Ribophagy is a crucial mechanism that maintains ribosome homeostasis in the cell by directing nonfunctional ribosomes to degradation via macroautophagy/autophagy. Impaired ribophagy may lead to ribosome quality control disorders and may consequently be associated with various diseases known as ribosomopathies. This topic has been actively studied over the past decade, but the complete mechanism of ribophagy is not fully understood. To study the mechanism of ribophagy, we performed a genome-wide CRISPR-Cas9-based screening using a fluorescent ribophagy reporter, which is a cell line with ribosomes carrying RPL29 fused with mCherry and GFP fluorescent proteins. Using the genome-wide Brunello library of guide RNAs, we identified the most promising targets for further study, including the ubiquitin ligase TRIM25, for which we have shown specific binding to the ribosome during ribophagy induction, leading to ubiquitination of the ribosome on the nascent peptide chain and degradation of the whole ribosome. Our findings also demonstrated that poly(I:C) treatment, which mimics viral infection, activates ribophagy in a TRIM25-dependent manner, suggesting the ribophagy pathway could be an antiviral defense mechanism. Taken together, we discovered a novel regulator of ribophagy, TRIM25, which provides new insights into the regulation of selective autophagy in the context of ribosomopathies.Abbreviations: CHX: cycloheximide; FACS: fluorescence-activated cell sorting; GFP: green fluorescent protein; MAGeCK: model-based Analysis of Genome-wide CRISPR-Cas9 Knockout; MOI: multiplicity of infection; poly(I:C): polyinosinic:polycytidylic acid; RFP: red fluorescent protein.

The dynamic equilibrium between synthesis and degradation of biomolecules is maintained by cells, however, with aging, this balance is disrupted, resulting in the onset of diseases, including diabetes and neurodegenerative diseases. A decrease in autophagy, a key cellular process that is involved in lysosome-mediated degradation of damaged or dysfunctional cellular components, may contribute to this imbalance. Autophagy is strictly regulated within the cell through multiple signaling pathways, e.g., through the AMPK-dependent pathway, which functions as a key sensor of cellular energy limitation. In this study, we assessed the autophagy/mitophagy activation ability of a small set of 1,3-diaza-2-oxophenoxazine derivatives and analogs using a fluorescent reporter assay and immunoblot analysis. The two lead compounds, AR493 and AR900, which exhibited the highest autophagy induction levels, were demonstrated to activate the AMPK-dependent pathway. The introduction of a 2'-hydroxyl group into AR493 had almost no influence on its activity, while subsequent attachment of a metabolizable masked phosphate group resulted in a notable increase in activity, although accompanied by substantial toxicity. When analyzing the specificity of the lead compounds to AMPK and its main upstream regulator SIRT1 on the corresponding knockout cell lines, AR493 demonstrated the greatest specificity of action to AMPK. Molecular docking revealed that AR493 binds to Site 2 of the AMPK γ-subunit, which may promote AMPK activation by two possible mechanisms: by preventing ATP binding to Site 3, thus favoring AMP binding; and by directly engaging the αRIM2 motif to stabilize its interaction with the γ-subunit.

