Small non-coding RNAs are key regulators of gene expression across eukaryotes. Piwi-interacting small RNAs (piRNAs) are a specific type of small non-coding RNAs, conserved across animals, which are best known as regulators of genome stability through their ability to target transposable elements for silencing. Despite the near ubiquitous presence of piRNAs in animal lineages, there are some examples where the piRNA pathway has been lost completely, most dramatically in nematodes where loss has occurred in at least four independent lineages. In this perspective I will provide an evaluation of the presence of piRNAs across animals, explaining how it is known that piRNAs are missing from certain organisms. I will then consider possible explanations for why the piRNA pathway might have been lost and evaluate the evidence in favor of each possible mechanism. While it is still impossible to provide definitive answers, these theories will prompt further investigations into why such a highly conserved pathway can nevertheless become dispensable in certain lineages. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.
RNA-binding proteins (RBPs) are powerful and versatile regulators in living creatures, playing fundamental roles in organismal development, metabolism, and various diseases by the regulation of gene expression at multiple levels. The requirements of deep research on RBP function have promoted the rapid development of RBP-RNA interplay detection methods. Recently, the detection method of fusing RNA modification enzymes (RME) with RBP of interest has become a hot topic. Here, we reviewed RNA modification enzymes in adenosine deaminases that act on RNA (ADAR), terminal nucleotidyl transferase (TENT), and activation-induced cytosine deaminase/ApoB mRNA editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family, regarding the biological function, biochemical activity, and substrate specificity originated from enzyme selves, their domains and partner proteins. In addition, we discussed the RME activity screening system, and the RME mutations with engineered enzyme activity. Furthermore, we provided a systematic overview of the basic principles, advantages, disadvantages, and applications of the RME-based and cross-linking and immunopurification (CLIP)-based RBP target profiling strategies, including targets of RNA-binding proteins identified by editing (TRIBE), RNA tagging, surveying targets by APOBEC-mediated profiling (STAMP), CLIP-seq, and their derivative technology. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > RNA Editing and Modification.
Selection of the correct start codon is critical for high-fidelity protein synthesis. In eukaryotes, this is typically governed by a multitude of initiation factors (eIFs), including eIF2·GTP that directly delivers the initiator tRNA (Met-tRNAi Met ) to the P site of the ribosome. However, numerous reports, some dating back to the early 1970s, have described other initiation factors having high affinity for the initiator tRNA and the ability of delivering it to the ribosome, which has provided a foundation for further work demonstrating non-canonical initiation mechanisms using alternative initiation factors. Here we provide a critical analysis of current understanding of eIF2A, eIF2D, and the MCT-1·DENR dimer, the evidence surrounding their ability to initiate translation, their implications in human disease, and lay out important key questions for the field. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Mechanisms Translation > Regulation.
Most eukaryotic mRNAs and different non-coding RNAs undergo a form of 3' end processing known as polyadenylation. Polyadenylation machinery is present in almost all organisms except few species. In bacteria, the machinery has evolved from PNPase, which adds heteropolymeric tails, to a poly(A)-specific polymerase. Differently, a complex machinery for accurate polyadenylation and several non-canonical poly(A) polymerases are developed in eukaryotes. The role of poly(A) tail has also evolved from serving as a degradative signal to a stabilizing modification that also regulates translation. In this review, we discuss poly(A) tail emergence in prokaryotes and its development into a stable, yet dynamic feature at the 3' end of mRNAs in eukaryotes. We also describe how appearance of novel poly(A) polymerases gives cells flexibility to shape poly(A) tail. We explain how poly(A) tail dynamics help regulate cognate RNA metabolism in a context-dependent manner, such as during oocyte maturation. Finally, we describe specific mRNAs in metazoans that bear stem-loops instead of poly(A) tails. We conclude with how recent discoveries about poly(A) tail can be applied to mRNA technology. This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Processing > 3' End Processing RNA Turnover and Surveillance > Regulation of RNA Stability.
