RNA Complex Research Articles

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.

Essential Considerations for Free Energy Calculations of RNA-Small Molecule Complexes: Lessons from the Theophylline-Binding RNA Aptamer.

Alchemical free energy calculations are widely used to predict the binding affinity of small molecule ligands to protein targets; however, the application of these methods to RNA targets has not been deeply explored. We systematically investigated how modeling decisions affect the performance of absolute binding free energy calculations for a relatively simple RNA model system: theophylline-binding RNA aptamer with theophylline and five analogs. The goal of this investigation was 2-fold: (1) understanding the performance levels we can expect from absolute free energy calculations for a simple RNA complex and (2) learning about practical modeling considerations that impact the success of RNA-binding predictions, which may be different from the best practices established for protein targets. We learned that magnesium ion (Mg2+) placement is a critical decision that impacts affinity predictions. When information regarding Mg2+ positions is lacking, implementing RNA backbone restraints is an alternative way of stabilizing the RNA structure that recapitulates prediction accuracy. Since mistakes in Mg2+ placement can be detrimental, omitting magnesium ions entirely and using RNA backbone restraints are attractive as a risk-mitigating approach. We found that predictions are sensitive to modeling experimental buffer conditions correctly, including salt type and ionic strength. We explored the effects of sampling in the alchemical protocol, choice of the ligand force field (GAFF2/OpenFF Sage), and water model (TIP3P/OPC) on predictions, which allowed us to give practical advice for the application of free energy methods to RNA targets. By capturing experimental buffer conditions and implementing RNA backbone restraints, we were able to compute binding affinities accurately (mean absolute error (MAE) = 2.2 kcal/mol, Pearson's correlation coefficient = 0.9, Kendall's τ = 0.7). We believe there is much to learn about how to apply free energy calculations for RNA targets and how to enhance their performance in prospective predictions. This study is an important first step for learning best practices and special considerations for RNA-ligand free energy calculations. Future studies will consider increasingly complicated ligands and diverse RNA systems and help the development of general protocols for therapeutically relevant RNA targets.

Hybrid RNA/DNA Concatemers and Self-Limited Complexes: Structure and Prospects for Therapeutic Applications

The development of new convenient tools for the design of multicomponent nucleic acid (NA) complexes is one of the challenges in biomedicine and NA nanotechnology. In this paper, we analyzed the formation of hybrid RNA/DNA concatemers and self-limited complexes by a pair of oligonucleotides using UV melting, circular dichroism spectroscopy, and a gel shift assay. Effects of the size of the linker between duplex-forming segments of the oligonucleotides on complexes’ shape and number of subunits were compared and systematized for RNA/DNA, DNA/DNA, and RNA/RNA assemblies. The data on complex types summarized here as heat maps offer a convenient tool for the design of NA constructs. General rules found for RNA/DNA, DNA/DNA, and RNA/RNA complexes allow not only designing complexes with desired structures but also purposefully transforming their geometry. The A-form of the double helix of the studied RNA/DNA complexes was confirmed by circular dichroism analysis. Moreover, we show for the first time efficient degradation of RNA in hybrid self-limited complexes by RNase H and imidazole. The results open up new prospects for the design of supramolecular complexes as tools for nanotechnology, nanomachinery, and biomedical applications.

Cas9-Mediated Poxvirus Recombinant Recovery (CASPRR) for Fast Recovery of Recombinant Vaccinia Viruses.

Generation of recombinant vaccinia viruses opens many avenues for poxvirus research; however current methods for virus production can be laborious. Traditional methods rely on recombination strategies that produce engineered viruses at a low frequency, which then need to be identified and isolated from a large background of parent virus. For this reason, marker and reporter genes are often included, but in many cases these require removal in subsequent steps and the entire process is relatively inefficient. Cas9-mediated selection is a technique that repurposes Cas9/guide RNA complexes to amplify chosen subsets of vaccinia viruses. We refer to this approach as Cas9-mediated poxvirus recombinant recovery (CASPRR). Transient introduction of appropriately guided Cas9 allows for recovery of marker-free recombinant viruses in just 5days, and requires no additional virus modification. Following three rounds of purification, pure virus stocks are obtained. A newer method, stable expression of Cas9 and guide RNAs in a permissive cell line, allows for additional process streamlining, removing cell type-specific concerns related to transient transfection of Cas9. Within this chapter, the protocol for CASPRR is described in both a transient and stable expression model. These methods can be utilized to accelerate recovery of recombinant vaccinia viruses and be applied to generation of vaccinia libraries or novel therapeutic agents.

