The lack of a sufficient number of validated miRNA targets severely hampers the understanding of their biological function. Even for the well-studied miR-155-5p, there are only 239 experimentally validated targets out of...The lack of a sufficient number of validated miRNA targets severely hampers the understanding of their biological function. Even for the well-studied miR-155-5p, there are only 239 experimentally validated targets out of 42,554 predicted targets. For a more complete assessment of the immune-related miR-155 targetome, we used an inverse correlation of time-resolved mRNA profiles and miR-155-5p expression of early CD4+ T cell activation to predict immune-related target genes. Using a high-throughput miRNA interaction reporter (HiTmIR) assay we examined 90 target genes and confirmed 80 genes as direct targets of miR-155-5p. Our study increases the current number of verified miR-155-5p targets approximately threefold and exemplifies a method for verifying miRNA targetomes as a prerequisite for the analysis of miRNA-regulated cellular networks.
Mutations in coding sequence and abnormal PKD1 expression levels contribute to the development of autosomal-dominant polycystic kidney disease, the most common genetic disorder. Regulation of PKD1 expression by factors...Mutations in coding sequence and abnormal PKD1 expression levels contribute to the development of autosomal-dominant polycystic kidney disease, the most common genetic disorder. Regulation of PKD1 expression by factors located in the promoter and 3´ UTR have been extensively studied. Less is known about its regulation by 5´ UTR elements. In this study, we investigated the effects of uORFs and uORF-affecting variants by combining bioinformatic analyses, luciferase reporter assays, RT-qPCR and immunoblotting experiments. Our analyses demonstrate that mRNA contains two evolutionarily conserved translation-inhibitory uORFs. uORF1 is translatable, and uORF2 is likely not translatable. The 5´ UTR and uORFs do not modulate downstream protein output under endoplasmic reticulum stress and oxidative stress conditions. Some of uORF-perturbing variants in the SNP database are predicted to affect gene translation. Luciferase reporter assays and RT-qPCR results reveal that rs2092942382 and rs1596636969 increase, while rs2092942900 decreases main gene translation without affecting transcription. Antisense oligos targeting the uORFs reduce luciferase protein levels without altering luciferase mRNA levels. Our results establish as a novel target of uORF-mediated translational regulation and mutations that perturb uORFs may dysregulate PKD1 protein level.
RNA-focused therapy and diagnostics have been making waves in molecular biology due to the advantages RNA has over DNA; for instance, the ability of RNA to target nearly any genetic component in the cell is a big step in...RNA-focused therapy and diagnostics have been making waves in molecular biology due to the advantages RNA has over DNA; for instance, the ability of RNA to target nearly any genetic component in the cell is a big step in treating disorders. Moreover, RNA-based diagnosis of diseases is only becoming increasingly popular, especially after the COVID-19 pandemic, which brought up the need for cost-effective and efficient diagnosing kits for the vast majority. RNA-based techniques also have close to no risk of genotoxicity and can efficiently target undruggable regions of the cell. RNA treatments have effectively shown the future of the medical industry in the past couple of decades, and they will only be seen to improve. This review paper provides an overview on the different techniques that use RNA-based approaches in the field of diagnostics and therapeutics.
Transfer RNA (tRNA) is one of the most abundant RNA types in cells, acting as an adaptor to bridge the genetic information in mRNAs with the amino acid sequence in proteins. Both tRNAs and small fragments processed from...Transfer RNA (tRNA) is one of the most abundant RNA types in cells, acting as an adaptor to bridge the genetic information in mRNAs with the amino acid sequence in proteins. Both tRNAs and small fragments processed from them play many nonconventional roles in addition to translation. tRNA molecules undergo various types of chemical modifications to ensure the accuracy and efficiency of translation and regulate their diverse functions beyond translation. In this review, we discuss the biogenesis and molecular mechanisms of tRNA modifications, including major tRNA modifications, writer enzymes, and their dynamic regulation. We also summarize the state-of-the-art technologies for measuring tRNA modification, with a particular focus on 2'-O-methylation (Nm), and discuss their limitations and remaining challenges. Finally, we highlight recent discoveries linking dysregulation of tRNA modifications with genetic diseases.
