Fusion protein therapeutics engineering is advancing to meet the need for novel medicine. Herein, we further characterize the development of novel RTA & PAP-S1 antiviral fusion proteins. In brief, RTA/PAP-S1 and PAP-S1/RTA fusion proteins were produced in both cell free and E. coli in vivo expression systems, purified by His-tag affinity chromatography, and protein synthesis inhibitory activity assayed by comparison to the production of a control protein, CalmL3. Results showed that the RTA/PAP-S1 fusion protein is amenable to standardized production and purification and has both increased potency and less toxicity compared to either RTA or PAP-S1 alone. Thus, this research highlights the developmental potential of novel fusion proteins with reduced cytotoxic risk and increased potency.

The castor plant (Ricinus communis L.) has been known since time immemorial in traditional medicine in the pharmacopeia of Mediterranean and eastern ancient cultures. Moreover, it is still used in folk medicine worldwide. Castor bean has been mainly recommended as anti-inflammatory, anthelmintic, anti-bacterial, laxative, abortifacient, for wounds, ulcers, and many other indications. Many cases of human intoxication occurred accidentally or voluntarily with the ingestion of castor seeds or derivatives. Ricinus toxicity depends on several molecules, among them the most important is ricin, a protein belonging to the family of ribosome-inactivating proteins. Ricin is the most studied of this category of proteins and it is also known to the general public, having been used for biocrimes in several cases. Here, the main steps of ricin research are reported with particular regards to its enzymatic activity, structure and cytotoxicity. Moreover, we discuss ricin toxicity for animals and humans, as well as the relation amongst bioterrorism and ricin and its impact on environmental toxicity. Ricin has also been of great utility to develop a number of immunotoxins specific for the elimination of unwanted cells, mainly cancer cells; some of these immunotoxins gave promising results also in clinical trials.

Ribosomal proteins (RPs) mediate protein synthesis and stability of the ribosomal complex. RPs and rRNA form ribosomal subunits and ri-bosomes, the cellular machinery for protein synthesis, a fundamental biological process related to cell growth and proliferation. The differ-ences between paralogs within the same RP family have led them to ac-quire extraribosomal functions involved in plants' growth, develop-ment, and biotic and abiotic stresses. From transcriptomic analysis, it has been revealed that ribosomal proteins have a crucial role during MTs development. The RPLs; RPL11, RPL29 and RPL40 interact with PEBP family members (Phosphatidylethanolamine-binding proteins), poten-tial activators in the MTs development. In potatoes, genome editing is a promising technology to introduce crop breeding traits. Recently, the edition of the genome through the CRISPR/Cas9 system has made it the most efficient and powerful genetic modification method. Based on this, the gene modulation by overexpression and silencing in RPL11, RPL29 and RPL40 will guide us to understand the effect on the microtuberiza-tion process and produce improved potato plants with the capability of growing under adverse environments (biotic and abiotic stresses). By understanding the molecular biology mechanisms that RPs govern, we can improve crops under today's changing climatic conditions.

The investigation of membrane proteins, key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage dependent anion channel (VDAC), a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function is proposed that quantifies the feasibility of formation of the translation initiation complex through the ribosome-mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome. The scoring function enables to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies. This scoring function is publicly available as webservice XenoExpressO at rna.tbi.univie.ac.at.

Multi-Alignment method coupled with phylogenetic analysis we disclosed the Nsp9 and Nsp10 non-structural proteins of Corona Virus as rRNA RlmH/K methyltransferases with similarities with bin recombinase and int-core integrase fold. Further, Nsp9 has similarities to S8 ribosomal protein and Nap10 has similarity to S10 ribosomal protein. Previously, we showed Nsp13, Nsp14, Nsp15 and Nsp16 are also different types of rRNA RlmE/N and Cfr-like methyltransferases-ribonuclease with RNA helicase domains. Two domains of Nsp13 astonishingly have similarities to ribosomal proteins L6 and L9. Taken together, Nsp9/10 and Nsp13-16 proteins could mimic host ribosome assembly and also could methylate rRNA of mitobibosome preventing mitochondrial protein synthesis and oxidative phosphorylation. Low ATP synthesis causes lowering blood pressure following coma but very ATP concentration (1-10nM) surely induces platelets aggregation through vWA, collagen and GpIIb/IIIa proteins followed by fibrin formation and blood clotting as recently have seen in the lung of many Corona virus infected patients. We have also postulated that two polyproteins itself resemble like 28S and 38S mitoribosome subunits and compete with rRNAs inhibiting the ribosome turnover and new protein synthesis due to their similarities with many ribosomal proteins. Such finding may be valuable in computer-based novel drug design against Corona virus.