Molecular basis of mRNA delivery to the bacterial ribosome.

Protein synthesis begins with the formation of a ribosome-messenger RNA (mRNA) complex. In bacteria, the small ribosomal subunit (30S) is recruited to many mRNAs through base pairing with the Shine-Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs, and RNA polymerase (RNAP) can promote the recruitment of the pioneering 30S. Here, we examined 30S recruitment to nascent mRNAs using cryo-electron microscopy, single-molecule fluorescence colocalization, and in-cell cross-linking mass spectrometry. We show that bS1 delivers the mRNA to the ribosome for SD duplex formation and 30S activation. Additionally, bS1 and RNAP stimulate translation initiation. Our work provides a mechanistic framework for how the SD duplex, ribosomal proteins, and RNAP cooperate in 30S recruitment to mRNAs and establish transcription-translation coupling.

RADIP technology comprehensively identifies H3K27me3-associated RNA-chromatin interactions.

Many RNAs associate with chromatin, either directly or indirectly. Several technologies for mapping regions where RNAs interact across the genome have been developed to investigate the function of these RNAs. Obtaining information on the proteins involved in these RNA-chromatin interactions is critical for further analysis. Here, we developed RADIP [RNA and DNA interacting complexes ligated and sequenced (RADICL-seq) with immunoprecipitation], a novel technology that combines RADICL-seq technology with chromatin immunoprecipitation to characterize RNA-chromatin interactions mediated by individual proteins. Building upon the foundational principles of RADICL-seq, RADIP extends its advantages by increasing genomic coverage and unique mapping rate efficiency compared to existing methods. To demonstrate its effectiveness, we applied an anti-H3K27me3 antibody to the RADIP technology and generated libraries from mouse embryonic stem cells (mESCs). We identified a multitude of RNAs, including RNAs from protein-coding genes and non-coding RNAs, that are associated with chromatin via H3K27me3 and that likely facilitate the spread of Polycomb repressive complexes over broad regions of the mammalian genome, thereby affecting gene expression, chromatin structures and pluripotency of mESCs. Our study demonstrates the applicability of RADIP to investigations of the functions of chromatin-associated RNAs.

AI-integrated network for RNA complex structure and dynamic prediction.

RNA complexes are essential components in many cellular processes. The functions of these complexes are linked to their tertiary structures, which are shaped by detailed interface information, such as binding sites, interface contact, and dynamic conformational changes. Network-based approaches have been widely used to analyze RNA complex structures. With their roots in the graph theory, these methods have a long history of providing insight into the static and dynamic properties of RNA molecules. These approaches have been effective in identifying functional binding sites and analyzing the dynamic behavior of RNA complexes. Recently, the advent of artificial intelligence (AI) has brought transformative changes to the field. These technologies have been increasingly applied to studying RNA complex structures, providing new avenues for understanding the complex interactions within RNA complexes. By integrating AI with traditional network analysis methods, researchers can build more accurate models of RNA complex structures, predict their dynamic behaviors, and even design RNA-based inhibitors. In this review, we introduce the integration of network-based methodologies with AI techniques to enhance the understanding of RNA complex structures. We examine how these advanced computational tools can be used to model and analyze the detailed interface information and dynamic behaviors of RNA molecules. Additionally, we explore the potential future directions of how AI-integrated networks can aid in the modeling and analyzing RNA complex structures.

REDalign: accurate RNA structural alignment using residual encoder-decoder network

BackgroundRNA secondary structural alignment serves as a foundational procedure in identifying conserved structural motifs among RNA sequences, crucially advancing our understanding of novel RNAs via comparative genomic analysis. While various computational strategies for RNA structural alignment exist, they often come with high computational complexity. Specifically, when addressing a set of RNAs with unknown structures, the task of simultaneously predicting their consensus secondary structure and determining the optimal sequence alignment requires an overwhelming computational effort of O(L6)\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$O(L^6)$$\\end{document} for each RNA pair. Such an extremely high computational complexity makes these methods impractical for large-scale analysis despite their accurate alignment capabilities.ResultsIn this paper, we introduce REDalign, an innovative approach based on deep learning for RNA secondary structural alignment. By utilizing a residual encoder-decoder network, REDalign can efficiently capture consensus structures and optimize structural alignments. In this learning model, the encoder network leverages a hierarchical pyramid to assimilate high-level structural features. Concurrently, the decoder network, enhanced with residual skip connections, integrates multi-level encoded features to learn detailed feature hierarchies with fewer parameter sets. REDalign significantly reduces computational complexity compared to Sankoff-style algorithms and effectively handles non-nested structures, including pseudoknots, which are challenging for traditional alignment methods. Extensive evaluations demonstrate that REDalign provides superior accuracy and substantial computational efficiency.ConclusionREDalign presents a significant advancement in RNA secondary structural alignment, balancing high alignment accuracy with lower computational demands. Its ability to handle complex RNA structures, including pseudoknots, makes it an effective tool for large-scale RNA analysis, with potential implications for accelerating discoveries in RNA research and comparative genomics.

