RNA editing enzymes: structure, biological functions and applications.

With the advancement of sequencing technologies and bioinformatics, over than 170 different RNA modifications have been identified. However, only a few of these modifications can lead to base pair changes, which are called RNA editing. RNA editing is a ubiquitous modification in mammalian transcriptomes and is an important co/posttranscriptional modification that plays a crucial role in various cellular processes. There are two main types of RNA editing events: adenosine to inosine (A-to-I) editing, catalyzed by ADARs on double-stranded RNA or ADATs on tRNA, and cytosine to uridine (C-to-U) editing catalyzed by APOBECs. This article provides an overview of the structure, function, and applications of RNA editing enzymes. We discuss the structural characteristics of three RNA editing enzyme families and their catalytic mechanisms in RNA editing. We also explain the biological role of RNA editing, particularly in innate immunity, cancer biogenesis, and antiviral activity. Additionally, this article describes RNA editing tools for manipulating RNA to correct disease-causing mutations, as well as the potential applications of RNA editing enzymes in the field of biotechnology and therapy.

Circulating nucleosomes as potential biomarkers for cancer diagnosis and treatment monitoring.

Nucleosomes play a crucial role in regulating gene expression through their composition and post-translational modifications. When cells die, intracellular endonucleases are activated and cleave chromatin into oligo- and mono-nucleosomes, which are then released into the body fluids. Studies have shown that the levels of nucleosomes are increased in serum and plasma in various cancer types, suggesting that analysis of circulating nucleosomes can provide an initial assessment of carcinogenesis. However, it should be noted that elevated serum nucleosome levels may not accurately diagnose certain tumor types, as increased cell death may occur in different pathological conditions. Nevertheless, detection of circulating nucleosomes and their histone modifications, along with specific tumor markers, can help diagnose certain types of cancer. Furthermore, monitoring changes in circulating nucleosome levels during chemotherapy or radiotherapy in patients with malignancies can provide valuable insights into clinical outcomes and therapeutic efficacy. The utilization of circulating nucleosomes as biomarkers is an exciting and emerging area of research, with the potential for early detection of various diseases and monitoring of treatment response. Integrating nucleosome-based biomarkers with existing ones may improve the specificity and sensitivity of current assays, offering the possibility of personalized precision medical treatment for patients.

Advances and opportunities in methods to study protein translation - A review.

It is generally believed that the regulation of gene expression involves protein translation occurring before RNA transcription. Therefore, it is crucial to investigate protein translation and its regulation. Recent advancements in biological sciences, particularly in the field of omics, have revolutionized protein translation research. These studies not only help characterize changes in protein translation during specific biological or pathological processes but also have significant implications in disease prevention and treatment. In this review, we summarize the latest methods in ribosome-based translation omics. We specifically focus on the application of fluorescence imaging technology and omics technology in studying overall protein translation. Additionally, we analyze the advantages, disadvantages, and application of these experimental methods, aiming to provide valuable insights and references to researchers studying translation.

Development of mitochondrial gene-editing strategies and their potential applications in mitochondrial hereditary diseases: a review.

Mitochondrial DNA (mtDNA) is a critical genome contained within the mitochondria of eukaryotic cells, with many copies present in each mitochondrion. Mutations in mtDNA often are inherited and can lead to severe health problems, including various inherited diseases and premature aging. The lack of efficient repair mechanisms and the susceptibility of mtDNA to damage exacerbate the threat to human health. Heteroplasmy, the presence of different mtDNA genotypes within a single cell, increases the complexity of these diseases and requires an effective editing method for correction. Recently, gene-editing techniques, including programmable nucleases such as restriction endonuclease, zinc finger nuclease, transcription activator-like effector nuclease, clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated 9 and base editors, have provided new tools for editing mtDNA in mammalian cells. Base editors are particularly promising because of their high efficiency and precision in correcting mtDNA mutations. In this review, we discuss the application of these techniques in mitochondrial gene editing and their limitations. We also explore the potential of base editors for mtDNA modification and discuss the opportunities and challenges associated with their application in mitochondrial gene editing. In conclusion, this review highlights the advancements, limitations and opportunities in current mitochondrial gene-editing technologies and approaches. Our insights aim to stimulate the development of new editing strategies that can ultimately alleviate the adverse effects of mitochondrial hereditary diseases.