Background/Objectives: The recent COVID-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for protocols for rapid development of efficient screening methods to search for the optimal mRNA vaccine structures against mutable viral agents. The unmatched success of mRNA vaccines by Pfizer and Moderna encoding the spike protein of SARS-CoV-2 confirms the potential of lipid nanoparticles for mRNA delivery for an accelerated development of new vaccines. The efficacy of vaccination and the production cost of mRNA-based vaccines largely depend on the composition of mRNA components, since the synthesis of an immunogenic protein requires precise and efficient translation in vivo. The composition of 5' and 3' UTR combinations of mRNA has a strong impact on the translation efficiency. The major objective of this study was to increase the probability of producing the immunogenic protein encoded by vaccine mRNA. For this purpose, we proposed to find a new combination of natural UTRs and, in parallel with that, to design and test the system for in vivo selection of translationally active UTRs. Methods: By using Ribo-Seq analysis, sets of candidate short UTRs were generated. These UTRs were tested both in cell cultures and in mice for effective production of secreted nanoluciferase (NLuc) and the S protein of SARS-CoV-2. A combination of the most effective UTRs was used to generate a prototype of an mRNA vaccine capable of inducing neutralizing antibodies against coronavirus. Results: The usefulness of the selected UTRs for vaccine development was tested by implicating the full-length coding sequence of SARS-CoV-2 S protein to produce the main immunogen. As a result, the system for functional screening of UTRs was created by using the NLuc gene. Conclusions: The proposed approach allows non-invasive quantitative assessment of the translational activity of UTRs in the blood serum of mice. By using the full-length sequence of SARS-CoV-2 S protein as a prototype, we demonstrated that the combination of UTRs selected using our luciferase-based reporter assay induces IgG titers and neutralization rates comparable to those obtained by using UTRs from commercial S-protein-based mRNA vaccines.

Spermatozoid's flagella assemble in transcriptionally silent spermatids and thus depend on posttranscriptional regulation of gene expression. Mutations in Nsun7 gene are known to cause male infertility in human and mice. We identified m5C-specific NSUN7 RNA methyltransferase as a protein present in elongated spermatids and interacting with RNAs specific for this type of spermatozoid's precursor cells. Inactivation of the Nsun7 gene in mice leads to upregulation of its RNA interactors, thus indicating that NSUN7 downregulates a set of RNAs in the elongated spermatids. A physiologic consequence of Nsun7 gene knockout is male infertility, which is mechanistically explained by the observed mispositioning of longitudinal columns relative to the axonemal microtubular doublets leading to a motility defect.

Translation of mRNAs is a tightly regulated process in gene expression. In mRNA, the 5' untranslated region (5' UTR) controls ribosome recruitment and frequently contains structured elements that modulate translation efficacy. This study investigates stable structural motifs within the 5' UTR of DDX23 mRNA, encoding a protein relevant for anticancer therapy, as potential regulators and targets for antisense oligonucleotides (ASOs). Despite bioinformatic predictions and transcriptomic validations suggesting RNA G-quadruplex (rG4) formation, comprehensive structural analysis using a light-up assay and CD, UV, and NMR spectroscopy revealed that most putative rG4-forming sequences do not fold into stable rG4 structures, although one of them exists in an equilibrium between rG4 and an alternative, likely hairpin, conformation. Reporter assays using a robust G4 stabilizer also argue against the significant regulatory role of rG4s in DDX23 mRNA translation. Instead, we identified and characterized a stable hairpin structure with potential regulatory function. Based on these findings, we designed fully locked nucleic acid (LNA)-modified ASOs to target this hairpin and regions flanking the upstream open reading frame (uORF) and start codon of the coding sequence. A reporter assay demonstrated that cap-proximal targeting achieved robust translation inhibition up to 80%. In contrast, targeting the efficiently translated uORF was ineffective, presumably due to steric hindrances from the ribosomal complex. The study yields crucial design principles for translation-regulating ASOs: avoid targeting regions shielded by efficient uORF translation and carefully tune ASO-RNA duplex stability to surpass endogenous structures without disrupting regulatory mechanisms. These findings provide insights into the regulation of DDX23 expression and establish a framework for developing ASO-based therapeutics with broad implications for mRNA targeting in anticancer applications.