Studies over the past three years have substantially expanded the involvements of eIF3 in mRNA translation. It now appears that this multi-subunit complex is involved in every possible form of mRNA translation, controlling every step of protein synthesis from initiation to elongation, termination and quality control in positive as well as negative fashion. Through the study of eIF3, we are beginning to appreciate protein synthesis as a highly integrated process coordinating protein production with protein folding, subcellular targeting, and degradation. At the same time, eIF3 subunits appear to have specific functions that probably vary between different tissues and individual cells. Considering the broad functions of eIF3 in protein homeostasis, it comes as little surprise that eIF3 is increasingly implicated in major human diseases and first attempts at therapeutically targeting eIF3 have been undertaken. Much remains to be learned, however, about subunit- and tissue-specific functions of eIF3 in protein synthesis and disease and their regulation by environmental conditions and posttranslational modifications.

Phylogenetic tree analysis is commonly used to understand the evolutionary distance between different species or strains of the same species. This work originally aimed to gain an understanding and visual depiction of the evolutionary distance in 23S rRNA gene of different Helicobacter pylori strains. Using an in-house MATLAB multiple sequence alignment and phylogenetic tree analysis software, the phylogenetic tree constructed for 23S rRNA gene reveals two major lineages with further subdivisions in each cluster. This suggests two major evolutionary forces driving the evolution of the 23S rRNA gene. In comparison, there is relatively short evolutionary distance in the 16S rRNA gene phylogenetic tree of H. pylori, suggesting lack of major evolutionary forces driving mutational changes of the gene. Overall, the data suggests that the large ribosome subunit which houses the 23S rRNA of H. pylori is undergoing significant conformational changes that may be a response to the frequent use of various antibiotics that target the ribosome of H. pylori.

Despite several decades of research, the physics underlying translation – protein synthesis at the ribosome – remains poorly studied. For instance, the mechanism coordinating various events occurring in distant parts of the ribosome is unknown. Very recently, we have suggested that this allosteric mechanism could be based on the transport of electric charges (electron holes) along RNA molecules and localization of these charges in the functionally important areas; this assumption was justified using tRNA as an example. In this study, we turn to the ribosome and show computationally that holes can also efficiently migrate within the whole ribosomal small subunit (SSU). The potential sites of charge localization in SSU are revealed, and it is shown that most of them are located in the functionally important areas of the ribosome – intersubunit bridges, Fe4S4 cluster and the pivot linking the SSU head to the body. As a result, we suppose that hole localization within the SSU can affect intersubunit rotation (ratcheting) and SSU head swiveling, in agreement with the scenario of electronic coordination of ribosome operation. We anticipate that our findings will improve the understanding of the translation process and advance the molecular biology and medicine.

