting the growth of S. aureus Proteomics and metabolomics revealed a unique mechanism of Bacillus self-resistance to Ami, based on a subtle equilibrium of its deactivation and activation by kinase AmiN and phosphatase AmiO, respectively. We developed uHT quantitative single-cell analysis to estimate antibiotic efficacy toward different microbiomes and used it to determine the activity spectra of Ami toward human and Siberian bear microbiota. Thus, uHT microfluidic droplet platform activity profiling is a powerful tool for discovering antibiotics and quantifying external influences on a microbiome.

Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

PURPOSE: High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. PROCEDURES: The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. RESULTS: For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. CONCLUSION: The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

The genome-wide transcription response to telomerase deficiency in the thermotolerant yeast Hansenula polymorpha DL-1.

BACKGROUND: In the course of replication of eukaryotic chromosomes, the telomere length is maintained due to activity of telomerase, the ribonucleoprotein reverse transcriptase. Abolishing telomerase function causes progressive shortening of telomeres and, ultimately, cell cycle arrest and replicative senescence. To better understand the cellular response to telomerase deficiency, we performed a transcriptomic study for the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1 lacking telomerase activity. RESULTS: Mutant strain of H. polymorpha carrying a disrupted telomerase RNA gene was produced, grown to senescence and analyzed by RNA-seq along with wild type strain. Telomere shortening induced a transcriptional response involving genes relevant to telomere structure and maintenance, DNA damage response, information processing, and some metabolic pathways. Genes involved in DNA replication and repair, response to environmental stresses and intracellular traffic were up-regulated in senescent H. polymorpha cells, while strong down-regulation was observed for genes involved in transcription and translation, as well as core histones. CONCLUSIONS: Comparison of the telomerase deletion transcription responses by Saccharomyces cerevisiae and H. polymorpha demonstrates that senescence makes different impact on the main metabolic pathways of these yeast species but induces similar changes in processes related to nucleic acids metabolism and protein synthesis. Up-regulation of a subunit of the TORC1 complex is clearly relevant for both types of yeast.

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity.

Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.

Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli.

Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5΄-UTR sequences and introduced random and designed mutations into natural and artificially selected 5΄-UTRs. Several distinct properties could be ascribed to a group of 5΄-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5΄-UTRs.

Mixed valence copper(I,II) binuclear complexes with unexpected structure: synthesis, biological properties and anticancer activity.

We have synthesized and characterized a panel of new binuclear mixed valence Cu(I,II) complexes containing substituted 2-alkylthio-5-arylmethylene-4H-imidazolin-4-ones with unusual structure. These complexes are shown to be cytotoxic for various cell lines. We have found that these compounds did not intercalate DNA, inhibited number of polymerases (telomerase predominantly), accumulated in the cell nucleus, and caused DNA degradation. Preliminary studies revealed that lead compound inhibited human breast adenocarcinoma growth in mice model.

Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique.

Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.

Distinctive properties of the 5'-untranslated region of human hsp70 mRNA.

A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.

Conversion of 48S translation preinitiation complexes into 80S initiation complexes as revealed by toeprinting.

A method of analysis of translation initiation complexes by toeprinting has recently acquired a wide application to investigate molecular mechanisms of translation initiation in eukaryotes. So far, this very fruitful approach was used when researchers did not aim to discriminate between patterns of toeprints for 48S and 80S translation initiation complexes. Here, using cap-dependent and internal ribosomal entry site (IRES)-dependent mRNAs, we show that the toeprint patterns for 48S and 80S complexes are distinct whether the complexes are assembled in rabbit reticulocyte lysate or from fully purified individual components. This observation allowed us to demonstrate for the first time a delay in the conversion of the 48S complex into the 80S complex for beta-globin and encephalomyocarditis virus (EMCV) RNAs, and to assess the potential of some 80S antibiotics to block polypeptide elongation. Besides, additional selection of the authentic initiation codon among three consecutive AUGs that follow the EMCV IRES was revealed at steps subsequent to the location of the initiation codon by the 40S ribosomal subunit.