Mesenchymal Stromal/Stem Cells (MSCs) are among the most frequently studied cell types in clinical trials, and their small extracellular vesicles (sEVs) are now being extensively investigated for therapeutic applications...Mesenchymal Stromal/Stem Cells (MSCs) are among the most frequently studied cell types in clinical trials, and their small extracellular vesicles (sEVs) are now being extensively investigated for therapeutic applications. The RNA cargo of MSC-sEVs, particularly miRNAs and mRNAs, is widely believed to be a key therapeutic component of these vesicles. In this review, we critically examine using first principles and peer-reviewed literature, whether MSC- extracellular vesicles (MSC-EVs) can deliver sufficient quantity of functional miRNA or mRNA to target compartments within recipient cells to elicit a pharmacological response. Several RNA sequencing studies reveal that miRNAs are underrepresented in the small RNA population of MSC-sEVs compared to the parent MSCs. Additionally, the majority of miRNAs are mature forms that are not associated with Argonaute (AGO) proteins, essential for their function in RNA-induced silencing complexes (RISCs). Compounding this, cellular uptake of EVs is generally inefficient, with less than 1% being internalized, and only a fraction of these reaching the cytosol. This suggests that EVs may not deliver miRNAs in sufficient quantities to meaningfully interact with AGO proteins, either through canonical or non-canonical pathways, or with other proteins like Toll-like receptors (TLRs). Further, MSC-sEV RNAs are generally small, with sizes less than 500 nucleotides indicating that any mRNA present is likely fragmented as the average mammalian mRNA is approximately 2000 nucleotides, a fact confirmed by RNA sequencing data. Together, these findings challenge the notion that RNA, particularly miRNAs and mRNAs, are primary therapeutic attributes of MSC-sEVs.
The crosstalk between the tumour immune microenvironment (TIME) and tumour cells promote immune evasion and resistance to immunotherapy in gastrointestinal (GI) tumours. Post-transcriptional regulation of genes is pivota...The crosstalk between the tumour immune microenvironment (TIME) and tumour cells promote immune evasion and resistance to immunotherapy in gastrointestinal (GI) tumours. Post-transcriptional regulation of genes is pivotal to GI tumours progression, and RNA-binding proteins (RBPs) serve as key regulators via their RNA-binding domains. RBPs may exhibit either anti-tumour or pro-tumour functions by influencing the TIME through the modulation of mRNAs and non-coding RNAs expression, as well as post-transcriptional modifications, primarily N6-methyladenosine (mA). Aberrant regulation of RBPs, such as HuR and YBX1, typically enhances tumour immune escape and impacts prognosis of GI tumour patients. Further, while targeting RBPs offers a promising strategy for improving immunotherapy in GI cancers, the mechanisms by which RBPs regulate the TIME in these tumours remain poorly understood, and the therapeutic application is still in its early stages. This review summarizes current advances in exploring the roles of RBPs in regulating genes expression and their effect on the TIME of GI tumours, then providing theoretical insights for RBP-targeted cancer therapies.
Circular RNAs (circRNAs) are a unique class of covalently closed single-stranded RNA molecules that play diverse roles in normal physiology and pathology. Among the major types of circRNA, exon-intron circRNA (EIciRNA) d...Circular RNAs (circRNAs) are a unique class of covalently closed single-stranded RNA molecules that play diverse roles in normal physiology and pathology. Among the major types of circRNA, exon-intron circRNA (EIciRNA) distinguishes itself by its sequence composition and nuclear localization. Recent RNA-seq technologies and computational methods have facilitated the detection and characterization of EIciRNAs, with features like circRNA intron retention (CIR) and tissue-specificity being characterized. EIciRNAs have been identified to exert their functions via mechanisms such as regulating gene transcription, and the physiological relevance of EIciRNAs has been reported. Within this review, we present a summary of the current understanding of EIciRNAs, delving into their identification and molecular functions. Additionally, we emphasize factors regulating EIciRNA biogenesis and the physiological roles of EIciRNAs based on recent research. We also discuss the future challenges in EIciRNA exploration, underscoring the potential for novel functions and functional mechanisms of EIciRNAs for further investigation.
Liver diseases are a significant global health issue, characterized by elevated levels of disorder and death. The substantial impact of ageing on liver diseases and their prognosis is evident. Multiple processes are invo...Liver diseases are a significant global health issue, characterized by elevated levels of disorder and death. The substantial impact of ageing on liver diseases and their prognosis is evident. Multiple processes are involved in the ageing process, which ultimately leads to functional deterioration of this organ. The process of liver ageing not only renders the liver more susceptible to diseases but also compromises the integrity of other organs due to the liver's critical function in metabolism regulation. A growing body of research suggests that long non-coding RNAs (lncRNAs) play a significant role in the majority of pathophysiological pathways. They regulate gene expression through a variety of interactions with microRNAs (miRNAs), messenger RNAs (mRNAs), DNA, or proteins. LncRNAs exert a major influence on the progression of age-related liver diseases through the regulation of cell proliferation, necrosis, apoptosis, senescence, and metabolic reprogramming. A concise overview of the current understanding of lncRNAs and their potential impact on the development of age-related liver diseases will be provided in this mini-review.