After axonal injury, chromatolysis (fragmentation of Nissl substance) occurs in both intrinsic neurons (whose processes are within the CNS) and extrinsic neurons (whose axons extend outside the CNS). Electron microscopy shows that chromatolysis involves fission of the rough endoplasmic reticulum. In intrinsic neurons (which do not regenerate axons) or in extrinsic neurons denied axon regeneration, chromatolysis is often accompanied by degranulation (loss of ribosomes from rough endoplasmic reticulum), disaggregation of polyribosomes and degradation of monoribosomes into dust-like particles. Ribosomes and rough endoplasmic reticulum may also be degraded in autophagic vacuoles by Ribophagy and Reticulophagy, respectively. In other words, chromatolysis is disruption of parts of the protein synthesis infrastructure. Whereas some neurons may show transient or no chromatolysis, severely injured neurons can remain chromatolytic and never again synthesise normal levels of protein; some may atrophy or die. What molecule(s) cause fragmentation or degranulation of rough endoplasmic reticulum, disaggregation of polyribosomes and degradation of monoribosomes? Ribonucleases can modify (and perhaps fragment) rough endoplasmic reticulum; various endoribonucleases can degrade mRNA causing polyribosomes to unchain and disperse; they can disassemble monoribosomes; Ribonuclease 5 can control rRNA synthesis and degrade tRNA; Ribonuclease T2 can degrade ribosomes, rough endoplasmic reticulum and RNA within autophagic vacuoles; and Ribonuclease IRE1α acts as a stress sensor within the endoplasmic reticulum. Regeneration might be improved after axonal injury by protecting the protein synthesis machinery from catabolism; targeting ribonucleases could be a profitable strategy.

Luteinizing hormone receptor (LHR) is a member of the seven-transmembrane (TM) receptor family. Several mutations in LHR have been identified in many mammalian species, leading to either constitutive activation or inactivation of the receptor. Mutations in highly conserved regions of the TM domain have been reported. In this study, we analyzed signal transduction by three constitutively active mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of eelLHR known as naturally occurring in human LHR . To directly assess the functional effects of these mutations, site-directed mutant receptors were transiently expressed in CHO-K1 cells and cAMP accumulation stimulated by recombinant eelLH (rec-eelLH) was measured by homogeneous time-resolved fluorescence (HTRF) assays. The cAMP response in cells expressing eelLHR wild-type (eLHR-WT) increased in a dose-dependent manner with rec-eelLH ligand stimulation. Cells expressing the activating eelLHR mutants, M410T, L469R, and D590Y, exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response, respectively. However, their maximal responses were approximately 73, 53, and 92%, respectively, of the maximal response of LHR-WT. The L469R mutant exhibited a particularly marked increase in cAMP production in the absence of agonist. The maximal responses of the inactivating mutants, D383N and Y546F, were 32 and 24% of LHR-WT, respectively. However, the inactivating mutations did not completely impair signal transduction. Thus, we report here the first characterization of activating and inactivating mutations in eelLHR and we show that these mutations have similar effects as those reported for mammalian LHRs. Moreover, eelLHR with activating mutations showed constitutive cAMP responses. These results provide important data on the signal transduction of constitutively active and inactive LHR mutants. Further studies should aim to identify the mechanism responsible for the significant increase in basal cAMP response in the constitutively activated eelLHR mutants.

Microsporidia are fungi-like parasites that have the smallest known eukaryotic genome, and for that reason they are used as a model to study the phenomenon of genome decay in parasitic forms of life. Similar to other intracellular parasites that reproduce asexually in an environment with alleviated natural selection, Microsporidia experience continuous genome decay driven by Muller's ratchet - an evolutionary process of irreversible accumulation of deleterious mutations, which leads to gene loss and miniaturization of cellular components. Particularly, Microsporidia have remarkably small ribosomes in which the rRNA is reduced to the minimal enzymatic core. To better understand the impact of Muller's ratchet on RNA and protein molecules in parasitic organisms, particularly regarding their ribosome structure, we have explored an apparent effect of Muller's ratchet on microsporidian ribosomal proteins. Through mass spectrometry, analysis of microsporidian genome sequences and analysis of ribosome structure from non-parasitic eukaryotes, we found that massive rRNA reduction in microsporidian ribosomes appears to annihilate binding sites for ribosomal proteins eL8, eL27, and eS31, suggesting that these proteins are no longer bound to the ribosome in microsporidian species. We then provided an evidence that protein eS31 is retained in Microsporidia due to its non-ribosomal function in ubiquitin biogenesis. To sum up, our study illustrates that while Microsporidia carry the same set of ribosomal proteins as non-parasitic eukaryotes, some of ribosomal proteins are no longer participating in protein synthesis in Microsporidia and they are preserved from genome decay by having extra-ribosomal functions.