With glucose being at the center of energy consumption and production, maintaining homeostasis of this simple sugar is of pivotal importance. Loss of glucose homeostasis results in altered blood glucose levels, that are frequently observed in type 2 diabetes (T2D) and obesity. T2D and obesity share pathophysiological mechanisms and genetic backgrounds. These conditions collectively impact over 500 million individuals worldwide, necessitating a deeper mechanistic understanding for effective therapeutic strategies. Recent studies have highlighted the involvement of abnormal alternative splicing (AS) and changes in splicing factors (SFs) in the development and progression of diabetic conditions, presenting AS and SFs as promising targets for therapy. This review focuses on the deregulation of AS (INSR, TCF7L2, and mTOR) and SF (Sam68) deregulation in diabetic conditions. In addition, we discuss the importance of mTOR signaling in diabetic conditions and its regulation by AS and SFs. Furthermore, we discuss current strategies aimed at targeting AS and SFs. Finally, we discuss research challenges, and unresolved questions in the field, and offer recommendations to enhance our comprehension of the significance of AS and SFs in the context of diabetes and obesity. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease
Nonsense-mediated mRNA decay (NMD) is a quality-control process that selectively degrades mRNAs having premature termination codon, upstream open reading frame, or unusually long 3'UTR. NMD detects such mRNAs and rapidly degrades them during initial rounds of translation in the eukaryotic cells. Since NMD is a translation-dependent cytoplasmic mRNA surveillance process, the noncoding RNAs were initially believed to be NMD-resistant. The sequence feature-based analysis has revealed that many putative long noncoding RNAs (lncRNAs) have short open reading frames, most of which have translation potential. Subsequent transcriptome-based molecular studies showed an association of a large set of such putative lncRNAs with translating ribosomes, and some of them produce stable and functionally active micropeptides. The translationally active lncRNAs typically have relatively longer and unprotected 3'UTR, which can induce their NMD-dependent degradation. This review defines the mechanism and regulation of NMD-dependent degradation of lncRNAs and its impact on biological processes related to the functions of lncRNAs or their encoded micropeptides. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability RNA in Disease and Development > RNA in Disease.
The precursor transfer RNAs (pre-tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5'-processing, 3'-processing, modifications at specific sites, if any, and 3'-CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre-tRNA sequences at the 5' end by the removal of phosphodiester linkages between nucleotides at position -1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless-position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion-mediated conserved catalytic reaction. While the canonical RNA-based ribonucleoprotein RNase P has been well-known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA-free protein-only RNase P in eukaryotes and RNA-free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA-based and RNA-free RNase P holoenzymes towards harnessing critical RNA-protein and protein-protein interactions in achieving conserved pre-tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications. This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.
The brain is a complex computing system composed of a multitude of interacting neurons. The computational outputs of this system determine the behavior and perception of every individual. Each brain cell expresses thousands of genes that dictate the cell's function and physiological properties. Therefore, deciphering the molecular expression of each cell is of great significance for understanding its characteristics and role in brain function. Additionally, the positional information of each cell can provide crucial insights into their involvement in local brain circuits. In this review, we briefly overview the principles of single-cell RNA sequencing and spatial transcriptomics, the potential issues and challenges in their data processing, and their applications in brain research. We further outline several promising directions in neuroscience that could be integrated with single-cell RNA sequencing, including neurodevelopment, the identification of novel brain microstructures, cognition and behavior, neuronal cell positioning, molecules and cells related to advanced brain functions, sleep-wake cycles/circadian rhythms, and computational modeling of brain function. We believe that the deep integration of these directions with single-cell and spatial RNA sequencing can contribute significantly to understanding the roles of individual cells or cell types in these specific functions, thereby making important contributions to addressing critical questions in those fields. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA in Disease and Development > RNA in Development RNA in Disease and Development > RNA in Disease.
Pericentromeric heterochromatin is mainly composed of satellite DNA sequences. Although being historically associated with transcriptional repression, some pericentromeric satellite DNA sequences are transcribed. The transcription events of pericentromeric satellite sequences occur in highly flexible biological contexts. Hence, the apparent randomness of pericentromeric satellite transcription incites the discussion about the attribution of biological functions. However, pericentromeric satellite RNAs have clear roles in the organization of nuclear structure. Silencing pericentromeric heterochromatin depends on pericentromeric satellite RNAs, that, in a feedback mechanism, contribute to the repression of pericentromeric heterochromatin. Moreover, pericentromeric satellite RNAs can also act as scaffolding molecules in condensate subnuclear structures (e.g., nuclear stress bodies). Since the formation/dissociation of nuclear condensates provides cell adaptability, pericentromeric satellite RNAs can be an epigenetic platform for regulating (sub)nuclear structure. We review current knowledge about pericentromeric satellite RNAs that, irrespective of the meaning of biological function, should be functionally addressed in regular and disease settings. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA in Disease and Development > RNA in Disease.
