RNA Loading Research Articles

Circular RNAs from bovine blastocysts can interact with miRNAs/tsRNAs from embryonic extracellular vesicles and regulate hatching

Circular RNAs (circRNAs) are endogenous biological macromolecules that regulate various biological processes including embryo development. However, little is known about which circRNAs are present in bovine preimplantation embryos and their respective roles. Here, we characterized the expression profile of circRNAs in bovine blastocysts for the first time. We detected 25,700 circRNAs in total, with 12,630 circRNAs uniquely expressed in blastocysts compared to degenerated embryos. CircRNA alternative splicing (AS) events were also found more frequently in blastocysts than in degenerated embryos (299 vs 258). Additionally, 410 circRNAs, among which 11 circRNAs with a high potential to encode polypeptides, were found differentially expressed between blastocysts and degenerated embryos. We further predicted and constructed a circRNA-miRNA-mRNA network, wherein differentially expressed circRNAs were shown to bind to bovine preimplantation embryo development-related miRNAs. Employing bioinformatic algorithms we found that differentially expressed circRNAs are associated with differentially expressed miRNAs and transfer RNA-derived small RNAs (tsRNAs) enclosed in embryonic extracellular vesicles (EVs). Furthermore, functional analysis revealed that knockdown of the evolutionarily conserved circAGO2 can inhibit blastocyst hatching. Overall, our study provides the first landscape of circRNAs in bovine preimplantation embryos and highlights the novel role of circRNAs as tsRNA binding partners influencing small RNA sorting and loading into EVs, with circAGO2 playing a regulatory role in bovine blastocyst hatching.

Ionizable nanoemulsions for RNA delivery into the central nervous system – importance of diffusivity

Lipid nanoparticles (LNPs) currently dominate the RNA delivery landscape; however their limited diffusivity hampers targeted tissue dissemination, and, hence, their capacity for intracellular drug delivery. This is especially relevant for tissues such as the central nervous system (CNS), where overcoming proactive brain barriers is crucial for the efficacy of genetic therapeutics. This research aimed to create ionizable nanoemulsions (iNEs), a new generation of RNA delivery systems with enhanced diffusivity. The developed iNEs (consisting of the combination of C12–200, DOPE, Vitamin E, and DMG-PEG) with a size below 100 nm, neutral surface charge, and high RNA loading capacity, showed excellent cell viability and transfection efficiency in various cellular models, including neurons, astrocytes, and microglia. Subsequently, iNEs containing mRNA GFP were tested for CNS transfection, highlighting their exceptional diffusivity and selective transfection of neurons following intra-parenchymal administration.

Single-Particle Spectroscopic Chromatography Reveals Heterogeneous RNA Loading and Size Correlations in Lipid Nanoparticles.

Lipid nanoparticles (LNP) have emerged as pivotal delivery vehicles for RNA therapeutics. Previous research and development usually assumed that LNPs are homogeneous in population, loading density, and composition. Such perspectives are difficult to examine due to the lack of suitable tools to characterize these physicochemical properties at the single-nanoparticle level. Here, we report an integrated spectroscopy-chromatography approach as a generalizable strategy to dissect the complexities of multicomponent LNP assembly. Our platform couples cylindrical illumination confocal spectroscopy (CICS) with single-nanoparticle free solution hydrodynamic separation (SN-FSHS) to simultaneously profile population identity, hydrodynamic size, RNA loading levels, and distributions of helper lipid and PEGylated lipid of LNPs at the single-particle level and in a high-throughput manner. Using a benchmark siRNA LNP formulation, we demonstrate the capability of this platform by distinguishing seven distinct LNP populations, quantitatively characterizing size distribution and RNA loading level in wide ranges, and more importantly, resolving composition-size correlations. This SN-FSHS-CICS analysis provides critical insights into a substantial degree of heterogeneity in the packing density of RNA in LNPs and size-dependent loading-size correlations, explained by kinetics-driven assembly mechanisms of RNA LNPs.

Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification.

Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV-liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.