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the “spacer” sequences. The canonical pathway for the removal of the ITS1 spacer, located between 18S and 5.8S rRNAs in the primary transcript, involves cleavages at the 3’ end of 18S rRNA and at two sites inside ITS1. The process generates a long and a short 5.8S rRNA that differ in the number of ITS1 nucleotides retained at the 5.8S 5’ end. Here we document a novel pathway that generates the long 5.8S for ITS1 while bypassing cleavage within ITS1. It entails a single endonuclease cut at the 3’-end of 18S rRNA followed by exonuclease Xrn1 degradation of ITS1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA; traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. In contrast, we report here that the MRP induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the switch may depend on RNase MRP processing RNA molecules other than pre-rRNA.

It was thought until the 1990s that the eukaryotic translation machinery was unable to translate a circular RNA. However internal ribosome entry sites (IRESs) and m⁶A-induced ribosome engagement sites (MIRESs) were discovered, promoting 5’end-independent translation initiation. Today a new family of so-called “non-coding” circular RNAs (circRNAs) has emerged, revealing the pivotal role of 5’end-independent translation. CircRNAs have a strong impact on translational control via their sponge function, and form a new mRNA family as they are translated into proteins with pathophysiological roles. While there is no more doubt about translation of covalently closed circRNA, the linearity of canonical mRNA is only theoretical: it has been shown for more than thirty years that polysomes exhibit a circular form and mRNA functional circularization has been demonstrated in the 1990s by the interaction of initiation factor eIF4G with poly(A) binding protein. More recently, additional mechanisms of 3’-5’ interaction have been reported, including m⁶A modification. Functional circularization enhances translation via ribosome recycling and acceleration of the translation initiation rate. This update of covalently and non-covalently circular mRNA translation landscape shows that RNA circular shape might be the rule for translation with an important impact on disease development and biotechnological applications.

Osteosarcoma (OSA) is the most common primary malignant bone tumor. More than 40% of patients with OSA have poor prognoses. We aimed to discover a biomarker for patient stratification and therapeutic targets for these high-risk patients. Using Single Sample Gene Set Enrichment Analysis (ssGSEA) and univariate Cox analysis, six hallmarks were identified as significant prognostic factors for overall survival (OS). Three were selected to construct a multivariate Cox model. Then, WGCNA, univariate Cox regression, Kaplan-Meier (KM) survival analyses, and multivariate Cox analyses were combined to filter promising candidates and establish a seven-gene signature to predict OS, whose prognostic value was validated internally and externally. Subsequently, Differential Expression Analysis was conducted between high- and low-risk patients, and the Robust Rank Aggregation algorithm was used to determine the robust DEGs. Metascape was used to perform pathway and process enrichment analyses as well as construct protein-protein interaction (PPI) networks. Finally, RPS28 was identified as an independent risk factor by using univariate and multivariate Cox regression, which was preliminarily validated as a promising therapeutic target by using RNA interference. In conclusion, we might contribute to optimizing risk stratification and an excellent therapeutic target for high-risk patients with OSA.

The evolution of release factors catalyzing the hydrolysis of the final peptidyl-tRNA bond and the release of the polypeptide from the ribosome has been a longstanding paradox. While the components of the translation apparatus are generally well-conserved across extant life, structurally-unrelated release factor peptidyl hydrolases (RF-PHs) emerged in the stems of the bacterial and archaeo-eukaryotic lineages. We analyze the diversification of RF-PH domains within the broader evolutionary framework of the translation apparatus. Thus, we reconstruct the possible state of translation termination in the Last Universal Common Ancestor with possible tRNA-like terminators. Further, evolutionary trajectories of the several auxiliary release factors in ribosome quality control (RQC) and rescue pathways point to multiple independent solutions to this problem and frequent transfers between superkingdoms including the recently-characterized ArfT, which is more widely-distributed across life than previously appreciated. The eukaryotic RQC system was pieced together from components with disparate provenance, which include the long-sought Vms1/ANKZF1 RF-PH of bacterial origin. We also uncover an under-appreciated evolutionary driver of innovation in rescue pathways: effectors deployed in biological conflicts that target the ribosome. At least three rescue pathways (centered on the prfH/RFH, baeRF-1, and C12orf65 RF-PH domains), were likely innovated in response to such conflicts.