More than 4,000 single nucleotide polymorphisms (SNP) variants have been identified in the human gene, however only a few have been studied in the context of protein function. The tandem zinc finger domain of ZFP36L2, a...More than 4,000 single nucleotide polymorphisms (SNP) variants have been identified in the human gene, however only a few have been studied in the context of protein function. The tandem zinc finger domain of ZFP36L2, an RNA binding protein, is the functional domain that binds to its target mRNAs. This protein/RNA interaction triggers mRNA degradation, controlling gene expression. We identified 32 non-synonymous SNPs (nsSNPs) in the tandem zinc finger domain of ZFP36L2 that could have possible deleterious impacts in humans. Using different bioinformatic strategies, we prioritized five among these 32 nsSNPs, namely rs375096815, rs1183688047, rs1214015428, rs1215671792 and rs920398592 to be validated. When we experimentally tested the functionality of these protein variants using gel shift assays, all five (Y154H, R160W, R184C, G204D, and C206F) resulted in a dramatic reduction in RNA binding compared to the WT protein. To understand the mechanistic effect of these variants on the protein/RNA interaction, we employed DUET, DynaMut and PyMOL to investigate structural changes in the protein. Additionally, we conducted Molecular Docking and Molecular Dynamics Simulations to fine tune the active behaviour of this biomolecular system at an atomic level. Our results propose atomic explanations for the impact of each of these five genetic variants identified.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) pandemic and is continuously spreading globally. The continuous emergence of new SARS-CoV-2 variants keeps p...The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) pandemic and is continuously spreading globally. The continuous emergence of new SARS-CoV-2 variants keeps posing threats, highlighting the need for fast-acting, mutation-resistant broad-spectrum therapeutics. Protein translation is vital for SARS-CoV-2 replication, producing early non-structural proteins for RNA replication and transcription, and late structural proteins for virion assembly. Targeted blocking of viral protein translation is thus a potential approach to developing effective anti-SARS-CoV-2 drugs. SARS-CoV-2, as an obligate parasite, utilizes the host's translation machinery. Translation-blocking strategies that target the SARS-CoV-2 mRNA, especially those that target its conserved elements are generally preferred. In this review, we discuss the current understanding of SARS-CoV-2 translation, highlighting the important conserved motifs and structures involved in its regulation. We also discuss the current strategies for blocking SARS-CoV-2 translation through viral RNA degradation or RNA element dysfunction.
Throughout the tree of life RNA-binding proteins play important roles, but they are poorly characterized in cyanobacteria. Overexpression of the predicted RNA-binding protein Ssr1238 in the cyanobacterium 6803 for 24 h...Throughout the tree of life RNA-binding proteins play important roles, but they are poorly characterized in cyanobacteria. Overexpression of the predicted RNA-binding protein Ssr1238 in the cyanobacterium 6803 for 24 h led to higher levels of RNase P RNA, tRNAs, and stress-related mRNAs. Co-immunoprecipitation of proteins followed by MS analysis and sequencing of UV crosslinked, co-immunoprecipitated RNA samples identified potential interaction partners of Ssr1238. The most enriched transcript was RNase P RNA, and RnpA, the protein component of RNase P, was among the most highly enriched proteins. A second highly enriched transcript is derived from gene , which encodes a central enzyme in cell wall remodelling during cell division. The data also showed a strong connection to the RNA maturation and modification system indicated by co-precipitation of RNA modifying enzymes, riboendonuclease E and enolase. Surprisingly, cyanophycin synthetase and urease were highly enriched as well. In conclusion, Ssr1238 specifically binds to two different transcripts and could be involved in the coordination of RNA maturation, translation, cell division, and aspects of nitrogen metabolism. Our results are consistent with recent findings that the YlxR protein functions as an RNase P modulator (RnpM), extending its proposed role to the phylum cyanobacteria, and suggesting additional functionalities.
