Spatially exploring RNA biology in archival formalin-fixed paraffin-embedded tissues.

The capability to spatially explore RNA biology in formalin-fixed paraffin-embedded (FFPE) tissues holds transformative potential for histopathology research. Here, we present pathology-compatible deterministic barcoding in tissue (Patho-DBiT) by combining in situ polyadenylation and computational innovation for spatial whole transcriptome sequencing, tailored to probe the diverse RNA species in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for 5 years. Furthermore, genome-wide single-nucleotide RNA variants can be captured to distinguish malignant subclones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis. Single-cell level Patho-DBiT dissects the spatiotemporal cellular dynamics driving tumor clonal architecture and progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to aid in clinical pathology evaluation.

RNA polymerase II dynamics and mRNA stability feedback scale mRNA amounts with cell size.

A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size.

SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.

Small RNAs are modified with N-glycans and displayed on the surface of living cells.

Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These "glycoRNAs" were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.

Transient Protein-RNA Interactions Guide Nascent Ribosomal RNA Folding.

Ribosome assembly is an efficient but complex and heterogeneous process during which ribosomal proteins assemble on the nascent rRNA during transcription. Understanding how the interplay between nascent RNA folding and protein binding determines the fate of transcripts remains a major challenge. Here, using single-molecule fluorescence microscopy, we follow assembly of the entire 3' domain of the bacterial small ribosomal subunit in real time. We find that co-transcriptional rRNA folding is complicated by the formation of long-range RNA interactions and that r-proteins self-chaperone the rRNA folding process prior to stable incorporation into a ribonucleoprotein (RNP) complex. Assembly is initiated by transient rather than stable protein binding, and the protein-RNA binding dynamics gradually decrease during assembly. This work questions the paradigm of strictly sequential and cooperative ribosome assembly and suggests that transient binding of RNA binding proteins to cellular RNAs could provide a general mechanism to shape nascent RNA folding during RNP assembly.

Spatial omics advances for in situ RNA biology.

Spatial regulation of RNA plays a critical role in gene expression regulation and cellular function. Understanding spatially resolved RNA dynamics and translation is vital for bringing new insights into biological processes such as embryonic development, neurobiology, and disease pathology. This review explores past studies in subcellular, cellular, and tissue-level spatial RNA biology driven by diverse methodologies, ranging from cell fractionation, in situ and proximity labeling, imaging, spatially indexed next-generation sequencing (NGS) approaches, and spatially informed computational modeling. Particularly, recent advances have been made for near-genome-scale profiling of RNA and multimodal biomolecules at high spatial resolution. These methods enabled new discoveries into RNA's spatiotemporal kinetics, RNA processing, translation status, and RNA-protein interactions in cells and tissues. The evolving landscape of experimental and computational strategies reveals the complexity and heterogeneity of spatial RNA biology with subcellular resolution, heralding new avenues for RNA biology research.

The expanding universe of PARP1-mediated molecular and therapeutic mechanisms.

ADP-ribosylation (ADPRylation) is a post-translational modification of proteins catalyzed by ADP-ribosyl transferase (ART) enzymes, including nuclear PARPs (e.g., PARP1 and PARP2). Historically, studies of ADPRylation and PARPs have focused on DNA damage responses in cancers, but more recent studies elucidate diverse roles in a broader array of biological processes. Here, we summarize the expanding array of molecular mechanisms underlying the biological functions of nuclear PARPs with a focus on PARP1, the founding member of the family. This includes roles in DNA repair, chromatin regulation, gene expression, ribosome biogenesis, and RNA biology. We also present new concepts in PARP1-dependent regulation, including PAR-dependent post-translational modifications, "ADPR spray," and PAR-mediated biomolecular condensate formation. Moreover, we review advances in the therapeutic mechanisms of PARP inhibitors (PARPi) as well as the progress on the mechanisms of PARPi resistance. Collectively, the recent progress in the field has yielded new insights into the expanding universe of PARP1-mediated molecular and therapeutic mechanisms in a variety of biological processes.

NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation.

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform (Eif4a2PTC ). NMD deficiency leads to translation of the truncated eIF4A2PTC protein. eIF4A2PTC elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs (Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions.

Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology.

Bacterial toxin-antitoxin (TA) modules are abundant genetic elements that encode a toxin protein capable of inhibiting cell growth and an antitoxin that counteracts the toxin. The majority of toxins are enzymes that interfere with translation or DNA replication, but a wide variety of molecular activities and cellular targets have been described. Antitoxins are proteins or RNAs that often control their cognate toxins through direct interactions and, in conjunction with other signaling elements, through transcriptional and translational regulation of TA module expression. Three major biological functions of TA modules have been discovered, post-segregational killing ("plasmid addiction"), abortive infection (bacteriophage immunity through altruistic suicide), and persister formation (antibiotic tolerance through dormancy). In this review, we summarize the current state of the field and highlight how multiple levels of regulation shape the conditions of toxin activation to achieve the different biological functions of TA modules.

The discovery of a catalytic RNA within RNase P and its legacy.

Sidney Altman's discovery of the processing of one RNA by another RNA that acts like an enzyme was revolutionary in biology and the basis for his sharing the 1989 Nobel Prize in Chemistry with Thomas Cech. These breakthrough findings support the key role of RNA in molecular evolution, where replicating RNAs (and similar chemical derivatives) either with or without peptides functioned in protocells during the early stages of life on Earth, an era referred to as the RNA world. Here, we cover the historical background highlighting the work of Altman and his colleagues and the subsequent efforts of other researchers to understand the biological function of RNase P and its catalytic RNA subunit and to employ it as a tool to downregulate gene expression. We primarily discuss bacterial RNase P-related studies but acknowledge that many groups have significantly contributed to our understanding of archaeal and eukaryotic RNase P, as reviewed in this special issue and elsewhere.

A systematic benchmark of machine learning methods for protein-RNA interaction prediction.

RNA-binding proteins (RBPs) are central actors of RNA post-transcriptional regulation. Experiments to profile-binding sites of RBPs in vivo are limited to transcripts expressed in the experimental cell type, creating the need for computational methods to infer missing binding information. While numerous machine-learning based methods have been developed for this task, their use of heterogeneous training and evaluation datasets across different sets of RBPs and CLIP-seq protocols makes a direct comparison of their performance difficult. Here, we compile a set of 37 machine learning (primarily deep learning) methods for in vivo RBP-RNA interaction prediction and systematically benchmark a subset of 11 representative methods across hundreds of CLIP-seq datasets and RBPs. Using homogenized sample pre-processing and two negative-class sample generation strategies, we evaluate methods in terms of predictive performance and assess the impact of neural network architectures and input modalities on model performance. We believe that this study will not only enable researchers to choose the optimal prediction method for their tasks at hand, but also aid method developers in developing novel, high-performing methods by introducing a standardized framework for their evaluation.

PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes.

The discovery of poly(ADP-ribose) >50 years ago opened a new field, leading the way for the discovery of the poly(ADP-ribose) polymerase (PARP) family of enzymes and the ADP-ribosylation reactions that they catalyze. Although the field was initially focused primarily on the biochemistry and molecular biology of PARP-1 in DNA damage detection and repair, the mechanistic and functional understanding of the role of PARPs in different biological processes has grown considerably of late. This has been accompanied by a shift of focus from enzymology to a search for substrates as well as the first attempts to determine the functional consequences of site-specific ADP-ribosylation on those substrates. Supporting these advances is a host of methodological approaches from chemical biology, proteomics, genomics, cell biology, and genetics that have propelled new discoveries in the field. New findings on the diverse roles of PARPs in chromatin regulation, transcription, RNA biology, and DNA repair have been complemented by recent advances that link ADP-ribosylation to stress responses, metabolism, viral infections, and cancer. These studies have begun to reveal the promising ways in which PARPs may be targeted therapeutically for the treatment of disease. In this review, we discuss these topics and relate them to the future directions of the field.

