Control of RNA Stability in Immunity.

Posttranscriptional control of mRNA regulates various biological processes, including inflammatory and immune responses. RNA-binding proteins (RBPs) bind cis-regulatory elements in the 3' untranslated regions (UTRs) of mRNA and regulate mRNA turnover and translation. In particular, eight RBPs (TTP, AUF1, KSRP, TIA-1/TIAR, Roquin, Regnase, HuR, and Arid5a) have been extensively studied and are key posttranscriptional regulators of inflammation and immune responses. These RBPs sometimes collaboratively or competitively bind the same target mRNA to enhance or dampen regulatory activities. These RBPs can also bind their own 3' UTRs to negatively or positively regulate their expression. Both upstream signaling pathways and microRNA regulation shape the interactions between RBPs and target RNA. Dysregulation of RBPs results in chronic inflammation and autoimmunity. Here, we summarize the functional roles of these eight RBPs in immunity and their associated diseases.

The molecular basis of tRNA selectivity by human pseudouridine synthase 3.

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

Gene- and Species-Specific Hox mRNA Translation by Ribosome Expansion Segments.

Ribosomes have been suggested to directly control gene regulation, but regulatory roles for ribosomal RNA (rRNA) remain largely unexplored. Expansion segments (ESs) consist of multitudes of tentacle-like rRNA structures extending from the core ribosome in eukaryotes. ESs are remarkably variable in sequence and size across eukaryotic evolution with largely unknown functions. In characterizing ribosome binding to a regulatory element within a Homeobox (Hox) 5' UTR, we identify a modular stem-loop within this element that binds to a single ES, ES9S. Engineering chimeric, "humanized" yeast ribosomes for ES9S reveals that an evolutionary change in the sequence of ES9S endows species-specific binding of Hoxa9 mRNA to the ribosome. Genome editing to site-specifically disrupt the Hoxa9-ES9S interaction demonstrates the functional importance for such selective mRNA-rRNA binding in translation control. Together, these studies unravel unexpected gene regulation directly mediated by rRNA and how ribosome evolution drives translation of critical developmental regulators.

High-resolution RNA tertiary structures in Zika virus stem-loop A for the development of inhibitory small molecules.

Flaviviruses such as Zika (ZIKV) and dengue virus (DENV) are positive-sense RNA viruses belonging to Flaviviridae The flavivirus genome contains a 5' end stem-loop promoter sequence known as stem-loop A (SLA) that is recognized by the flavivirus polymerase NS5 during viral RNA synthesis and 5' guanosine cap methylation. The crystal structures of ZIKV and DENV SLAs show a well-defined fold, consisting of a bottom stem, side loop, and top stem-loop, providing unique interaction sites for small molecule inhibitors to disrupt the promoter function. To facilitate the identification of small molecule binding sites in flavivirus SLA, we determined high-resolution structures of the bottom and top stems of ZIKV SLA, which contain a single U- or G-bulge, respectively. Both bulge nucleotides exhibit multiple orientations, from folded back on the adjacent nucleotide to flipped out of the helix, and are stabilized by stacking or base triple interactions. These structures suggest that even a single unpaired nucleotide can provide flexibility to RNA structures, and its conformation is mainly determined by the stabilizing chemical environment. To facilitate discovery of small molecule inhibitors that interfere with the functions of ZIKV SLA, we screened and identified compounds that bind to the bottom and top stems of ZIKV SLA.

The role of secondary structures in the functioning of 3' untranslated regions of mRNA: A review of functions of 3' UTRs' secondary structures and hypothetical involvement of secondary structures in cytoplasmic polyadenylation in Drosophila.