Extracellular vesicles (EVs) are membrane-bound particles released by cells that play vital roles in intercellular communication by transporting diverse biologically active molecules, including RNA molecules, including m...Extracellular vesicles (EVs) are membrane-bound particles released by cells that play vital roles in intercellular communication by transporting diverse biologically active molecules, including RNA molecules, including mRNA, miRNA, lncRNA, and other regulatory RNAs. These RNA types are protected within the lipid bilayer of EVs, ensuring their stability and enabling long-distance cellular interactions. Notably, EVs play roles in infection, where pathogens and host cells use EV-mediated RNA transfer to influence immune responses and disease outcomes. For example, bacterial EVs play a crucial role in infection by modulating host immune responses and facilitating pathogen invasion. This review explores the complex interactions between EV-associated RNA and host-pathogen dynamics in bacteria, parasites, and fungi, aiming to uncover molecular mechanisms in infectious diseases and potential therapeutic targets.
As a typical RNA virus, SARS-CoV-2 is subjected to RNA editing in host cells. While some researchers believe that a traditional variant calling pipeline retrieves all true-positive RNA editing events from the transcripto...As a typical RNA virus, SARS-CoV-2 is subjected to RNA editing in host cells. While some researchers believe that a traditional variant calling pipeline retrieves all true-positive RNA editing events from the transcriptome, others argue that conventional methods identify many false-positive sites. Here, I describe several additional and experimental approaches to validate the authenticity of RNA editing in SARS-CoV-2. These approaches include requiring strand-specific sequencing, analysis of hyperedited reads, linkage analysis, orthogonal methods like mass spectrometry, and the use of ADAR-deficient host cells. These findings may improve future analyses on the identification of RNA editing, especially in RNA viruses.
General RNA chaperones are RNA-binding proteins (RBPs) that interact transiently and non-specifically with RNA substrates and assist in their folding into their native state. In bacteria, these chaperones impact both cod...General RNA chaperones are RNA-binding proteins (RBPs) that interact transiently and non-specifically with RNA substrates and assist in their folding into their native state. In bacteria, these chaperones impact both coding and non-coding RNAs and are particularly important for large, structured RNAs which are prone to becoming kinetically trapped in misfolded states. Currently, due to the limited number of well-characterized examples and the lack of a consensus structural or sequence motif, it is difficult to identify general RNA chaperones in bacteria. Here, we adapted a previously published RNA regional accessibility probing assay to screen genome wide for intracellular factors in affecting RNA folding, among which we aimed to uncover novel RNA chaperones. Through this method, we identified eight proteins whose deletion gives changes in regional accessibility within the exogenously expressed group I intron ribozyme. Furthermore, we purified and measured properties of two of these proteins, YagL and PepA, which were especially attractive as general chaperone candidates. We showed that both proteins bind RNA and that YagL accelerates native refolding of the ribozyme from a long-lived misfolded state. Further dissection of YagL showed that a putative helix-turn-helix (HTH) domain is responsible for most of its RNA-binding activity, but only the full protein shows chaperone activity. Altogether, this work expands the current repertoire of known general RNA chaperones in bacteria.
SRP9/SRP14 is a protein heterodimer that plays a critical role in the signal recognition particle through its interaction with the scaffolding signal recognition particle RNA (7SL). SRP9/SRP14 binding to 7SL is mediated...SRP9/SRP14 is a protein heterodimer that plays a critical role in the signal recognition particle through its interaction with the scaffolding signal recognition particle RNA (7SL). SRP9/SRP14 binding to 7SL is mediated through a conserved structural motif that is shared with the primate-specific Alu RNA. Alu RNA are transcription products of Alu elements, a retroelement that comprises ~10% of the human genome. Alu RNA are involved in myriad biological processes and are dysregulated in several human disease states. This review focuses on the roles SRP9/SRP14 has in regulating Alu RNA diversification, maturation, and function. The diverse mechanisms through which SRP9/SRP14 regulates Alu RNA exemplify the breadth of protein-mediated regulation of non-coding RNA.