SARS-CoV-2 wastewater surveillance in the Czech Republic: Spatial and temporal differences in SARS-CoV-2 RNA concentrations and relationship to clinical data and wastewater parameters

This study presents the results of systematic wastewater monitoring of SARS-CoV-2 RNA and basic wastewater parameters from four different wastewater treatment plants (WWTPs) in the Czech Republic over the 2020–2022 epidemic. Two-step reverse-transcription quantitative PCR targeting genes encoding the N and Nsp12 proteins was employed to detect SARS-CoV-2 RNA loading in 420 wastewater samples. The results obtained were used to evaluate the potential of wastewater analysis for describing the epidemiological situation in cities of different sizes and determining temporal differences based on the prevailing SARS-CoV-2 variant. Strong correlations between the number of active and hospitalised COVID‐19 cases in each WWTP catchment area and the concentration of SARS-CoV-2 RNA detected in the wastewater clearly demonstrated the suitability of this wastewater-based epidemiological approach for WWTPs of different sizes and characteristics, despite differences in SARS-CoV-2 variant waves, with some WWTPs showing high predictive potential. This study demonstrated on the data from the Czech Republic that targeted systematic monitoring of wastewater provides sufficiently robust data for surveillance of viral loads in sample populations, and thus contributes to preventing the spread of infection and subsequent introduction of appropriate measures.

Extracellular Vesicles: Tiny Messengers for Mighty RNA Delivery

Extracellular vesicles (EVs) encompass a diverse array of cell-derived vesicles, originating either from the endosomal compartment (exosomes) or generated through shedding from the cell membrane. These lipid bilayer nanovesicles carry a diverse cargo consisting of nucleic acids, various macromolecules, and growth factors, capable of being assimilated by nearby or distant cells through biofluids, thereby triggering a wide range of cellular responses. Given their distinctive biological characteristics and crucial roles in intercellular communication, EVs have garnered significant attention, especially concerning potential clinical applications. Inheriting cargo from their parent cells, EVs present promising resources for diverse disease biomarkers. Research elucidating the specific impacts of cargo on target cells has sparked enthusiasm for their therapeutic potential. Compelling evidence indicates that RNA cargo housed within EVs can modulate gene expression and influence cellular functions in recipient cells. However, despite significant progress, numerous aspects of EV biology remain obscure, encompassing selective cargo-loading mechanisms that yield distinct compositions from source cells, variability in size and content, and undisclosed pathways governing uptake and cargo fate in recipient cells. A thorough understanding of core EV mechanisms—such as generation, trafficking, and payload delivery—is essential for their effective clinical utilization. This review explores the current understanding of RNA loading and transportation within EVs, shedding light on the advancements made toward clinical applications.

YBX1 Regulates Satellite II RNA Loading into Small Extracellular Vesicles and Promotes the Senescent Phenotype.

Senescent cells secrete inflammatory proteins and small extracellular vesicles (sEVs), collectively termed senescence-associated secretory phenotype (SASP), and promote age-related diseases. Epigenetic alteration in senescent cells induces the expression of satellite II (SATII) RNA, non-coding RNA transcribed from pericentromeric repetitive sequences in the genome, leading to the expression of inflammatory SASP genes. SATII RNA is contained in sEVs and functions as an SASP factor in recipient cells. However, the molecular mechanism of SATII RNA loading into sEVs is unclear. In this study, we identified Y-box binding protein 1 (YBX1) as a carrier of SATII RNA via mass spectrometry analysis after RNA pull-down. sEVs containing SATII RNA induced cellular senescence and promoted the expression of inflammatory SASP genes in recipient cells. YBX1 knockdown significantly reduced SATII RNA levels in sEVs and inhibited the propagation of SASP in recipient cells. The analysis of the clinical dataset revealed that YBX1 expression is higher in cancer stroma than in normal stroma of breast and ovarian cancer tissues. Furthermore, high YBX1 expression was correlated with poor prognosis in breast and ovarian cancers. This study demonstrated that SATII RNA loading into sEVs is regulated via YBX1 and that YBX1 is a promising target in novel cancer therapy.