Upstream open reading frames (uORFs) are a widespread class of translated regions (translons) occurring in 5′ leaders of mRNAs and serving critical roles in post-transcriptional regulation. However, their specific biological activities in human cells remains to be fully elucidated. Here, we conducted a genome-wide CRISPR-Cas9 loss-of-function screen of 978 uORFs identified with ribosome profiling, across human cell lines of distinct origin (HAP1, A549 and HEK293T). A total of 155 uORFs were identified as being essential for cell proliferation. These uORFs showed a high cell-type specificity, with only a few being universally essential. Subsequent analysis has revealed that the primary reason underlying the uORF essentiality is not encoded micropeptides, but rather cis -regulatory mechanisms. Moreover, uORFs located within short 5′ UTRs were disproportionately sensitive to frameshift-inducing indels, which frequently lead to the uORF extension and overlap with the coding region (CDS), resulting in translational repression. Finally, by intersecting regions of essential uORF with ClinVar and dbSNP datasets, we identified naturally occurring variants with the potential to disrupt their function and contribute to disease phenotypes. These findings highlight a pervasive and underappreciated layer of translational control in human cells and establish uORFs as critical cis -regulatory elements with potential relevance to human health. ### Competing Interest Statement Pavel V Baranov is a co-founder and a shareholder of Eirna Bio. The remaining authors declare no competing interests. Russian Science Foundation, https://ror.org/03y2gwe85, 23-14-00058

Long non‐coding RNAs (lncRNAs) are transcripts with a length more than 200 nt, which do not encode proteins and act just as RNA molecules. In general, lncRNAs have much more distinct tissue specificity than proteins, as they usually realize more peculiar regulatory functions. Their expression levels are often altered in a response to stress conditions, metabolic changes, development of different diseases, and carcinogenesis. Cancer‐associated lncRNAs are widely considered as perspective and useful biomarkers. Thus, development of clinical tests, which include tissue‐specific and cancer‐specific lncRNAs, might significantly contribute to cancer diagnostics and/or prognosis of the disease. A number of lncRNAs is known to be dysregulated in liver tumors and considered as probable biomarkers. However, most of them are rather universally well‐known lncRNAs associated with various cancers. In the present review, we aimed to shed light on other lncRNAs with preferential expression in liver and/or liver tumors, for example, LINC01554, LINC01093, LINC01348, LINC02428, FAM99B, etc. We summarized recent discoveries unveiling their dysregulation in liver malignancies and related cellular mechanisms in which they are involved and considered their significance as probable liver cancer biomarkers.

Objectives: Oocyte quality is crucial for female fertility, but the underlying molecular mechanisms remain unclear. This study investigates the non-canonical role of the telomerase RNA protein (TERP), whose function in oogenesis is unknown, in safeguarding female gamete quality. Methods: We used gain-of-function (AT) and loss-of-function (D7) mutant mouse lines to assess oocyte quality via morphological and molecular analyses. Key methods included immunofluorescence of meiotic spindles, Western blotting for the autophagy marker LC3B, and qRT-PCR to quantify the perinatal ovarian reserve. Results: Both AT and D7 mutations caused severe meiotic spindle abnormalities, including aberrant morphology and increased size. The D7 mutation, in particular, led to impaired cytoplasmic maturation and reduced autophagy levels in oocytes. Furthermore, loss of TERP function resulted in an abnormally large ovarian reserve in newborn females, which correlated with decreased expression of autophagy and lysosomal markers in the newborn ovary. Conclusions: This study establishes a novel, non-canonical function for TERP as a crucial regulator of oocyte quality. TERP dysregulation compromises meiotic integrity and oocyte maturation by disrupting lysosome-dependent autophagy.

This work focuses on the search for new small molecules for anticancer therapy using the fluorescent cells cocultivation test (FCCT). This method allows the control of the specificity of the action of compounds from the earliest stages of drug development. For the FCCT, labeled MCF7' breast cancer cells and noncancerous breast MCF10A cells are cocultured. Screening of 2025 compounds in the above system and previously developed coculture of A549 with VA13 yields 16 selectively cytotoxic molecules. The results are confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for seven of these molecules. Few are known as potential antitumor agents: angelicin, coumarin, and colchicine derivatives. However, the structures of macrocycle 1, pyrazole-azepinoindole derivative 2, and complex heterocyclic derivative 3 are not described as anticancer compounds according to the PubChem and SciFinder databases. Structure-activity relationships are investigated for 2 and its derivatives. The indole with a caprolactam ring (tetrahydro-azepinoindolone core) together with the pyrazolyl at the third position is the key element of the pharmacophore. The optimized pyrazole-azepinoindole derivative 23 shows SI = 18 for HCT116 versus VA-13 on the expanded array of cell lines. Its effect is mainly mediated by the G1 arrest of the cell cycle.