Bulk increases in nucleobase oxidation, most commonly manifesting as the guanine (G) nucleobase modification 8-oxo-7,8-dihydroguanine (8-oxoG), have been linked to several disease pathologies. Elucidating the effects of...Bulk increases in nucleobase oxidation, most commonly manifesting as the guanine (G) nucleobase modification 8-oxo-7,8-dihydroguanine (8-oxoG), have been linked to several disease pathologies. Elucidating the effects of RNA oxidation on cellular homoeostasis is limited by a lack of effective tools for detecting specific regions modified with 8-oxoG. Building on a previously published method for studying 8-oxoG in DNA, we developed ChLoRox-Seq, which works by covalently functionalizing 8-oxoG sites in RNA with biotin. Importantly, this method enables antibody-free enrichment of 8-oxoG-containing RNA fragments for Next Generation Sequencing-based detection of modified regions transcriptome-wide. We demonstrate the high specificity of ChLoRox-Seq for functionalizing 8-oxoG over unmodified nucleobases in RNA and benchmark this specificity to a commonly used antibody-based approach. Key advantages of ChLoRox-Seq include: (1) heightened resolution of RNA oxidation regions (e.g. exon-level) and (2) lower experimental costs. By applying ChLoRox-Seq to mRNA extracted from human lung epithelial cells (BEAS-2B) after exposure to environmentally relevant stress, we observe that 8-oxoG modifications tend to cluster in regions that are G-rich and within mRNA transcripts possessing longer 5' UTR and CDS regions. These findings provide new insight into the complex mechanisms that bias the accumulation of RNA oxidation across the transcriptome. Notably, our analysis suggests the possibility that most mRNA oxidation events are probabilistically driven and that mRNAs that possess more favourable intrinsic properties are prone to incur oxidation events at elevated rates. ChLoRox-Seq can be readily applied in future studies to identify regions of elevated RNA oxidation in any cellular model of interest.
Circular RNAs (circRNAs) exhibit a wide range of activities that allow them to participate in numerous cellular processes and make them relevant in a variety of diseases. In this regard, a key process in which circRNAs a...Circular RNAs (circRNAs) exhibit a wide range of activities that allow them to participate in numerous cellular processes and make them relevant in a variety of diseases. In this regard, a key process in which circRNAs are involved, and which is the focus of this article, is DNA damage repair (DDR). This study aims to illustrate how circRNAs influence different DNA repair pathways, with particular emphasis on the underlying mechanisms. In addition, the potential medical applications of this knowledge are discussed, particularly in the diagnosis, prognosis and treatment of diseases. In this sense, circRNAs were found to play a crucial role in DNA repair processes by regulating the expression and activity of proteins involved in various DNA repair pathways. They influence the expression of DNA repair proteins by interacting with their mRNAs, sponging miRNAs that target these mRNAs, regulating transcription factors that bind to their promoters, modulating upstream signalling pathways, and affecting mRNA translation. Furthermore, circRNAs regulate the activity of DNA repair proteins by interacting directly with them, sequestering them in specific cellular compartments and controlling activation signalling or upstream DDR signalling.
Next-generation sequencing has revolutionized cancer genomics by enabling high-throughput mutation screening yet detecting fusion genes reliably remains challenging. Long-read sequencing offers potential for accurate fus...Next-generation sequencing has revolutionized cancer genomics by enabling high-throughput mutation screening yet detecting fusion genes reliably remains challenging. Long-read sequencing offers potential for accurate fusion transcript identification, though challenges persist. In this study, we present an optimized workflow using nanopore sequencing technology to precisely identify fusion transcripts. Our approach encompasses a tailored library preparation protocol, data processing, and fusion gene analysis pipeline. We evaluated the performance using Universal Human Reference RNA and human adenocarcinoma cell lines. Our optimized nanopore sequencing workflow generated high-quality full-length transcriptome data characterized by an extended length distribution and comprehensive transcript coverage. Validation experiments confirmed novel fusion events with potential clinical relevance. Our protocol aims to mitigate biases and enhance accuracy, facilitating increased adoption in clinical diagnostics. Continued advancements in long-read sequencing promise deeper insights into fusion gene biology and improved cancer diagnostics.
In an RNA world, the emergence of template-specific self-replication and catalysis necessitated the presence of motifs facilitating reliable recognition between RNA molecules. What did these motifs entail, and how did th...In an RNA world, the emergence of template-specific self-replication and catalysis necessitated the presence of motifs facilitating reliable recognition between RNA molecules. What did these motifs entail, and how did they evolve into the proteinaceous RNA recognition entities observed today? Direct observation of these primordial entities is hindered by rapid degradation over geological time scales. To overcome this challenge, researchers employ diverse approaches, including scrutiny of conserved sequences and structural motifs across extant organisms and employing directed evolution experiments to generate RNA molecules with specific catalytic abilities. In this review, we delve into the theme of ribonucleotide recognition across key periods of early Earth's evolution. We explore scenarios of RNA interacting with small molecules and examine hypotheses regarding the role of minerals and metal ions in enabling structured ribonucleotide recognition and catalysis. Additionally, we highlight instances of RNA-protein mimicry in interactions with other RNA molecules. We propose a hypothesis where RNA initially recognizes small molecules and metal ions/minerals, with subsequent mimicry by proteins leading to the emergence of proteinaceous RNA binding domains.