GCN1 is recognized as a factor that is essential for the activation of GCN2, which is a sensor of amino acid starvation. This function is evolutionarily conserved from yeast to higher eukaryotes. However, recent studies have revealed non-canonical functions of GCN1 that are independent of GCN2, such as its participation in cell proliferation, apoptosis, and the immune response, beyond the borders of species. Although it is known that GCN1 and GCN2 interact with ribosomes to accomplish amino acid starvation sensing, recent studies have reported that GCN1 binds to disomes (i.e. ribosomes that collide each other), thereby regulating both the co-translational quality control and stress response. We propose that GCN1 regulates ribosome-mediated signaling by dynamically changing its partners among RWD domain-possessing proteins via unknown mechanisms. We recently demonstrated that GCN1 is essential for cell proliferation and whole-body energy regulation in mice. However, the manner in which ribosome-initiated signaling via GCN1 is related to various physiological functions warrants clarification. GCN1-mediated mechanisms and its interaction with other quality control and stress response signals should be important for proteostasis during aging and neurodegenerative diseases, and may be targeted for drug development.

We analyzed signal transduction of three constitutively activating mutants (M410T, L469R, and D590Y) and two inactivating mutants (D417N and Y558F) of the eel luteinizing hormone receptor (eel LHR), known to occur in human LHR. The objective of this study was to assess the functional effects of these mutations in signal transduction and cell surface loss of receptor. Mutant receptors were transiently expressed in Chinese hamster ovary (CHO-K1) cells. Eel LH-stimulated accumulation of cyclic adenosine monophosphate (cAMP) was measured by homogeneous time-resolved fluorescence (HTRF) assays. The loss of receptors from the cells surface was measured using an enzyme-linked immunosorbent assay (ELISA) in human embryonic kidney (HEK) 293 cells. The cAMP response in cells expressing the wild type eel LHR was increased in a dose-dependent manner using eel LH ligand stimulation. Compared with the wild type, cells expressing the activating mutants (M410T, L469R, and D590Y), exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response without agonist stimulation, respectively. Their maximal responses to agonist stimulation were approximately 65%, 52%, and 98%, respectively, of those of the wild type. The inactivating mutants (D417N and Y558F) did not completely impair signal transduction, and their maximal responses were only 33% and25 % of those of wild type. These data clearly showed that the eel LHR-L469R and D590Y, activating mutants enhanced the rate of the loss of cell surface receptors following treatment with eel LH. Thus, the loss of cell surface receptors in cells expressing mutant eel LHRs was consistent with the eel LH agonist-induced production of cAMP. Our results suggested that the activation of the eel LHR requires appropriate loss of LHR-ligand complexes from the cell surface.

In the current work, in continuation of our recent research [1] we synthesized and studied new chimeric compounds comprising the ribosome-targeting antibiotic chloramphenicol (CHL) and the membrane-penetrating cation triphenylphosphonium (TPP) connected by alkyl linkers of different lengths. Using various biochemical assays, we showed that these CAM-Cn-TPP compounds bind to the bacterial ribosome, inhibit protein synthesis in vitro and in vivo in a way similar to that of the parent CHL, and significantly decrease membrane potential. Similar to CAM-C4-TPP, the mode of action of CAM-C10-TPP and CAM-C14-TPP on bacterial ribosomes differ from that of CHL. By simulating the dynamics of complexes of CAM-Cn-TPP with bacterial ribosomes, we have proposed a possible explanation for the specificity of the action of these analogs on the translation process. CAM-C10-TPP and CAM-C14-TPP stronger inhibit the growth of the Gram-positive bacteria in comparison to the CHL and suppress some strains of CHL-resistant bacteria. Thus, we have shown that TPP derivatives of CHL are dual-acting compounds that target the ribosomes and the cellular membranes of bacteria. The TPP fragment of CAM-Cn-TPP compounds contributes to the inhibitory effect on bacteria. Moreover, since the mitochondria of eukaryotic cells have qualities similar to those of their prokaryotic ancestors, we demonstrate the possibility of targeting chemoresistant cancer cells with these compounds.