Molecular mechanisms of eukaryotic translation fidelity and their associations with diseases.

Converting genetic information into functional proteins is a complex, multi-step process, with each step being tightly regulated to ensure the accuracy of translation, which is critical to cellular health. In recent years, advances in modern biotechnology, especially the development of cryo-electron microscopy and single-molecule techniques, have enabled a clearer understanding of the mechanisms of protein translation fidelity. Although there are many studies on the regulation of protein translation in prokaryotes, and the basic elements of translation are highly conserved in prokaryotes and eukaryotes, there are still great differences in the specific regulatory mechanisms. This review describes how eukaryotic ribosomes and translation factors regulate protein translation and ensure translation accuracy. However, a certain frequency of translation errors does occur in translation, so we describe diseases that arise when the rate of translation errors reaches or exceeds a threshold of cellular tolerance.

A male germ-cell-specific ribosome controls male fertility.

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the male germ-cell-specific protein RPL39L, the paralogue of core ribosome (RibosomeCore) protein RPL39. Deletion of RibosomeST in mice causes defective sperm formation, resulting in substantially reduced fertility. Our comparison of single-particle cryo-electron microscopy structures of ribosomes from mouse kidneys and testes indicates that RibosomeST features a ribosomal polypeptide exit tunnel of distinct size and charge states compared with RibosomeCore. RibosomeST predominantly cotranslationally regulates the folding of a subset of male germ-cell-specific proteins that are essential for the formation of sperm. Moreover, we found that specialized functions of RibosomeST were not replaceable by RibosomeCore. Taken together, identification of this sperm-specific ribosome should greatly expand our understanding of ribosome function and tissue-specific regulation of protein expression pattern in mammals.

BRAF V600E protect from cell death via inhibition of the mitochondrial permeability transition in papillary and anaplastic thyroid cancers.

BRAF T1799A mutation is the most common genetic variation in thyroid cancer, resulting in the production of BRAF V600E mutant protein reported to make cells resistant to apoptosis. However, the mechanism by which BRAF V600E regulates cell death remains unknown. We constructed BRAF V600E overexpression and knockdown 8505C and BCPAP papillary and anaplastic thyroid cancer cell to investigate regulatory mechanism of BRAF V600E in cell death induced by staurosporine (STS). Induced BRAF V600E expression attenuated STS-induced papillary and anaplastic thyroid cancer death, while BRAF V600E knockdown aggravated it. TMRM and calcein-AM staining showed that opening of the mitochondrial permeability transition pore (mPTP) during STS-induced cell death could be significantly inhibited by BRAF V600E. Moreover, our study demonstrated that BRAF V600E constitutively activates mitochondrial ERK (mERK) to inhibit GSK-3-dependent CypD phosphorylation, thereby making BRAF V600E mutant tumour cells more resistant to mPTP opening. In the mitochondria of BRAF V600E mutant cells, there was an interaction between ERK1/2 and GSKa/ß, while upon BRAF V600E knockdown, interaction of GSKa/ß to ERK was decreased significantly. These results show that in thyroid cancer, BRAF V600E regulates the mitochondrial permeability transition through the pERK-pGSK-CypD pathway to resist death, providing new intervention targets for BRAF V600E mutant tumours.

Translational Control of COVID-19 and Its Therapeutic Implication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, which has broken out worldwide for more than two years. However, due to limited treatment, new cases of infection are still rising. Therefore, there is an urgent need to understand the basic molecular biology of SARS-CoV-2 to control this virus. SARS-CoV-2 replication and spread depend on the recruitment of host ribosomes to translate viral messenger RNA (mRNA). To ensure the translation of their own mRNAs, the SARS-CoV-2 has developed multiple strategies to globally inhibit the translation of host mRNAs and block the cellular innate immune response. This review provides a comprehensive picture of recent advancements in our understanding of the molecular basis and complexity of SARS-CoV-2 protein translation. Specifically, we summarize how this viral infection inhibits host mRNA translation to better utilize translation elements for translation of its own mRNA. Finally, we discuss the potential of translational components as targets for therapeutic interventions.

Mitochondrial Ca2+ Homeostasis: Emerging Roles and Clinical Significance in Cardiac Remodeling.