3' untranslated regions (3' UTRs) of mRNAs have many functions, including mRNA processing and transport, translational regulation, and mRNA degradation and stability. These different functions require cis-elements in 3' UTRs that can be either sequence motifs or RNA structures. Here we review the role of secondary structures in the functioning of 3' UTRs and discuss some of the trans-acting factors that interact with these secondary structures in eukaryotic organisms. We propose potential participation of 3'-UTR secondary structures in cytoplasmic polyadenylation in the model organism Drosophila melanogaster. Because the secondary structures of 3' UTRs are essential for post-transcriptional regulation of gene expression, their disruption leads to a wide range of disorders, including cancer and cardiovascular diseases. Trans-acting factors, such as STAU1 and nucleolin, which interact with 3'-UTR secondary structures of target transcripts, influence the pathogenesis of neurodegenerative diseases and tumor metastasis, suggesting that they are possible therapeutic targets.

Transcription elongation rate affects nascent histone pre-mRNA folding and 3' end processing.

Transcription elongation rate influences cotranscriptional pre-mRNA maturation, but how such kinetic coupling works is poorly understood. The formation of nonadenylated histone mRNA 3' ends requires recognition of an RNA structure by stem-loop-binding protein (SLBP). We report that slow transcription by mutant RNA polymerase II (Pol II) caused accumulation of polyadenylated histone mRNAs that extend past the stem-loop processing site. UV irradiation, which decelerates Pol II elongation, also induced long poly(A)+ histone transcripts. Inhibition of 3' processing by slow Pol II correlates with failure to recruit SLBP to histone genes. Chemical probing of nascent RNA structure showed that the stem-loop fails to fold in transcripts made by slow Pol II, thereby explaining the absence of SLBP and failure to process 3' ends. These results show that regulation of transcription speed can modulate pre-mRNA processing by changing nascent RNA structure and suggest a mechanism by which alternative processing could be controlled.

LeishIF4E2 is a cap-binding protein that plays a role in Leishmania cell cycle progression.

Leishmania encode six paralogs of the cap-binding protein eIF4E and five eIF4G candidates, forming unique complexes. Two cap-binding proteins, LeishIF4E1 and LeishIF4E2, do not bind any identified LeishIF4Gs, thus their roles are intriguing. Here, we combine structural prediction, proteomic analysis, and interaction assays to shed light on LeishIF4E2 function. A nonconserved C-terminal extension was identified through structure prediction and sequence alignment. m7 GTP-binding assays involving both recombinant and transgenic LeishIF4E2 with and without the C-terminal extension revealed that this extension functions as a regulatory gate, modulating the cap-binding activity of LeishIF4E2. The interactomes of the two LeishIF4E2 versions were investigated, highlighting the role of the C-terminal extension in binding to SLBP2. SLBP2 is known to interact with a stem-loop structure in the 3' UTRs of histone mRNAs. Consistent with the predicted inhibitory effect of SLBP2 on histone expression in Xenopus laevis, a hemizygous deletion mutant of LeishIF4E2, exhibited an upregulation of several histones. We therefore propose that LeishIF4E2 is involved in histone expression, possibly through its interaction between SLBP2 and LeishIF4E2, thus affecting cell cycle progression. In addition, cell synchronization showed that LeishIF4E2 expression decreased during the S-phase, when histones are known to be synthesized. Previous studies in T. brucei also highlighted an association between TbEIF4E2 and SLBP2, and further reported on an interaction between TbIF4E2 and S-phase-abundant mRNAs. Our results show that overexpression of LeishIF4E2 correlates with upregulation of cell cycle and chromosome maintenance proteins. Along with its effect on histone expression, we propose that LeishIF4E2 is involved in cell cycle progression.

Breaking self-regulation of Regnase-1 promotes its own protein expression.