Dual targeted extracellular vesicles regulate oncogenic genes in advanced pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) tumours carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to target high solid tumour burden like PDAC. Here, we report a dual targeted extracellular vesicle (dtEV) carrying high loads of therapeutic RNA that effectively suppresses large PDAC tumours in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation release abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dtEVs suppress large orthotopic PANC-1 and patient derived xenograft tumours and metastasis in mice and extended animal survival. Our work presents a clinically accessible and scalable way to produce abundant EVs for delivering multiple gene cargoes to large solid tumours.

Biomimetic Mineralized CRISPR/Cas RNA Nanoparticles for Efficient Tumor-Specific Multiplex Gene Editing.

CRISPR/Cas9 systems have great potential to achieve sophisticated gene therapy and cell engineering by editing multiple genomic loci. However, to achieve efficient multiplex gene editing, the delivery system needs adequate capacity to transfect all CRISPR/Cas9 RNA species at the required stoichiometry into the cytosol of each individual cell. Herein, inspired by biomineralization in nature, we develop an all-in-one biomimetic mineralized CRISPR/Cas9 RNA delivery system. This system allows for precise control over the coencapsulation ratio between Cas9 mRNA and multiple sgRNAs, while also exhibiting a high RNA loading capacity. In addition, it enhances the storage stability of RNA at 4 °C for up to one month, and the surface of the nanoparticles can be easily functionalized for precise targeting of RNA nanoparticles in vivo at nonliver sites. Based on the above characteristics, as a proof-of-concept, our system was able to achieve significant gene-editing at each target gene (Survivin: 31.9%, PLK1: 24.41%, HPV: 23.2%) and promote apoptosis of HeLa cells in the mouse model, inhibiting tumor growth without obvious off-target effects in liver tissue. This system addresses various challenges associated with multicomponent RNA delivery in vivo, providing an innovative strategy for the RNA-based CRISPR/Cas9 gene editing.

Oversized liposomes boost macrophage-targeted RNA delivery to regulate macrophage polarity

Oversized liposomes boost macrophage-targeted RNA delivery to regulate macrophage polarity

Conformational dynamics of the RNA binding channel regulates loading and translocation of the DEAH-box helicase Prp43.

The DEAH-box helicase Prp43 has essential functions in pre-mRNA splicing and ribosome biogenesis, remodeling structured RNAs. To initiate unwinding, Prp43 must first accommodate a single-stranded RNA segment into its RNA binding channel. This allows translocation of the helicase on the RNA. G-patch (gp) factors activate Prp43 in its cellular context enhancing the intrinsically low ATPase and RNA unwinding activity. It is unclear how the RNA loading process is accomplished by Prp43 and how it is regulated by its substrates, ATP and RNA, and the G-patch partners. We developed single-molecule (sm) FRET reporters on Prp43 from Chaetomium thermophilum to monitor the conformational dynamics of the RNA binding channel in Prp43 in real-time. We show that the channel can alternate between open and closed conformations. Binding of Pfa1(gp) and ATP shifts the distribution of states towards channel opening, facilitating the accommodation of RNA. After completion of the loading process, the channel remains firmly closed during successive cycles of ATP hydrolysis, ensuring stable interaction with the RNA and processive translocation. Without Pfa1(gp), it remains predominantly closed preventing efficient RNA loading. Our data reveal how the ligands of Prp43 regulate the structural dynamics of the RNA binding channel controlling the initial binding of RNA.

Bioengineered Bacteriophage-Like Nanoparticles as RNAi Therapeutics to Enhance Radiotherapy against Glioblastomas.