Mitochondria are the sites of oxidative metabolism in eukaryotes where the metabolites of sugars, fats, and amino acids are oxidized to harvest energy. Notably, mitochondria store Ca2+ and work in synergy with organelles such as the endoplasmic reticulum and extracellular matrix to control the dynamic balance of Ca2+ concentration in cells. Mitochondria are the vital organelles in heart tissue. Mitochondrial Ca2+ homeostasis is particularly important for maintaining the physiological and pathological mechanisms of the heart. Mitochondrial Ca2+ homeostasis plays a key role in the regulation of cardiac energy metabolism, mechanisms of death, oxygen free radical production, and autophagy. The imbalance of mitochondrial Ca2+ balance is closely associated with cardiac remodeling. The mitochondrial Ca2+ uniporter (mtCU) protein complex is responsible for the uptake and release of mitochondrial Ca2+ and regulation of Ca2+ homeostasis in mitochondria and consequently, in cells. This review summarizes the mechanisms of mitochondrial Ca2+ homeostasis in physiological and pathological cardiac remodeling and the regulatory effects of the mitochondrial calcium regulatory complex on cardiac energy metabolism, cell death, and autophagy, and also provides the theoretical basis for mitochondrial Ca2+ as a novel target for the treatment of cardiovascular diseases.

Mitochondrial Protein Translation: Emerging Roles and Clinical Significance in Disease.

Mitochondria are one of the most important organelles in cells. Mitochondria are semi-autonomous organelles with their own genetic system, and can independently replicate, transcribe, and translate mitochondrial DNA. Translation initiation, elongation, termination, and recycling of the ribosome are four stages in the process of mitochondrial protein translation. In this process, mitochondrial protein translation factors and translation activators, mitochondrial RNA, and other regulatory factors regulate mitochondrial protein translation. Mitochondrial protein translation abnormalities are associated with a variety of diseases, including cancer, cardiovascular diseases, and nervous system diseases. Mutation or deletion of various mitochondrial protein translation factors and translation activators leads to abnormal mitochondrial protein translation. Mitochondrial tRNAs and mitochondrial ribosomal proteins are essential players during translation and mutations in genes encoding them represent a large fraction of mitochondrial diseases. Moreover, there is crosstalk between mitochondrial protein translation and cytoplasmic translation, and the imbalance between mitochondrial protein translation and cytoplasmic translation can affect some physiological and pathological processes. This review summarizes the regulation of mitochondrial protein translation factors, mitochondrial ribosomal proteins, mitochondrial tRNAs, and mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) in the mitochondrial protein translation process and its relationship with diseases. The regulation of mitochondrial protein translation and cytoplasmic translation in multiple diseases is also summarized.

Defective mitochondrial ISCs biogenesis switches on IRP1 to fine tune selective mitophagy.

Both iron metabolism and mitophagy, a selective mitochondrial degradation process via autolysosomal pathway, are fundamental for the cellular well-being. Mitochondria are the major site for iron metabolism, especially the biogenesis of iron-sulfur clusters (ISCs) via the mitochondria-localized ISCs assembly machinery. Here we report that mitochondrial ISCs biogenesis is coupled with receptor-mediated mitophagy in mammalian cells. Perturbation of mitochondrial ISCs biogenesis, either by depleting iron with the iron chelator or by knocking down the core components of the mitochondrial ISCs assembly machinery, triggers FUNDC1-dependent mitophagy. IRP1, one of the cellular iron sensors to maintain iron homeostasis, is crucial for iron stresses induced mitophagy. Knockdown of IRP1 disturbed iron stresses induced mitophagy. Furthermore, IRP1 could bind to a newly characterized IRE in the 5' untranslated region of the Bcl-xL mRNA and suppress its translation. Bcl-xL is an intrinsic inhibitory protein of the mitochondrial phosphatase PGAM5, which catalyzes the dephosphorylation of FUNDC1 for mitophagy activation. Alterations of the IRP1/Bcl-xL axis navigate iron stresses induced mitophagy. We conclude that ISCs serve as physiological signals for mitophagy activation, thus coupling mitophagy with iron metabolism.

Conformational activation of ribosome recycling by intra- and inter-molecular dynamics of RRF.