ObjectiveMany long noncoding RNAs (lncRNAs) are associated with liver cancers, mainly hepatocellular carcinoma (HCC) and to a smaller extent intrahepatic cholangiocarcinoma (CCA). Most of such lncRNAs show similar dysregulation patterns when the two types of tumors are compared, suggesting that these aberrations are characteristic features of these liver tumor types. In the present study, we aimed to identify some candidate lncRNAs that are associated specifically with CCA.MethodsAccording to The Cancer Genome Atlas data, we chose LINC00941, ABHD11-AS1, and CASC8 as promising biomarkers dysregulated in CCA but unaffected in HCC. We first verified their upregulation in an existing transcriptomic dataset for CCA patients. Next, we estimated expression levels of these three lncRNAs by reverse-transcription quantitative PCR in a group of paired (tumorous/adjacent) postsurgery tissue samples from 110 patients with various liver lesions: CCA, HCC, combined HCC-CCA, or benign liver tumors.ResultsSignificant upregulation of LINC00941 and ABHD11-AS1 was noted in most of the investigated CCA samples, whereas in HCC samples, increased expression of these two lncRNAs was observed only in some types of cases (mainly characterized by an advanced tumor stage). In contrast, CASC8 manifested extremely low expression and no diagnostic potential in all the tested liver samples. Analyzing expression correlations of lncRNAs with candidate genes, we obtained strong evidence for LINC00941-mediated upregulation of CAPRIN2 in CCA.ConclusionsFor the first time, we show the upregulation of LINC00941 and ABHD11-AS1 in CCA and report their good potential as diagnostic biomarkers for this type of liver tumor.

Gene expression is a complex process regulated at multiple levels in eukaryotic cells. Translation frequently represents a pivotal step in the control of gene expression. Among the stages of translation, initiation is particularly important, as it governs ribosome recruitment and the efficiency of protein synthesis. The 5′ untranslated region (5′ UTR) of mRNA plays a key role in this process, often exhibiting a complicated and structured landscape. Numerous eukaryotic mRNAs possess long 5′ UTRs that contain diverse regulatory elements, including RNA secondary structures, specific nucleotide motifs, and chemical modifications. These structural features can independently modulate translation through their intrinsic properties or by serving as platforms for trans-acting factors such as RNA-binding proteins. The dynamic nature of 5′ UTR elements allows cells to fine-tune translation in response to environmental and cellular signals. Understanding these mechanisms is not only fundamental to molecular biology but also holds significant biomedical potential. Insights into 5′ UTR-mediated regulation could drive advancements in synthetic biology and mRNA-based targeted therapies. This review outlines the current knowledge of the structural elements of the 5′ UTR, the interplay between them, and their combined functional impact on translation.

Ribosome biogenesis is essential for the functioning of living cells. In higher eukaryotes, this multistep process is tightly controlled and involves a variety of specialized proteins and RNAs. This pool of so-called ribosome biogenesis factors includes diverse proteins with enzymatic and structural functions. Some of them have homologs in yeast S. cerevisiae, and their function can be inferred from the structural and biochemical data obtained for the yeast counterparts. The functions of human proteins RPF1 and ESF1 remain largely unclear, although RPF1 has been recently shown to participate in 60S biogenesis. Both proteins have drawn our attention since they contribute to the early stages of ribosome biogenesis, which are far less studied than the later stages. In this study, we employed the loss-of-function shRNA/siRNA-based approach to the human cell line HEK293 to determine the role of RPF1 and ESF1 in ribosome biogenesis. Downregulating RPF1 and ESF1 significantly changed the pattern of RNA products derived from 47S pre-rRNA. Our findings demonstrate that RPF1 and ESF1 are associated with different pre-ribosomal particles, pre-60S, and pre-40S particles, respectively. Our results allow for speculation about the particular steps of pre-rRNA processing, which highly rely on the RPF1 and ESF1 functions. We suggest that both factors are not directly involved in pre-rRNA cleavage but rather help pre-rRNA to acquire the conformation favoring its cleavage.