This study comprehensively evaluates the DNA/RNA Defend Pro (DRDP) sample collection buffer designed to inactivate and stabilize patient samples. The primary objectives encompassed assessing DRDP's efficacy in ensuring sample stability, facilitating extraction-free polymerase chain reaction (PCR), and ensuring compatibility with rapid antigen testing.Ninety-five diagnostic nasopharyngeal swab samples tested for influenza A/B, RSV A/B, and/or SARS-CoV-2 were diluted 1/10 with DRDP and anonymized. Stability testing involved incubating 31 positive samples at 4 °C, 20 °C, and 37 °C for 7 days, with regular extraction-free nucleic acid amplification testing (NAAT).Results demonstrated that DRDP guaranteed viral RNA stability at all temperatures for influenza, SARS-CoV-2 and RSV showing stability up to 7 days at 4 °C. DRDP-diluted samples exhibited robust compatibility with direct PCR, as 72 out of 88 samples positive for cobas Liat also tested positive for direct PCR. Non-concordant results could be explained by the 200-fold lower input in the extraction-free NAAT.Compared to NAAT, rapid antigen testing demonstrated lower sensitivity, as expected, while maintaining perfect specificity. Clear cutoffs between positive and negative rapid antigen testing results were observed in both cobas Liat and direct PCR analyses, validating DRDP's compatibility with these techniques. In conclusion, DRDP is an effective stabilizing medium compatible with direct PCR and rapid antigen testing and shows great potential for optimizing diagnostic processes, particularly in resource-limited or time-sensitive scenarios.

In addition to the canonical loss-of-function mutations, mutations in proteins may additionally result in gain-of-function through the binary activation of cryptic ‘structural capacitance elements’. Our previous bioinformatic analysis allowed us to propose a new mechanism of protein evolution - structural capacitance – that arises via the generation of new elements of microstructure upon mutations that cause a disorder-to-order (DO) transition in previously disordered regions of proteins. Here we propose that the DO transition is a necessary follow-on from expected early codon-anticodon and tRNA acceptor stem-amino acid usage, via the accumulation of structural capacitance elements - reservoirs of disorder in proteins. We develop this argument further to posit that structural capacitance is an inherent consequence of the evolution of the genetic code.

Many metabolic processes at the molecular level support both viral attack strategies and human defenses during Covid-19. This knowledge is of vital importance in the design of antiviral drugs. In this study, we extracted 18 articles (2021-2023) from PubMed reporting the discovery of hub-nodes specific for the liver during covid-19, identifying 142 hub-nodes. They are highly connected proteins from which to get deep functional information on viral strategies when used as functional seeds. Therefore, we evaluated the functional and structural significance of each of them to endorse their reliable use as seeds. After filtering, the remaining 111 hubs were used to get by STRING an enriched interactome of 1111 nodes (13,494 interactions). It shows the viral strategy in the liver is to attack the entire cytoplasmic translational system, including ribosomes, to take control of protein bio-synthesis. We used the SARS2-Human-Proteome Interaction Database (33,791 interactions), de-signed by us with BioGRID data to implement a reverse-engineering process that identified human proteins actively interacting with viral proteins. The results show 57% of human liver proteins di-rectly involved in Covid, a strong impairment of the ribosome and spliceosome, an antiviral defense mechanism against cellular stress of the p53 system, and, surprisingly, a viral capacity for multiple protein attacks against single human proteins that reveal underlying evolutionary-topological molecular mechanisms. Viral behavior over time suggests different molecular strategies for different organs.