Ribosome recycling is the final step of the cyclic process of translation, where the post-termination complex (PoTC) is disassembled by the concerted action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in the sub-second time range. Since, however, both the RRF and PoTC display highly dynamic action during this process, it is difficult to assess the molecular details of the interactions between the factors and the ribosome that are essential for rapid subunit separation. Here we characterized the molecular dynamics of RRF and PoTC by combined use of molecular dynamics simulations, single molecule fluorescence detection and single-particle cryo-EM analysis, with time resolutions in the sub-millisecond to minute range. We found that RRF displays two-layer dynamics: intra- and inter-molecular dynamics during ribosome splitting. The intra-molecular dynamics exhibits two different configurations of RRF: 'bent' and 'extended'. A single-site mutant of RRF increases its propensity to the 'extended' conformation and leads to a higher binding affinity of RRF to the PoTC. The inter-molecular dynamics between RRF and EF-G in the PoTC reveals that the domain IV of EF-G pushes against the domain II of RRF, triggering the disruption of the major inter-subunit bridge B2a, and catalyzes the splitting.

Genome wide analyses uncover allele-specific RNA editing in human and mouse.

RNA editing is one of the most common RNA level modifications that potentially generate amino acid changes similar to those resulting from genomic nonsynonymous mutations. However, unlike DNA level allele-specific modifications such as DNA methylation, it is currently unknown whether RNA editing displays allele-specificity across tissues and species. Here, we analyzed allele-specific RNA editing in human tissues and from brain tissues of heterozygous mice generated by crosses between divergent mouse strains and found a high proportion of overlap of allele-specific RNA editing sites between different samples. We identified three allele-specific RNA editing sites cause amino acid changes in coding regions of human and mouse genes, whereas their associated SNPs yielded synonymous differences. In vitro cellular experiments confirmed that sequences differing at a synonymous SNP can have differences in a linked allele-specific RNA editing site with nonsynonymous implications. Further, we demonstrate that allele-specific RNA editing is influenced by differences in local RNA secondary structure generated by SNPs. Our study provides new insights towards a better comprehension of the molecular mechanism that link SNPs with human diseases and traits.

SmProt: a database of small proteins encoded by annotated coding and non-coding RNA loci.

Small proteins is the general term for proteins with length shorter than 100 amino acids. Identification and functional studies of small proteins have advanced rapidly in recent years, and several studies have shown that small proteins play important roles in diverse functions including development, muscle contraction and DNA repair. Identification and characterization of previously unrecognized small proteins may contribute in important ways to cell biology and human health. Current databases are generally somewhat deficient in that they have either not collected small proteins systematically, or contain only predictions of small proteins in a limited number of tissues and species. Here, we present a specifically designed web-accessible database, small proteins database (SmProt, http://bioinfo.ibp.ac.cn/SmProt), which is a database documenting small proteins. The current release of SmProt incorporates 255 010 small proteins computationally or experimentally identified in 291 cell lines/tissues derived from eight popular species. The database provides a variety of data including basic information (sequence, location, gene name, organism, etc.) as well as specific information (experiment, function, disease type, etc.). To facilitate data extraction, SmProt supports multiple search options, including species, genome location, gene name and their aliases, cell lines/tissues, ORF type, gene type, PubMed ID and SmProt ID. SmProt also incorporates a service for the BLAST alignment search and provides a local UCSC Genome Browser. Additionally, SmProt defines a high-confidence set of small proteins and predicts the functions of the small proteins.

Mammalian elongation factor 4 regulates mitochondrial translation essential for spermatogenesis.

Elongation factor 4 (EF4) is a key quality-control factor in translation. Despite its high conservation throughout evolution, EF4 deletion in various organisms has not yielded a distinct phenotype. Here we report that genetic ablation of mitochondrial EF4 (mtEF4) in mice causes testis-specific dysfunction in oxidative phosphorylation, leading to male infertility. Deletion of mtEF4 accelerated mitochondrial translation at the cost of producing unstable proteins. Somatic tissues overcame this defect by activating mechanistic (mammalian) target of rapamycin (mTOR), thereby increasing rates of cytoplasmic translation to match rates of mitochondrial translation. However, in spermatogenic cells, the mTOR pathway was downregulated as part of the developmental program, and the resulting inability to compensate for accelerated mitochondrial translation caused cell-cycle arrest and apoptosis. We detected the same phenotype and molecular defects in germline-specific mtEF4-knockout mice. Thus, our study demonstrates cross-talk between mtEF4-dependent quality control in mitochondria and cytoplasmic mTOR signaling.