Cationic and ionizable amphiphiles based on di-hexadecyl ester of <I>L</I>-glutamic acid for liposomal transport of RNA

Various RNAs are among the most promising and actively developed therapeutic agents for the treatment of tumors, infectious diseases and a number of other pathologies associated with the dysfunction of specific genes. Some nanocarriers are used for the effective delivery of RNAs to target cells, including liposomes based on cationic and/or ionizable amphiphiles. Cationic amphiphiles contain a protonated amino group and exist as salts in an aqueous environment. Ionizable amphiphiles are a new generation of cationic lipids that exhibit reduced toxicity and immunogenicity and undergo ionization only in the acidic environment of the cell. In this work we developed a scheme for the preparation and carried out the synthesis of new cationic and ionizable amphiphiles based on natural amino acids (L-glutamic acid, glycine, beta-alanine, and gamma-aminobutyric acid). Cationic and ionizable liposomes were formed based on the obtained compounds, mixed with natural lipids (phosphatidylcholine and cholesterol), and their physicochemical characteristics (particle size, zeta potential, and storage stability) were determined. Average diameter of particles stable for 5–7 days did not exceed 100 nm. Zeta potential of cationic and ionizable liposomes was about 30 and 1 mV, respectively. The liposomal particles were used to form complexes with RNA molecules. Such RNA complexes were characterized by atomic force microscopy and their applicability for nucleic acid transport was determined.

Integrated Biochemical and Computational Methods for Deciphering RNA-Processing Codes.

RNA processing involves steps such as capping, splicing, polyadenylation, modification, and nuclear export. These steps are essential for transforming genetic information in DNA into proteins and contribute to RNA diversity and complexity. Many biochemical methods have been developed to profile and quantify RNAs, as well as to identify the interactions between RNAs and RNA-binding proteins (RBPs), especially when coupled with high-throughput sequencing technologies. With the rapid accumulation of diverse data, it is crucial to develop computational methods to convert the big data into biological knowledge. In particular, machine learning and deep learning models are commonly utilized to learn the rules or codes governing the transformation from DNA sequences to intriguing RNAs based on manually designed or automatically extracted features. When precise enough, the RNA codes can be incredibly useful for predicting RNA products, decoding the molecular mechanisms, forecasting the impact of disease variants on RNA processing events, and identifying driver mutations. In this review, we systematically summarize the biochemical and computational methods for deciphering five important RNA codes related to alternative splicing, alternative polyadenylation, RNA localization, RNA modifications, and RBP binding. For each code, we review the main types of experimental methods used to generate training data, as well as the key features, strategic model structures, and advantages of representative tools. We also discuss the challenges encountered in developing predictive models using large language models and extensive domain knowledge. Additionally, we highlight useful resources and propose ways to improve computational tools for studying RNA codes.

Activity-assembled nBAF complex mediates rapid immediate early gene transcription by regulating RNA polymerase II productive elongation

Signal-dependent RNA polymerase II (RNA Pol II) productive elongation is an integral component of gene transcription, including that of immediate early genes (IEGs) induced by neuronal activity. However, it remains unclear how productively elongating RNA Pol II overcomes nucleosomal barriers. Using RNAi, three degraders, and several small-molecule inhibitors, we show that the mammalian switch/sucrose non-fermentable (SWI/SNF) complex of neurons (neuronal BRG1/BRM-associated factor or nBAF) is required for activity-induced transcription of neuronal IEGs, including Arc. The nBAF complex facilitates promoter-proximal RNA Pol II pausing and signal-dependent RNA Pol II recruitment (loading) and, importantly, mediates productive elongation in the gene body via interaction with the elongation complex and elongation-competent RNA Pol II. Mechanistically, RNA Pol II elongation is mediated by activity-induced nBAF assembly (especially ARID1A recruitment) and its ATPase activity. Together, our data demonstrate that the nBAF complex regulates several aspects of RNA Pol II transcription and reveal mechanisms underlying activity-induced RNA Pol II elongation. These findings may offer insights into human maladies etiologically associated with mutational interdiction of BAF functions.