The cellular stress response corresponds to the molecular changes that cell undergoes in response to various environmental stimuli. It induces drastic changes in the regulation of gene expression, at transcriptional and post-transcriptional levels. Actually, translation is strongly affected with a blockade of the classical cap-dependent mechanism, whereas alternative mechanisms are activated to support translation of specific mRNAs. One of the major mechanisms involved in stress-activated translation is the internal ribosome entry site (IRES)-driven initiation. IRESs, first discovered in viral mRNAs, are present in cellular mRNAs coding for master regulators of cell responses, whose expression must be tightly controlled. IRESs allow translation of these mRNAs in response to different stresses, including DNA damage, amino-acid starvation, hypoxia or endoplasmic reticulum stress, as well as to physiological stimuli such as cell differentiation or synapse network formation. Importantly, cellular mRNA IRESs are regulated by IRES trans-acting factor (ITAFs), exerting their action by at least nine different mechanisms. This review presents an update of the reported ITAFs regulating cellular mRNA translation and of the different mechanisms allowing them to control translation initiation in specific conditions. The impact of ITAFs on coordinated expression of mRNA families and consequences in cell physiology and diseases are also highlighted.

Triphenylphosphonium (TPP) derivatives of a decapeptide related to sequences of bactenecin 7 (Bac7) and oncocin (Onc112) were synthesized and studied as potential antimicrobial compounds using various biochemical and microbiological assays. It was shown that while the reduction of the Bac7 length to 10 a.a. residues dramatically decreases the affinity to bacterial ribosomes, the modification of peptide with alkyl-TPP moieties leads to an increase in the affinity. New analogs which structure combines a decapeptide related to Bac7 and Onc112 — Bac(1-10, R/Y) — and TPP, attached to the C-terminal amino acid residue through alkylamide linkers, inhibit translation in vitro and are found to be more selective inhibitors of bacterial translation as compared with eukaryotic translation, than Onc112 and Bac7. Triphenylphosphonium analogs of a decapeptide related to Bac7 and Onc112 suppress the growth of both gram-negative bacteria, similar to Onc112 and Bac7, and gram-positive ones as alkyl-TPP derivatives, and also act against some resistant laboratory strains. Bac(1-10, R/Y)-C2-TPP, containing a short alkylamide linker between the decapeptide and TPP, is transferred into the E. coli cells via the SbmA transporter protein. TPP derivatives of the decapeptide Bac(1-10, R/Y) containing either decylamide or ethylamide linker cause B. subtilis membrane depolarization, similar to alkyl-TPP. The Bac(1-10, R/Y)-C2-TPP analog is proved to be non-toxic for mammalian cells by MTT-test.

Ribosomes are macromolecular complexes important to protein translation, and thus, essential to life. Comprising an ensemble of ribosomal proteins and RNA molecules, ribosomes are conserved in structure and function across all domains of life, but recent structural studies have revealed differentiated structures of ribosomes from bacterial, archaea and eukaryotes. Additionally, unique ribosomal protein mass fingerprints have been found for individual species; thereby, indicating that ribosomes are differentiated in structure amongst different species. Given that structure defines function, differentiated function likely exists amongst ribosomes of different species, which could manifest as differences in translation efficiency that could impact on cell growth rate. But ribosomal proteins also hold phylogenetic significance in informing the evolutionary trajectory of each species. Such ribosomal proteins are thus not highly conserved and offers sufficient sequence space for the evolution of differentiated structure and function in different species. Using ribosomal proteins that hold phylogenetic significance as templates, this study sought to understand the mutational and conformational limits that define functional ribosomes. Specifically, ribosomal proteins in Bacillus subtilis that hold phylogenetic cues would be mutated through error-prone polymerase chain reaction to generate variants that are subsequently transformed into Escherichia coli. To help assess the functional properties of the heterologous ribosomal proteins, endogenous ribosomal protein genes would be inactivated by multiplex CRISPR interference. Since variants in ribosomal proteins would likely impact on ribosome function and translation efficiency, live/dead screening on LB agar would be effective as a preliminary screen for functional mutants. These mutants would subsequently be inoculated into liquid LB medium in 96 well plates to quantify relative growth rates between different strains harbouring different heterologous variants of ribosomal proteins. Plasmids containing different ribosomal protein mutants would be extracted from each functional strain and subjected to Sanger sequencing for determining the specific mutations involved. Collection of such mutations would provide a comprehensive mutational map that define the limits of ribosomal protein sequence space important to ribosome function. Furthermore, biochemical isolation of ribosomal proteins and their structural characterization by X-ray crystallography or cryo-electron microscopy would further illuminate the structural significance of each mutation on ribosome structure and function; thereby, elucidating the structural tolerance space for functional ribosomes. Overall, generating a diverse pool of mutant ribosomal proteins in viability assays followed by sequencing and structural characterization would help define the mutational and conformational limits of a functional and efficient ribosome.