The RNA-binding protein (RBP) Regnase-1 is an endonuclease that regulates immune responses by modulating target mRNA stability. Regnase-1 degrades a group of inflammation-associated mRNAs, which contributes to a balanced immune response and helps prevent autoimmune diseases. Regnase-1 also cleaves its own mRNA by binding stem-loop (SL) RNA structures in its 3'UTR. To understand how this autoregulation is important for immune responses, we generated mice with a 2-bp genome deletion in the target SL of the Regnase-1 3'-untranslated region (3'UTR). Deletion of these nucleotides inhibited SL formation and limited Regnase-1-mediated mRNA degradation. Mutant mice had normal hematopoietic cell differentiation. Biochemically, mutation of the 3'UTR SL increased Regnase-1 mRNA stability and enhanced both Regnase-1 mRNA and protein levels in mouse embryonic fibroblasts (MEFs). The expression of Il6, a Regnase-1 target gene, was constitutively suppressed at steady-state in mutant MEFs. Additionally, Regnase-1 protein expression in mutant MEFs was significantly elevated compared to that in wild-type MEFs at steady state and upon proinflammatory cytokine stimulation. These data suggest a negative feedback mechanism for Regnase-1 expression and represent a unique mouse model to probe Regnase-1 overexpression in vivo.

Structure of the dengue virus RNA promoter.

Dengue virus, a single-stranded positive sense RNA virus, is the most prevalent mosquito-borne pathogen in the world. Like all RNA viruses, it uses conserved structural elements within its genome to control essential replicative steps. A 70 nt stem-loop RNA structure (called SLA), found at the 5'-end of the genome of all flaviviruses, functions as the promoter for viral replication. This highly conserved structure interacts with the viral polymerase NS5 to initiate RNA synthesis. Here, we report the NMR structure of a monomeric SLA from dengue virus serotype 1, assembled to high-resolution from independently folded structural elements. The DENV1 SLA has an L-shaped structure, where the top and side helices are coaxially stacked, and the bottom helix is roughly perpendicular to them. Because the sequence is highly conserved among different flavivirus genomes, it is very likely that the three-dimensional fold and local structure of SLA are also conserved among flaviviruses and required for efficient replication. This work provides structural insight into the dengue promoter and provides the foundation for the discovery of new antiviral drugs that target this essential replicative step.

A conserved arginine in NS5 binds genomic 3' stem-loop RNA for primer-independent initiation of flavivirus RNA replication.

The commitment to replicate the RNA genome of flaviviruses without a primer involves RNA-protein interactions that have been shown to include the recognition of the stem-loop A (SLA) in the 5' untranslated region (UTR) by the nonstructural protein NS5. We show that DENV2 NS5 arginine 888, located within the carboxy-terminal 18 residues, is completely conserved in all flaviviruses and interacts specifically with the top-loop of 3'SL in the 3'UTR which contains the pentanucleotide 5'-CACAG-3' previously shown to be critical for flavivirus RNA replication. We present virological and biochemical data showing the importance of this Arg 888 in virus viability and de novo initiation of RNA polymerase activity in vitro. Based on our binding studies, we hypothesize that ternary complex formation of NS5 with 3'SL, followed by dimerization, leads to the formation of the de novo initiation complex that could be regulated by the reversible zipping and unzipping of cis-acting RNA elements.

A versatile and efficient method for detecting tRNA-derived fragments.

Recently, it has been discovered surprisingly that tRNA can be cleaved into specific small fragments under certain conditions. Most importantly, these tRNA-derived fragments (tRFs) participate in the regulation of gene expression, playing pivotal roles in various physiological and pathological processes and thus attracting widespread attention. Detecting tRF expression in tissues and cells often involves using tRF-specific stem-loop primers for reverse transcription. However, the high specificity offered by this method limits it to transcribing only one specific tRF sequence per reaction, necessitating separate reverse transcription and qPCR steps for multiple tRFs, leading to substantially increased time and resource consumption. This becomes especially challenging in precious samples with limited RNA availability. To address these issues, there is an urgent need for a universal and cost-effective tRF identification method. This study introduces a versatile tRF detection approach based on the uniform polyadenylation of all tRFs, allowing reverse transcription with a universal oligo(dT) primer. This method enables simultaneous reverse transcription of all target tRFs in one reaction, greatly facilitating subsequent qPCR analysis. Furthermore, it demonstrates exceptional sensitivity and specificity, offering significant value in tRF-related research.