RNA regulatory mechanisms that control antiviral innate immunity.

From the initial sensing of viral nucleotides by pattern recognition receptors, through the induction of type I and III interferons (IFN), upregulation of antiviral effector proteins, and resolution of the inflammatory response, each step of innate immune signaling is under tight control. Though innate immunity is often associated with broad regulation at the level of gene transcription, RNA-centric post-transcriptional processes have emerged as critical mechanisms for ensuring a proper antiviral response. Here, we explore the diverse RNA regulatory mechanisms that modulate the innate antiviral immune response, with a focus on RNA sensing by RIG-I-like receptors (RLR), interferon (IFN) and IFN signaling pathways, viral pathogenesis, and host genetic variation that contributes to these processes. We address the post-transcriptional interactions with RNA-binding proteins, non-coding RNAs, transcript elements, and modifications that control mRNA stability, as well as alternative splicing events that modulate the innate immune antiviral response.

Frontiers in plant RNA research in ICAR2023: from lab to innovative agriculture.

The recent growth in global warming, soil contamination, and climate instability have widely disturbed ecosystems, and will have a significant negative impact on the growth of plants that produce grains, fruits and woody biomass. To conquer this difficult situation, we need to understand the molecular bias of plant environmental responses and promote development of new technologies for sustainable maintenance of crop production. Accumulated molecular biological data have highlighted the importance of RNA-based mechanisms for plant stress responses. Here, we report the most advanced plant RNA research presented in the 33rd International Conference on Arabidopsis Research (ICAR2023), held as a hybrid event on June 5-9, 2023 in Chiba, Japan, and focused on "Arabidopsis for Sustainable Development Goals". Six workshops/concurrent sessions in ICAR2023 targeted plant RNA biology, and many RNA-related topics could be found in other sessions. In this meeting report, we focus on the workshops/concurrent sessions targeting RNA biology, to share what is happening now at the forefront of plant RNA research.

A layperson encounter, on the "modified" RNA world.

A chance conversation with a nonscientist about the mRNA-COVID vaccines, conveyed here, reminded the author of our enduring responsibility to accurately portray science to the public.

Roadblocks and fast tracks: How RNA binding proteins affect the viral RNA journey in the cell.

As obligate intracellular parasites with limited coding capacity, RNA viruses rely on host cells to complete their multiplication cycle. Viral RNAs (vRNAs) are central to infection. They carry all the necessary information for a virus to synthesize its proteins, replicate and spread and could also play essential non-coding roles. Regardless of its origin or tropism, vRNA has by definition evolved in the presence of host RNA Binding Proteins (RBPs), which resulted in intricate and complicated interactions with these factors. While on one hand some host RBPs recognize vRNA as non-self and mobilize host antiviral defenses, vRNA must also co-opt other host RBPs to promote viral infection. Focusing on pathogenic RNA viruses, we will review important scenarios of RBP-vRNA interactions during which host RBPs recognize, modify or degrade vRNAs. We will then focus on how vRNA hijacks the largest ribonucleoprotein complex (RNP) in the cell, the ribosome, to selectively promote the synthesis of its proteins. We will finally reflect on how novel technologies are helping in deepening our understanding of vRNA-host RBPs interactions, which can be ultimately leveraged to combat everlasting viral threats.

An 'Arms Race' between the Nonsense-mediated mRNA Decay Pathway and Viral Infections.

The Nonsense-mediated mRNA Decay (NMD) pathway is an RNA quality control pathway conserved among eukaryotic cells. While historically thought to predominantly recognize transcripts with premature termination codons, it is now known that the NMD pathway plays a variety of roles, from homeostatic events to control of viral pathogens. In this review we highlight the reciprocal interactions between the host NMD pathway and viral pathogens, which have shaped both the host antiviral defense and viral pathogenesis.