Pulsed EPR Methods in the Angstrom to Nanometre Scale Shed Light on the Conformational Flexibility of a Fluoride Riboswitch.

Riboswitches control gene regulation upon external stimuli such as environmental factors or ligand binding. The fluoride sensing riboswitch from Thermotoga petrophila is a complex regulatory RNA proposed to be involved in resistance to F- cytotoxicity. The details of structure and dynamics underpinning the regulatory mechanism are currently debated. Here we demonstrate that a combination of pulsed electron paramagnetic resonance (ESR/EPR) spectroscopies, detecting distances in the angstrom to nanometre range, can probe distinct regions of conformational flexibility in this riboswitch. PELDOR (pulsed electron-electron double resonance) revealed a similar preorganisation of the sensing domain in three forms, i.e. the free aptamer, the Mg2+-bound apo, and the F--bound holo form. 19F ENDOR (electron-nuclear double resonance) was used to investigate the active site structure of the F--bound holo form. Distance distributions without a priori structural information were compared with in silico modelling of spin label conformations based on the crystal structure. While PELDOR, probing the periphery of the RNA fold, revealed conformational flexibility of the RNA backbone, ENDOR indicated low structural heterogeneity at the ligand binding site. Overall, the combination of PELDOR and ENDOR with sub-angstrom precision gave insight into structural organisation and flexibility of a riboswitch, not easily attainable by other biophysical techniques.

Puls EPR Methoden im Angstöm‐ bis Nanometerbereich geben Aufschluss über die konformationelle Flexibilität eines Fluorid‐Riboschalters

AbstractRiboschalter steuern die Genregulation in Reaktion auf externe Reize wie Umweltfaktoren oder Ligandenbindung. Der fluoriderkennende Riboschalter aus Thermotoga petrophila ist eine komplexe regulatorische RNA, von der angenommen wird, an der Resistenz gegen die zytotoxische Wirkung von Fluorid beteiligt zu sein. Details der Struktur und Dynamik, die dem regulatorischen Mechanismus zugrunde liegen, werden derzeit diskutiert. Hier zeigen wir, dass mit einer Kombination aus Puls Elektronen‐Paramagnetischer‐Resonanz (ESR/EPR) Spektroskopien, die Abstände im Angström‐ bis Nanometerbereich detektiert, spezifische Bereiche konformationeller Flexibilität in diesem Riboschalter untersucht werden können. PELDOR (pulsed electron‐electron double resonance) zeigte eine ähnliche Präorganisation der Sensordomäne in drei Formen: der freien Aptamerform, der Mg2+‐gebundenen apo‐Form und der F−‐gebundenen holo‐Form. 19F ENDOR (electron‐nuclear double resonance) wurde verwendet, um die Struktur des aktiven Zentrums der F−‐gebundenen holo‐Form zu untersuchen. Abstandsverteilungen ohne vorherige strukturelle Informationen wurden mit auf der Kristallstruktur basierenden in silico Modellierungen der Spinmarkerkonformationen verglichen. Während PELDOR, welches die Peripherie der RNA Faltung untersucht, die konformationelle Flexibilität des RNA Rückgrats aufdeckte, zeigte ENDOR eine geringe strukturelle Heterogenität der Ligandenbindungstasche. Die Kombination aus PELDOR und ENDOR lieferte sub‐Angström‐präzise Einblicke in die strukturelle Organisation und Flexibilität eines Riboschalters, die mit anderen biophysikalischen Techniken nur schwer zu erreichen sind.

Alternative Splicing: A Potential Therapeutic Target in Hematological Malignancies.