The myriad roles of RNA structure in the flavivirus life cycle.

As positive-sense RNA viruses, the genomes of flaviviruses serve as the template for all stages of the viral life cycle, including translation, replication, and infectious particle production. Yet, they encode just 10 proteins, suggesting that the structure and dynamics of the viral RNA itself helps shepherd the viral genome through these stages. Herein, we highlight advances in our understanding of flavivirus RNA structural elements through the lens of their impact on the viral life cycle. We highlight how RNA structures impact translation, the switch from translation to replication, negative- and positive-strand RNA synthesis, and virion assembly. Consequently, we describe three major themes regarding the roles of RNA structure in flavivirus infections: 1) providing a layer of specificity; 2) increasing the functional capacity; and 3) providing a mechanism to support genome compaction. While the interactions described herein are specific to flaviviruses, these themes appear to extend more broadly across RNA viruses.

Polycations as Aptamer-Binding Modulators for Sensitive Fluorescence Anisotropy Assay of Aflatoxin B1.

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer-fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems.

4-Aminoquinolines modulate RNA structure and function: Pharmacophore implications of a conformationally restricted polyamine.

RNA structure plays an important role in regulating cellular function and there is a significant emerging interest in targeting RNA for drug discovery. Here we report the identification of 4-aminoquinolines as modulators of RNA structure and function. Aminoquinolines have a broad range of pharmacological activities, but their specific mechanism of action is often not fully understood. Using electrophoretic mobility shift assays and enzymatic probing we identified 4-aminoquinolines that bind the stem-loop II motif (s2m) of SARS-CoV-2 RNA site-specifically and induce dimerization. Using fluorescence-based RNA binding and T-box riboswitch functional assays we identified that hydroxychloroquine binds the T-box riboswitch antiterminator RNA element and inhibits riboswitch function. Based on its structure and riboswitch dose-response activity we identified that the antagonist activity of hydroxychloroquine is consistent with it being a conformationally restricted analog of the polyamine spermidine. Given the known role that polyamines play in RNA function, the identification of an RNA binding ligand with the pharmacophore of a conformationally restricted polyamine has significant implications for further elucidation of RNA structure-function relationships and RNA-targeted drug discovery.

Stem-loop RT-qPCR system for multiplex miRNA profiling and its application in wound healing-specific biomarker identification.

MiRNAs are biomarkers widely used in research but their clinical application is still challenging due to their low expression levels. Current methods for miRNA detection involve separate transcription and quantification for each target, which is costly and unsuitable for large sample sizes. This study provides a strategy for designing and screening miRNA-specific stem-loop reverse transcription (RT) primers, which enable the simultaneous transcription of three miRNAs and U6, and the concurrent detection of miRNA and U6 in the same transcript using TaqMan probes labeled with different dyes. The strategy was successfully employed to establish multiplex RT-PCR and dual-quantitative PCR (qPCR) quantification systems for 21 differentially expressed miRNAs during wound healing. The corresponding system can accurately quantify the cell culture samples containing miR-7a-5p mimic, miR-7a-5p inhibitor, or negative control. In summary, our results demonstrate that this strategy could efficiently accomplish the design, screening, and analysis of stem-loop RT primers for multiplex miRNA detection. Compared with the commercially customized miRNA assay kits, our system showed a higher degree of automation, more accurate qPCR assay capabilities, and lower assay costs, which could provide practical value for clinical diagnosis.

Multiple integrations of a sense transgene, including a tandem inverted repeat confer stable RNA-silencing mediated virus resistance under different abiotic and biotic conditions.

In a previous study, tobacco plants, transformed with a sense construct of the 57K domain of the replicase gene of tobacco rattle virus (TRV), provided resistance against genetically distant isolates of the virus. In this work, 57K-specific siRNAs were detected with RT-qPCR solely in the resistant line verifying the RNA-silencing base of the resistance. The integration sites of the transgene into the plant genome were identified with inverse-PCR. Moreover, the resistance against TRV was practically unaffected by low temperature conditions and the presence of heterologous viruses. The mechanism of the resistance was further examined by a gene expression analysis that showed increased transcript levels of genes with a key-role in the RNA silencing pathway and the basal antiviral defence. This work provides a comprehensive characterization of the robust virus resistance obtained by a sense transgene and underlines the usefulness of transgenic plants obtained by such a strategy.