Telomeres – special DNA-protein structures at the ends of linear eukaryotic chromosomes define the proliferation potential of cells. Extremely short telomeres promote DNA damage response and cell death to eliminate cells potentially accumulated mutations after multiple divisions. However, telomere elongation is associated with increased proliferative potential of special types of cells, such as stem and germ cells, permanently, and is activated temporally in processes of activation of immune response, regeneration processes. Activation of mechanisms of telomere lengthening coupled with increased proliferation and with requirements of cells in energy and building resourses. To obtain necessary nutrients cells stimulate metabolism by switching of oxidative phosphorylation program to glycolysis. In this review we focused on the interconnection of metabolism program and telomere lengthening mechanisms known for the situation of the programmed activation of proliferation such as germ cells maturation, early embryonic development program and immune response activation. Here, we propose that it is possible to reprogram metabolism in order to regulate the telomere length, proliferative activity of cells that may be important for the development of approaches to regeneration, immune response modulation and cancer therapy and further investigations in this area are necessary to improve the understanding and manipulating of molecular mechanisms used for regulation of proliferation.

Telomeres—special DNA–protein structures at the ends of linear eukaryotic chromosomes—define the proliferation potential of cells. Extremely short telomeres promote a DNA damage response and cell death to eliminate cells that may have accumulated mutations after multiple divisions. However, telomere elongation is associated with the increased proliferative potential of specific cell types, such as stem and germ cells. This elongation can be permanent in these cells and is activated temporally during immune response activation and regeneration processes. The activation of telomere lengthening mechanisms is coupled with increased proliferation and the cells’ need for energy and building resources. To obtain the necessary nutrients, cells are capable of finely regulating energy production and consumption, switching between catabolic and anabolic processes. In this review, we focused on the interconnection between metabolism programs and telomere lengthening mechanisms during programmed activation of proliferation, such as in germ cell maturation, early embryonic development, neoplastic lesion growth, and immune response activation. It is generally accepted that telomere disturbance influences biological processes and promotes dysfunctionality. Here, we propose that metabolic conditions within proliferating cells should be involved in regulating telomere lengthening mechanisms, and telomere length may serve as a marker of defects in cellular functionality. We propose that it is possible to reprogram metabolism in order to regulate the telomere length and proliferative activity of cells, which may be important for the development of approaches to regeneration, immune response modulation, and cancer therapy. However, further investigations in this area are necessary to improve the understanding and manipulation of the molecular mechanisms involved in the regulation of proliferation, metabolism, and aging.

High-throughput ribosome profiling demonstrates the translation of thousands of small open reading frames located in the 5′ untranslated regions of messenger RNAs (upstream ORFs). Upstream ORF can both perform a regulatory function by influencing the translation of the downstream main ORF and encode a small functional protein or microprotein. In this work, we showed that the 5′ untranslated region of the PRPF19 mRNA encodes an upstream ORF that is translated in human cells. Inactivation of this upstream ORF reduces the viability of human cells.

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.

Telomerase is a ribonucleoprotein complex, the main components of which are telomerase RNA and reverse transcriptase. Previously, it was shown in our laboratory that human telomerase RNA contains an open reading frame starting at adenine in position 176. The open reading frame encodes the hTERP protein, and the deletion of nucleotides 184–188 of human telomerase RNA disrupts the open reading frame and leads to the absence of hTERP. Human telomerase RNA has a conserved structure, changes in which affect telomerase activity. In this work, we have shown that the deletion of nucleotides 184–188 of telomerase RNA does not affect the functioning of telomerase.