Retroelement-derived RNA and its role in the brain.

Comprising ~40% of the human genome, retroelements are mobile genetic elements which are transcribed into RNA, then reverse-transcribed into DNA and inserted into a new site in the genome. Retroelements are referred to as "genetic parasites", residing among host genes and relying on host machinery for transcription and evolutionary propagation. The healthy brain has the highest expression of retroelement-derived sequences compared to other somatic tissue, which leads to the question: how does retroelement-derived RNA influence human traits and cellular states? While the functional importance of upregulating retroelement expression in the brain is an active area of research, RNA species derived from retroelements influence both self- and host gene expression by contributing to chromatin remodeling, alternative splicing, somatic mosaicism and translational repression. Here, we review the emerging evidence that the functional importance of RNA derived from retroelements is multifaceted. Retroelements can influence organismal states through the seeding of epigenetic states in chromatin, the production of structured RNA and even catalytically active ribozymes, the generation of cytoplasmic ssDNA and RNA/DNA hybrids, the production of viral-like proteins, and the generation of somatic mutations. Comparative sequencing suggests that retroelements can contribute to intraspecies variation through these mechanisms to alter transcript identity and abundance. In humans, an increasing number of neurodevelopmental and neurodegenerative conditions are associated with dysregulated retroelements, including Aicardi-Goutieres syndrome (AGS), Rett syndrome (RTT), Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), multiple sclerosis (MS), schizophrenia (SZ), and aging. Taken together, these concepts suggest a larger functional role for RNA derived from retroelements. This review aims to define retroelement-derived RNA, discuss how it impacts the mammalian genome, as well as summarize data supporting phenotypic consequences of this unique RNA subset in the brain.

Investigation of tRNA-based relatedness within the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum: a comparative analysis.

The PVC superphylum is a diverse group of prokaryotes that require stringent growth conditions. RNA is a fascinating molecule to find evolutionary relatedness according to the RNA World Hypothesis. We conducted tRNA gene analysis to find evolutionary relationships in the PVC phyla. The analysis of genomic data (P = 9, V = 4, C = 8) revealed that the number of tRNA genes varied from 28 to 90 in Planctomycetes and Chlamydia, respectively. Verrucomicrobia has whole genomes and the longest scaffold (3 + 1), with tRNA genes ranging from 49 to 53 in whole genomes and 4 in the longest scaffold. Most tRNAs in the E. coli genome clustered with homologs, but approximately 43% clustered with tRNAs encoding different amino acids. Planctomyces, Akkermansia, Isosphaera, and Chlamydia were similar to E. coli tRNAs. In a phylum, tRNAs coding for different amino acids clustered at a range of 8 to 10%. Further analysis of these tRNAs showed sequence similarity with Cyanobacteria, Proteobacteria, Viridiplantae, Ascomycota and Basidiomycota (Eukaryota). This indicates the possibility of horizontal gene transfer or, otherwise, a different origin of tRNA in PVC bacteria. Hence, this work proves its importance for determining evolutionary relatedness and potentially identifying bacteria using tRNA. Thus, the analysis of these tRNAs indicates that primitive RNA may have served as the genetic material of LUCA before being replaced by DNA. A quantitative analysis is required to test these possibilities that relate the evolutionary significance of tRNA to the origin of life.

The Role of microRNAs in Epigenetic Regulation of Signaling Pathways in Neurological Pathologies.

In recent times, there has been a significant increase in researchers' interest in the functions of microRNAs and the role of these molecules in the pathogenesis of many multifactorial diseases. This is related to the diagnostic and prognostic potential of microRNA expression levels as well as the prospects of using it in personalized targeted therapy. This review of the literature analyzes existing scientific data on the involvement of microRNAs in the molecular and cellular mechanisms underlying the development of pathologies such as Alzheimer's disease, cerebral ischemia and reperfusion injury, and dysfunction of the blood-brain barrier.