Leukemia represents the most prevalent malignancy in children, constituting 30% of childhood cancer cases, with acute lymphoblastic leukemia (ALL) being particularly heterogeneous. This paper explores the role of alternative splicing in leukemia, highlighting its significance in cancer development and progression. Aberrant splicing is often driven by mutations in splicing-factor genes, which can lead to the production of variant proteins that contribute to oncogenesis. The spliceosome, a complex of small nuclear RNAs and proteins, facilitates RNA splicing, a process critical for generating diverse mRNA and protein products from single genes. Mutations in splicing factors, such as U2AF1, SF3B1, SRSF2, ZRSR2, and HNRNPH1, are frequently observed across various hematological malignancies and are associated with poor prognosis and treatment resistance. This research underscores the necessity of understanding the mechanisms of RNA splicing dysregulation in order to develop targeted therapies to correct these aberrant processes, thereby improving outcomes for patients with leukemia and related disorders.

Ferroptosis-associated alterations in diabetes following ischemic stroke: Insights from RNA sequencing

ObjectiveFerroptosis is an iron-dependent form of programmed cell death associated with lipid peroxidation. Though diabetes worsens cerebral injury and clinical outcomes in stroke, it is poorly understood whether ferroptosis contributes to diabetes-exacerbated stroke. This study aimed to identify ferroptosis-associated differentially expressed genes in ischemic stroke under diabetic condition and then explore their roles using comprehensive bioinformatics analyses. MethodsType 1 diabetes (T1D) model was established in male mice at 8–10 weeks of age by one intraperitoneal injection of streptozotocin (110 mg/kg). Ischemic stroke was induced by a transient 45-minute middle cerebral artery occlusion and evaluated three days thereafter. Ischemic brain cortex was dissected 24 h after the reperfusion and subjected to bulk tissue RNA sequencing followed by bioinformatics analysis and verification of key findings via quantitative real-time PCR. ResultsEnlarged infarct size was seen in diabetic, as compared with non-diabetic mice, in conjunction with worsened neurological behaviors. Both body and spleen weights were reduced in diabetic as compared with non-diabetic mice. There was a trend for reduced survival rate in diabetic mice following the stroke. In RNA sequencing analysis, we identified 1299 differentially expressed genes in ischemic brain between diabetic and non-diabetic mice, with upregulation and downregulation for 732 and 567 genes, respectively. Among these genes, 27 genes were associated with ferroptosis. Further analysis reveals that solute carrier family 25 member 28(SLC25A28) and sterol carrier protein 2(SCP2) were the top genes associated with ferroptosis in diabetic mice following ischemic stroke. In several bioinformatics analyses, we found SLC25A28, one of the top ferroptosis-related genes, is involved in several metabolic and regulatory pathways as well as the regulatory complexity of microRNAs and circular RNAs, which demonstrates the potential role of SLC25A28 in diabetes-exacerbated stroke. Drug network analysis suggests SLC25A28 as a potential therapeutic target for ameliorating ischemic injury in diabetes. ConclusionsOur bulk RNA sequencing and bioinformatics analyses show that altered ferroptosis signaling pathway was associated with the exacerbation of experimental stroke injury under diabetic condition. Especially, additional investigation into the mechanisms of SLC25A28 and SCP2 in diabetes-exacerbated stroke will be explored in the future study.

RNA Nanotechnology for Codelivering High-Payload Nucleoside Analogs to Cancer with a Synergetic Effect.

Nucleoside analogs are potent inhibitors for cancer treatment, but the main obstacles to their application in humans are their toxicity, nonspecificity, and lack of targeted delivery tools. Here, we report the use of RNA four-way junctions (4WJs) to deliver two nucleoside analogs, floxuridine (FUDR) and gemcitabine (GEM), with high payloads through routine and simple solid-state RNA synthesis and nanoparticle assembly. The design of RNA nanotechnology for the co-delivery of nucleoside analogs and the chemotherapeutic drug paclitaxel (PTX) resulted in synergistic effects and high efficacy in the treatment of Triple-Negative Breast Cancer (TNBC). The 4WJ-drug complexes were confirmed to have efficient tumor spontaneous targeting and no toxicity because the motility of RNA nanoparticles has been previously shown to enable these RNA-drug complexes to spontaneously accumulate in tumor blood vessels. The negative charge of RNA enables those RNA complexes that are not targeted to tumor vasculature to circulate in the blood and enter the urine through the kidney glomerulus, without accumulating in organs, therefore being nontoxic. Drug incorporation into RNA 4WJ can be precisely controlled with a defined loading amount, location, and ratio. The incorporation of nucleoside analogs into 4WJ only requires one step using nucleoside analogue phosphoramidites during solid-phase RNA synthesis, without the need for additional conjugation and purification processes.