Translation of the genetic code into proteins is the main process across all life and ribosomes are ancient cellular machines uniquely enabling this information transformation. We provide a brief overview of the recent advances in linking the ribosomal structure and evolution. Based on these insights into ribosomal organisation across time, we propose that early replication and protein biosynthesis functions were inseparable and in fact were performed by the same ancient RNA molecule, the riboreplisome. Riboreplisome hypothesis helps to address issues of non-Darwinian evolution and complicated starting point that are characteristic to the RNA world, protein world and RNA:protein mixed co-development theories. We suggest that the riboreplisome is the missing link and a molecular machine connecting chemical and biological evolution paths, by being capable of basic genetic and feature selection functions in a cell- or cell-free setting. The riboreplisome hypothesis allows ease of sequential, genetically uninterrupted emergence and sophistication of the genetic code and its decoding machinery, and provides plausible explanations to the origins of the three main RNA types involved in the decoding: the ribosomal, transfer and messenger RNA. Furthermore, riboreplisome can help explaining the co-evolution of aminoacylation machinery, the driving force behind selective gene transcription and expression, and the cell-like compartmentalisation. While we may never find the original riboreplisome again, we might continue to discover different molecular remnants of its prior existence across the existing biological RNA, which, once identified or resurrected, can be useful in synthetic biology applications.

Ribosome assembly is one of the most fundamental processes in gene expression and has served as a playground to investigate the molecular mechanisms of how protein-RNA complexes (RNPs) assemble. The bacterial ribosome is composed of around 50 ribosomal proteins several of which are co-transcriptionally assembled on a ~4,500 nucleotides long pre-rRNA transcript that is further processed and modified during transcription, the entire process taking around 2 minutes in vivo and assisted by dozens of assembly factors. How this complex molecular process works so efficiently to produce an active ribosome has been investigated over decades and has resulted in the development of a plethora of novel approaches that can also be used to study the assembly of other RNPs. Here we review biochemical, structural and biophysical methods that have been developed and integrated to provide a detailed and quantitative understanding of this complex and intricate molecular process of assembly. We also discuss emerging cutting-edge approaches that could be used in the future to study how transcription, rRNA processing, cellular factors and the native cellular environment shape ribosome assembly and RNP assembly at large.

Cisplatin (CDDP), carboplatin (CP), and oxaliplatin (OXP) are three platinating agents clinically approved worldwide for use against a variety of cancers. They are canonically known as DNA damage inducers; however, that is only one of their mechanisms of cytotoxicity. CDDP mediates its effects through DNA damage-induced transcription inhibition and apoptotic signalling. In addition, CDDP targets the endoplasmic reticulum (ER) to induce ER-stress, the mitochondria via mitochondrial DNA damage leading to ROS production, and the plasma membrane and cytoskeletal components. CP acts in a similar fashion to CDDP by inducing DNA damage, mitochondrial damage, and ER stress. Additionally, CP is also able to upregulate micro-RNA activity, enhancing intrinsic apoptosis. OXP, on the other hand, at first induces damage to all the same targets as CDDP and CP, yet it is also capable of inducing immunogenic cell death via ER stress and can decrease ribosome biogenesis through its nucleolar effects. In this comprehensive review, we provide detailed mechanisms of action for the three platinating agents, going beyond their nuclear effects to include their cytoplasmic impact within cancer cells. In addition, we cover their current clinical use and limitations, including side effects and mechanisms of resistance.