Disruptions in spatiotemporal gene expression can result in atypical brain function. Specifically, autism spectrum disorder (ASD) is characterized by abnormalities in pre-mRNA splicing. Abnormal splicing patterns have been identified in the brains of individuals with ASD, and mutations in splicing factors have been found to contribute to neurodevelopmental delays associated with ASD. Here we review studies that shed light on the importance of splicing observed in ASD and that explored the intricate relationship between splicing factors and ASD, revealing how disruptions in pre-mRNA splicing may underlie ASD pathogenesis. We provide an overview of the research regarding all splicing factors associated with ASD and place a special emphasis on five specific splicing factors-HNRNPH2, NOVA2, WBP4, SRRM2, and RBFOX1-known to impact the splicing of ASD-related genes. In the discussion of the molecular mechanisms influenced by these splicing factors, we lay the groundwork for a deeper understanding of ASD's complex etiology. Finally, we discuss the potential benefit of unraveling the connection between splicing and ASD for the development of more precise diagnostic tools and targeted therapeutic interventions. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Evolution and Genomics > Computational Analyses of RNA RNA-Based Catalysis > RNA Catalysis in Splicing and Translation.
Post-transcriptional mRNA modifications play diverse roles in gene expression and RNA function. In many cases, RNA modifications function by altering how cellular machinery such as RNA binding proteins (RBPs) interact with RNA substrates. For instance, N6-methyladenosine (m6A) is recognized by the well-characterized YTH domain-containing family of "reader" proteins. For other mRNA modifications, similar global readers of modification status have not been clearly defined. Rather, most interactions between RBPs and RNA modifications have a more complicated dependence on sequence context and binding modality. The current handful of studies that demonstrate modifications impacting protein binding likely represent only a fraction of the full landscape. In this review, we dissect the known instances of RNA modifications altering RBP binding, specifically m6A, N1-methyladenosine (m1A), 5-methylcytosine (m5C), pseudouridine (Ψ), and internal N7-methylguanosine. We then review the biochemical properties of these and other identified mRNA modifications including dihydrouridine (D), N4-acetylcytosine (ac4C), and 2'-O-Methylation (Nme). We focus on how these properties would be likely to impact RNA:RBP interactions, including by changes to hydrogen bond potential, base-stacking efficiency, and RNA conformational preferences. The effects of RNA modifications on secondary structure have been well-studied, and we briefly discuss how structural effects imparted by modifications can lead to protein binding changes. Finally, we discuss strategies for uncovering as-yet-to-be identified modification-sensitive RBP:RNA Interactions. Coordinating future efforts to intersect the epitranscriptome and the RNA-protein interactome will illuminate the rules governing RNA modification recognition and the mechanisms responsible for the biological consequences of mRNA modification. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > RNA Editing and Modification.
Circular RNAs (circRNAs), characterized by their closed-loop structure, have emerged as significant transcriptomic regulators, with roles spanning from microRNA sponging to modulation of gene expression and potential peptide coding. The discovery and functional analysis of circRNAs have been propelled by advancements in both experimental and bioinformatics tools, yet the field grapples with challenges related to their detection, isoform diversity, and accurate quantification. This review navigates through the evolution of circRNA research methodologies, from early detection techniques to current state-of-the-art approaches that offer comprehensive insights into circRNA biology. We examine the limitations of existing methods, particularly the difficulty in differentiating circRNA isoforms and distinguishing circRNAs from their linear counterparts. A critical evaluation of various bioinformatics tools and novel experimental strategies is presented, emphasizing the need for integrated approaches to enhance our understanding and interpretation of circRNA functions. Our insights underscore the dynamic and rapidly advancing nature of circRNA research, highlighting the ongoing development of analytical frameworks designed to address the complexity of circRNAs and facilitate the assessment of their clinical utility. As such, this comprehensive overview aims to catalyze further advancements in circRNA study, fostering a deeper understanding of their roles in cellular processes and potential implications in disease. This article is categorized under: RNA Methods > RNA Nanotechnology RNA Methods > RNA Analyses in Cells RNA Methods > RNA Analyses In Vitro and In Silico.