Imino chemical shift assignments of tRNAAsp, tRNAVal and tRNAPhe from Escherichia coli

Transfer RNAs (tRNAs) are an essential component of the protein synthesis machinery. In order to accomplish their cellular functions, tRNAs go through a highly controlled biogenesis process leading to the production of correctly folded tRNAs. tRNAs in solution adopt the characteristic L-shape form, a stable tertiary conformation imperative for the cellular stability of tRNAs, their thermotolerance, their interaction with protein and RNA complexes and their activity in the translation process. The introduction of post-transcriptional modifications by modification enzymes, the global conformation of tRNAs, and their cellular stability are highly interconnected. We aim to further investigate this existing link by monitoring the maturation of bacterial tRNAs in E. coli extracts using NMR. Here, we report on the 1H, 15N chemical shift assignment of the imino groups and some amino groups of unmodified and modified E. coli tRNAAsp, tRNAVal and tRNAPhe, which are essential for characterizing their maturation process using NMR spectroscopy.

Functions and applications of RNA interference and small regulatory RNAs.

Small regulatory RNAs play a variety of crucial roles in eukaryotes, influencing gene regulation, developmental timing, antiviral defense, and genome integrity via a process termed RNA interference (RNAi). This process involves Argonaute/small RNA (AGO/sRNA) complexes that target transcripts via sequence complementarity and modulate gene expression and epigenetic modifications. RNAi is a highly conserved gene regulatory phenomenon that recognizes self- and non-self nucleic acids, thereby defending against invasive sequences. Since its discovery, RNAi has been widely applied in functional genomic studies and a range of practical applications. In this review, we focus on the current understanding of the biological roles of the RNAi pathway in transposon silencing, fertility, developmental regulation, immunity, stress responses, and acquired transgenerational inheritance. Additionally, we provide an overview of the applications of RNAi technology in biomedical research, agriculture, and therapeutics.

The biogenesis, regulation and functions of transitive siRNA in plants.

Small RNA (sRNA)-mediated RNA interference (RNAi) is a sequence-specific gene silencing mechanism that modulates gene expression in eukaryotes. As core molecules of RNAi, various sRNAs are encoded in the plant genome or derived from invading RNA molecules, and their biogenesis depends on distinct genetic pathways. Transitive small interfering RNAs (siRNAs), which are sRNAs produced from double-strand RNA (dsRNA) in a process that depends on RNA-dependent RNA polymerases (RDRs), can amplify and spread silencing signals to additional transcripts, thereby enabling a phenomenon termed "transitive RNAi". Members of this class of siRNAs function in various biological processes ranging from development to stress adaptation. In Arabidopsis thaliana, two RDRs participate in the generation of transitive siRNAs, acting cooperatively with various siRNA generation-related factors, such as the RNA-induced silencing complex (RISC) and aberrant RNAs. Transitive siRNAs are produced in diverse subcellular locations and structures under the control of various mechanisms, highlighting the intricacies of their biogenesis and functions. In this review, we discuss recent advances in understanding the molecular events of transitive siRNA biogenesis and its regulation, with a particular focus on factors involved in RDR recruitment. We aim to provide a comprehensive description of the generalized mechanism governing the biogenesis of transitive siRNAs. Additionally, we present an overview of the diverse biological functions of these siRNAs and raise some pressing questions in this area for further investigation.

Direct delivery of Cas9 or base editor protein and guide RNA complex enables genome editing in the retina

Genome editing by CRISPR-Cas holds promise for the treatment of retinal dystrophies. For therapeutic gene editing, transient delivery of CRISPR-Cas9 is preferable to viral delivery which leads to long-term expression with potential adverse consequences. Cas9 protein and its guide RNA, delivered as ribonucleoprotein (RNP) complexes, have been successfully delivered into the retinal pigment epithelium in vivo. However, the delivery into photoreceptors, the primary focus in retinal dystrophies, has not been achieved. Here, we investigate the feasibility of direct RNP delivery into photoreceptors and RPE cells. We demonstrate that Cas9 or adenine-base editors complexed with guide RNA, can enter retinal cells without the addition of any carrier compounds. Once in the retinal cells, editing rates vary based on the efficacy of the guide RNA and the specific location edited within the genes. Cas9 RNP delivery at high concentrations, however, leads to outer retinal toxicity. This underscores the importance of improving delivery efficiency for potential therapeutic applications in the future.