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus.

RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3' untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections.

A Fluorescence Resonance Energy Transfer Aptasensor for Aflatoxin B1 Based on Ligand-Induced ssDNA Displacement.

In this study, a fluorescence resonance energy transfer (FRET)-based aptasensor for the detection of aflatoxin B1 (AFB1) was designed using a carboxyfluorescein (FAM)-labeled aptamer and short complementary DNA (cDNA) labeled with low molecular quencher RTQ1. The sensing principle was based on the detection of restored FAM-aptamer fluorescence due to the ligand-induced displacement of cDNA in the presence of AFB1, leading to the destruction of the aptamer/cDNA duplex and preventing the convergence of FAM and RTQ1 at the effective FRET distance. Under optimal sensing conditions, a linear correlation was obtained between the fluorescence intensity of the FAM-aptamer and the AFB1 concentration in the range of 2.5-208.3 ng/mL with the detection limit of the assay equal to 0.2 ng/mL. The assay time was 30 min. The proposed FRET aptasensor has been successfully validated by analyzing white wine and corn flour samples, with recovery ranging from 76.7% to 91.9% and 84.0% to 86.5%, respectively. This work demonstrates the possibilities of labeled cDNA as an effective and easily accessible tool for sensitive AFB1 detection. The homogeneous FRET aptasensor is an appropriate choice for contaminant screening in complex matrices.

Stem-loop binding protein and metal carcinogenesis.

Pre-mRNA processing of the replication-dependent canonical histone mRNAs requires an endonucleolytic cleavage immediately after a conserved stem loop structure which occurs before RNA Pol II encounters any poly(A) signal. Thus, in contrast to all other eukaryotic mRNAs, the canonical histone mRNAs are not polyadenylated in their 3' ends. The binding of stem-loop binding protein (SLBP) to the stem loop structure of the histone mRNAs is required for this process. SLBP is also involved in regulation of histone mRNA nuclear export, degradation, and translation. Depletion of SLBP has been shown to induce polyadenylation of histone mRNAs and alteration of histone protein levels, which are considered to contribute to the observed aberrant cell cycle progress and genomic instability resulting from the loss of SLBP function. Recent studies have demonstrated that some heavy metal carcinogens, including arsenic and nickel, can induce the loss of SLBP and the gain of polyadenylation of canonical histone mRNAs. Polyadenylated canonical histone H3 can result in abnormal transcription, cell cycle arrest, genomic instability, and cell transformation, which links SLBP depletion and subsequent histone mRNA misprocessing to cancer. This review seeks to briefly summarize what is known about regulation of SLBP expression, consequences of SLBP depletion, its roles in cancer-related end points, with particular focus on metal-induced SLBP depletion and the potential of SLBP depletion as a new mechanism for metal-induced carcinogenesis.

Temperature Sensing in Plants: On the Dawn of Molecular Thermosensor Research.

Although many studies on plant growth and development focus on the effects of light, a growing number of studies dissect plant responses to temperature and the underlying signaling pathways. The identity of plant thermosensing molecules (thermosensors) acting upstream of the signaling cascades in temperature responses was elusive until recently. During the past six years, a set of plant thermosensors has been discovered, representing a major turning point in the research on plant temperature responses and signaling. Here, we review these newly discovered plant thermosensors, which can be classified as sensors of warmth or cold. We compare between plant thermosensors and those from other organisms and attempt to define the subcellular thermosensing compartments in plants. In addition, we discuss the notion that photoreceptive thermosensors represent a novel class of thermosensors, the roles of which have yet to be described in non-plant systems.