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.
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.
Abstract
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.
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.
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
resulted in repeated isolation of
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
,
, and
species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated
strains efficiently inhibit the growth of both Gram-positive
and Gram-negative
in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing
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.
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.
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.
•E. coli ribosomal protein S6 is oligoglutamylated by RimK.•Modification happens only in the stationary phase.•Assembled 30S subunit but not free S6 protein is modified.•A model is proposed for the regulation of S6 modification.
Summary
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.
Understanding the principles that determine mRNA translation efficiency is of primary value for deciphering translational control of gene expression and for optimization of protein synthesis in biotechnology. In this work we combined Flowseq method with randomization of a region within the spacer between the Shine‐Dalgarno box and AUG start codon to decipher an influence of this mRNA part on translation efficiency.
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.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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