Small RNAs (sRNAs) with sizes ranging from 15 to 50 nucleotides (nt) are critical regulators of gene expression control. Prior studies have shown that sRNAs are involved in a broad range of biological processes, such as organ development, tumorigenesis, and epigenomic regulation; however, emerging evidence unveils a hidden layer of diversity and complexity of endogenously encoded sRNAs profile in eukaryotic organisms, including novel types of sRNAs and the previously unknown post-transcriptional RNA modifications. This underscores the importance for accurate, unbiased detection of sRNAs in various cellular contexts. A multitude of high-throughput methods based on next-generation sequencing (NGS) are developed to decipher the sRNA expression and their modifications. Nonetheless, distinct from mRNA sequencing, the data from sRNA sequencing suffer frequent inconsistencies and high variations emanating from the adapter contaminations and RNA modifications, which overall skew the sRNA libraries. Here, we summarize the sRNA-sequencing approaches, and discuss the considerations and challenges for the strategies and methods of sRNA library construction. The pros and cons of sRNA sequencing have significant implications for implementing RNA fragment footprinting approaches, including CLIP-seq and Ribo-seq. We envision that this review can inspire novel improvements in small RNA sequencing and RNA fragment footprinting in future. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.
Spatially resolved transcriptomics has been dramatically transforming biological and medical research in various fields. It enables transcriptome profiling at single-cell, multi-cellular, or sub-cellular resolution, while retaining the information of geometric localizations of cells in complex tissues. The coupling of cell spatial information and its molecular characteristics generates a novel multi-modal high-throughput data source, which poses new challenges for the development of analytical methods for data-mining. Spatial transcriptomic data are often highly complex, noisy, and biased, presenting a series of difficulties, many unresolved, for data analysis and generation of biological insights. In addition, to keep pace with the ever-evolving spatial transcriptomic experimental technologies, the existing analytical theories and tools need to be updated and reformed accordingly. In this review, we provide an overview and discussion of the current computational approaches for mining of spatial transcriptomics data. Future directions and perspectives of methodology design are proposed to stimulate further discussions and advances in new analytical models and algorithms. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Evolution and Genomics > Computational Analyses of RNA RNA Export and Localization > RNA Localization.
RNAs are meticulously controlled by proteins. Through direct and indirect associations, every facet in the brief life of an mRNA is subject to regulation. RNA-binding proteins (RBPs) permeate biology. Here, we focus on their roles in pain. Chronic pain is among the largest challenges facing medicine and requires new strategies. Mounting pharmacologic and genetic evidence obtained in pre-clinical models suggests fundamental roles for a broad array of RBPs. We describe their diverse roles that span RNA modification, splicing, stability, translation, and decay. Finally, we highlight opportunities to expand our understanding of regulatory interactions that contribute to pain signaling. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Regulation RNA in Disease and Development > RNA in Disease.
Cell death plays a crucial role in various physiological and pathological processes. Until recently, programmed cell death was mainly attributed to caspase-dependent apoptosis. However, emerging evidence suggests that caspase-independent cell death (CICD) mechanisms also contribute significantly to cellular demise. We and others have reported and functionally characterized numerous long noncoding RNAs (lncRNAs) that modulate caspase-dependent apoptotic pathways potentially in a pathway-dependent manner. However, the interplay between lncRNAs and CICD pathways has not been comprehensively documented. One major reason for this is that most CICD pathways have been recently discovered with some being partially characterized at the molecular level. In this review, we discuss the emerging evidence that implicates specific lncRNAs in the regulation and execution of CICD. We summarize the diverse mechanisms through which lncRNAs modulate different forms of CICD, including ferroptosis, necroptosis, cuproptosis, and others. Furthermore, we highlight the intricate regulatory networks involving lncRNAs, protein-coding genes, and signaling pathways that orchestrate CICD in health and disease. Understanding the molecular mechanisms and functional implications of lncRNAs in CICD may unravel novel therapeutic targets and diagnostic tools for various diseases, paving the way for innovative strategies in disease management and personalized medicine. This article is categorized under: RNA in Disease and Development > RNA in Disease.