Mitochondrial ribosome assembly is a complex multi-step process involving many additional factors. Ribosome formation differs in various groups of organisms. However, there are universal steps of assembly and conservative factors that have been retained in evolutionarily distant taxa. METTL17, the object of the current study, is one of these conservative factors involved in mitochondrial ribosome assembly. It is present in both bacteria and the mitochondria of eukaryotes, in particular mice and humans. In this study, we tested a hypothesis of putative METTL17 methyltransferase activity. MALDI-TOF mass spectrometry was used to evaluate the methylation of a putative METTL17 target - a 12S rRNA region interacting with METTL17 during mitochondrial ribosome assembly. The investigation of METTL17 and other mitochondrial ribosome assembly factors is of both fundamental and practical significance, because defects in mitochondrial ribosome assembly are often associated with human mitochondrial diseases.

The biogenesis of ribosomes requires tightly controlled transcription and processing of pre-rRNA which comprises ribosomal RNAs forming the core of large and small ribosomal subunits. Early steps of the pre-rRNA processing and assembly of the ribosomal subunits require a large set of proteins that perform folding and nucleolytic cleavage of pre-rRNAs in the nucleoli. Structure and functions of proteins involved in the pre-rRNA processing have been extensively studied in the budding yeast S. cerevisiae. Functional characterization of their human homologues is complicated by the complexity of mammalian ribosomes and increased number of protein factors involved in the ribosomal biogenesis. Homologues of human nucleolar protein SURF6 from yeast and mouse, Rrp14 and Surf6, respectively, had been shown to be involved in the early steps of pre-rRNA processing. Rrp14 works as RNA chaperone in complex with proteins Ssf1 and Rrp15. Human SURF6 knockdown and overexpression were used to clarify a role of SURF6 in the early steps of pre-rRNA processing in human cell lines HeLa and HTC116. By analyzing the abundance of the rRNA precursors in cells with decreased level or overexpression of SURF6, we demonstrated that human SURF6 is involved in the maturation of rRNAs from both small and large ribosomal subunits. Changes in the SURF6 level caused by knockdown or overexpression of the protein do not result in the death of HeLa cells in contrast to murine embryonic fibroblasts, but significantly alter the distribution of cells among the phases of the cell cycle. SURF6 knockdown in both p53 sufficient and p53 deficient HCT116 human cancer cells results in elongation of G0/G1 and shortening of G2/M phase. This surprising result suggests p53 independence of SURF6 effects on the cell cycle and possible multiple functions of SURF6. Our data point to the shift from pathway 1 to pathway 2 of the rRNA biogenesis caused by the SURF6 knockdown and its likely association with p53 pathway.

Telomerase RNA has long been considered to be a noncoding component of telomerase. However, the expression of the telomerase RNA gene is not always associated with telomerase activity. The existence of distinct TERC gene expression products possessing different functions were demonstrated recently. During biogenesis, hTR is processed by distinct pathways and localized in different cell compartments, depending on whether it functions as a telomerase complex component or facilitates antistress activities as a noncoding RNA, in which case it is either processed in the mitochondria or translated. In order to identify the factors responsible for the appearance and localization of the exact isoform of hTR, we investigated the roles of the factors regulating transcription DSIF (Spt5) and NELF-E; exosome-attracting factors ZCCHC7, ZCCHC8, and ZFC3H1; ARS2, which attracts processing and transport factors; and transport factor PHAX during the biogenesis of hTR. The data obtained revealed that ZFC3H1 participates in hTR biogenesis via pathways related to the polyadenylated RNA degradation mechanism. The data revealed essential differences that are important for understanding hTR biogenesis and that are interesting for further investigations of new, therapeutically significant targets.