EF4 disengages the peptidyl-tRNA CCA end and facilitates back-translocation on the 70S ribosome.

EF4 catalyzes tRNA back-translocation through an unknown mechanism. We report cryo-EM structures of Escherichia coli EF4 in post- and pretranslocational ribosomes (Post- and Pre-EF4) at 3.7- and 3.2-Å resolution, respectively. In Post-EF4, peptidyl-tRNA occupies the peptidyl (P) site, but the interaction between its CCA end and the P loop is disrupted. In Pre-EF4, the peptidyl-tRNA assumes a unique position near the aminoacyl (A) site, denoted the A site/EF4 bound (A/4) site, with a large displacement at its acceptor arm. Mutagenesis analyses suggest that a specific region in the EF4 C-terminal domain (CTD) interferes with base-pairing between the peptidyl-tRNA 3'-CCA and the P loop, whereas the EF4 CTD enhances peptidyl-tRNA interaction at the A/4 site. Therefore, EF4 induces back-translocation by disengaging the tRNA's CCA end from the peptidyl transferase center of the translating ribosome.

HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions.

Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coli HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock-induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock. These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions.

New insights into the enzymatic role of EF-G in ribosome recycling.

During translation, elongation factor G (EF-G) plays a catalytic role in tRNA translocation and a facilitative role in ribosome recycling. By stabilizing the rotated ribosome and interacting with ribosome recycling factor (RRF), EF-G was hypothesized to induce the domain rotations of RRF, which subsequently performs the function of splitting the major intersubunit bridges and thus separates the ribosome into subunits for recycling. Here, with systematic mutagenesis, FRET analysis and cryo-EM single particle approach, we analyzed the interplay between EF-G/RRF and post termination complex (PoTC). Our data reveal that the two conserved loops (loop I and II) at the tip region of EF-G domain IV possess distinct roles in tRNA translocation and ribosome recycling. Specifically, loop II might be directly involved in disrupting the main intersubunit bridge B2a between helix 44 (h44 from the 30S subunit) and helix 69 (H69 from the 50S subunit) in PoTC. Therefore, our data suggest a new ribosome recycling mechanism which requires an active involvement of EF-G. In addition to supporting RRF, EF-G plays an enzymatic role in destabilizing B2a via its loop II.

EF-G catalyzes tRNA translocation by disrupting interactions between decoding center and codon-anticodon duplex.

During translation, elongation factor G (EF-G) catalyzes the translocation of tRNA2-mRNA inside the ribosome. Translocation is coupled to a cycle of conformational rearrangements of the ribosomal machinery, and how EF-G initiates translocation remains unresolved. Here we performed systematic mutagenesis of Escherichia coli EF-G and analyzed inhibitory single-site mutants of EF-G that preserved pretranslocation (Pre)-state ribosomes with tRNAs in A/P and P/E sites (Pre-EF-G). Our results suggest that the interactions between the decoding center and the codon-anticodon duplex constitute the barrier for translocation. Catalysis of translocation by EF-G involves the factor's highly conserved loops I and II at the tip of domain IV, which disrupt the hydrogen bonds between the decoding center and the duplex to release the latter, hence inducing subsequent translocation events, namely 30S head swiveling and tRNA2-mRNA movement on the 30S subunit.

The paradox of elongation factor 4: highly conserved, yet of no physiological significance?

LepA [EF4 (elongation factor 4)] is a highly conserved protein found in nearly all known genomes. EF4 triggers back-translocation of the elongating ribosome, causing the translation machinery to move one codon backwards along the mRNA. Knockout of the corresponding gene in various bacteria results in different phenotypes; however, the physiological function of the factor in vivo is unclear. Although functional research on Guf1 (GTPase of unknown function 1), the eukaryotic homologue of EF4, showed that it plays a critical role under suboptimal translation conditions in vivo, its detailed mechanism has yet to be identified. In the present review we briefly cover recent advances in our understanding of EF4, including in vitro structural and biochemical studies, and research on its physiological role in vivo. Lastly, we present a hypothesis for back-translocation and discuss the directions future EF4 research should focus on.