Protein-only RNase P (PRORP) is an essential enzyme responsible for the 5' maturation of precursor tRNAs (pre-tRNAs). PRORPs are classified into three categories with unique molecular architectures, although all three classes of PRORPs share a mechanism and have similar active sites. Single subunit PRORPs, like those found in plants, have multiple isoforms with different localizations, substrate specificities, and temperature sensitivities. Most recently, Arabidopsis thaliana PRORP2 was shown to interact with TRM1A and B, highlighting a new potential role between these enzymes. Work with At PRORPs led to the development of a ribonuclease that is being used to protect against plant viruses. The mitochondrial RNase P complex, found in metazoans, consists of PRORP, TRMT10C, and SDR5C1, and has also been shown to have substrate specificity, although the cause is unknown. Mutations in mitochondrial tRNA and mitochondrial RNase P have been linked to human disease, highlighting the need to continue understanding this complex. The last class of PRORPs, homologs of Aquifex RNase P (HARPs), is found in thermophilic archaea and bacteria. This most recently discovered type of PRORP forms a large homo-oligomer complex. Although numerous structures of HARPs have been published, it is still unclear how HARPs bind pre-tRNAs and in what ratio. There is also little investigation into the substrate specificity and ideal conditions for HARPs. Moving forward, further work is required to fully characterize each of the three classes of PRORP, the pre-tRNA binding recognition mechanism, the rules of substrate specificity, and how these three distinct classes of PRORP evolved. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.
Leukodystrophies are a class of rare heterogeneous disorders which affect the white matter of the brain, ultimately leading to a disruption in brain development and a damaging effect on cognitive, motor and social-communicative development. These disorders present a great clinical heterogeneity, along with a phenotypic overlap and this could be partially due to contributions from environmental stimuli. It is in this context that there is a great need to investigate what other factors may contribute to both disease insurgence and phenotypical heterogeneity, and novel evidence are raising the attention toward the study of epigenetics and transcription mechanisms that can influence the disease phenotype beyond genetics. Modulation in the epigenetics machinery including histone modifications, DNA methylation and non-coding RNAs dysregulation, could be crucial players in the development of these disorders, and moreover an aberrant RNA maturation process has been linked to leukodystrophies. Here, we provide an overview of these mechanisms hoping to supply a closer step toward the analysis of leukodystrophies not only as genetically determined but also with an added level of complexity where epigenetic dysregulation is of key relevance. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNA RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.
Noncoding RNAs (ncRNAs), previously referred to as "dark matter" and now recognized as "bright star," fulfill a variety of biological functions and are also emerging as potential biomarkers and therapies in oncology-focused clinical trials. Systems medicine has become a holist paradigm to study the human body, which organically integrates data from basic research and clinical practice to improve our understanding and treatment of complex diseases. It is an interdisciplinary approach that brings systems biology and bioinformatics closer to clinical medicine. The development of high-throughput techniques and the increasing adoption of computational methods across healthcare domains have accelerated and aided the tangible clinical applications of ncRNAs. Various resources and methodologies for ncRNAs enable a comprehensive analysis of heterogeneous and interlinked data, thereby providing new insights into systems medicine. In this review, we summarize the resources and models for ncRNA medical research, aiming to provide a technical manual for personalized and precision medicine at the system level. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico
Spatial transcriptomics (ST) is featured by high-throughput gene expression profiling within their native cell and tissue context, offering a means to investigate gene regulatory networks in tissue microenvironment. In situ sequencing (ISS) is an imaging-based ST technology that simultaneously detects hundreds to thousands of genes at subcellular resolution. As a highly reproducible and robust technique, ISS has been widely adapted and undergone a series of technical iterations. As the interest in ISS-based spatial transcriptomic analysis grows, scalable and integrated data analysis workflows are needed to facilitate the applications of ISS in different research fields. This review presents the state-of-the-art bioinformatic toolkits for ISS data analysis, which covers the upstream and downstream analysis workflows, including image analysis, cell segmentation, clustering, functional enrichment, detection of spatially variable genes and cell clusters, spatial cell-cell interactions, and trajectory inference. To assist the community in choosing the right tools for their research, the application of each tool and its compatibility with ISS data are reviewed in detailed. Finally, future perspectives and challenges concerning how to integrate heterogeneous tools into a user-friendly analysis pipeline are discussed. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico.