Since glioblastomas (GBMs) are radioresistant malignancies and most GBM recurrences occur in radiotherapy, increasing the effectiveness of radiotherapy by gene-silencing has recently attracted attention. However, the difficulty in precisely tuning the composition and RNA loading in nanoparticles leads to batch-to-batch variations of the RNA therapeutics, thus significantly restricting their clinical translation. Here, we bioengineer bacteriophage Qβ particles with a designed broccoli light-up three-way junction (b-3WJ) RNA scaffold (contains two siRNA/miRNA sequences and one light-up aptamer) packaging for the silencing of genes in radioresistant GBM cells. The in vitro results demonstrate that the cleavage of de novo designed b-3WJ RNA by Dicer enzyme can be easily monitored in real-time using fluorescence microscopy, and the TrQβ@b-3WJLet-7gsiEGFR successfully knocks down EGFR and IKKα simultaneously and thereby inactivates NF-κB signaling to inhibit DNA repair. Delivery of TrQβ@b-3WJLet-7gsiEGFR through convection-enhanced delivery (CED) infusion followed by 2Gy X-ray irradiation demonstrated that the median survival was prolonged to over 60 days compared with the 2Gy X-ray irradiated group (median survival: 31 days). Altogether, the results of this study could be critical for the design of RNAi-based genetic therapeutics, and CED infusion serves as a powerful delivery system for promoting radiotherapy against GBMs without evidence of systemic toxicity.

Abstract 3761: Mediators of RNA sorting and export in non-small cell lung cancer derived extracellular vesicles

Abstract Extracellular vesicle (EV) mediated transfer of biologically functional molecules between cancer cells and the surrounding environment significantly impacts cancer progression. Our previous study established that non-small cell lung cancer (NSCLC) cell-derived EV-RNA is an important mediator of this communication, since it significantly augments the invasive potential of recipient non-tumorigenic cells (BEAS2Bs). This led us to determine the EV-RNA cargo that is specific to NSCLC EVs. Our RNA sequencing analysis revealed preferential enrichment of specific repertoires of RNA in NSCLC EVs amongst which miR-10b, miR-100 and miR-155 show synergistic effects with regard to an invasive phenotype elicited in recipient BEAS2B cells. Interestingly, our RNA sequencing analysis also revealed a global decrease in specific subsets of microRNAs (miRNAs) in NSCLC (H358 and Calu6) cells in comparison to non-tumorigenic BEAS2B cells. In contrast, the dysregulated miRNAs were found to be exclusively abundant in the respective NSCLC cell derived EVs. This suggests a precise and active mechanism of RNA sorting and loading into EVs. A cohort of dysregulated miRNAs was shortlisted, and their cellular and EV expression was validated using RT-qPCR and flow cytometry analysis by conjugating each one of the candidates to a fluorophore. After confirming that inclusion of a fluorophore to the RNA does not disrupt RNA sorting into the EVs, we transfected the cohort of shortlisted miRNAs including miR-122, miR-451a, miR-486, miR-150, miR-200b, and miR-142 each conjugated with a separate fluorophore and evaluated the dynamics of export. As a control, one cell-retained miRNA was also transfected with the pool of candidate miRNAs and the fluorescence of transfected cells was monitored at different times. Our results indicate that of all miRNAs, the fluorescence specific to miR-122 and miR-150 diminishes in less than 72 hours while others are retained longer, indicating that all miRNAs are not sorted or exported similarly. EV-RNA release was validated by treating the cells with an inhibitor of EV biogenesis i.e., nSMase inhibitor Gw4869. The inhibitor caused the cells to retain the candidate fluorescence corresponding to the respective EV-RNAs. To investigate sequence similarities between the miRNAs exclusively enriched in EVs of NSCLC cells, multiple sequence alignment analysis (STREME) was conducted, and specific motif sequences overrepresented in EVs and lost in the cells were shortlisted. Multiple lines of evidence indicate that dysregulated RNA subsets are not only causal agents of cancer progression but also contribute to poor patient outcome. Hence, there is a critical need to evaluate EV export of these RNAs as a potential mechanism of RNA dysregulation in cancer. Resulting data from this study is expected to not only reveal mechanistic details of EV-RNA export but will also inform future therapeutic targets. Citation Format: Humna Hasan, Nadia A. Lanman, Sagar Utturkar, Jeremiah J. Jauch, Ikjot S. Sohal, Zulaida Soto-Vargas, Andrea L. Kasinski. Mediators of RNA sorting and export in non-small cell lung cancer derived extracellular vesicles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3761.

Broken up but still living together: how ARGONAUTE's retention of cleaved fragments explains its role during chromatin modification.

Throughout the eukaryotic kingdoms, small RNAs direct chromatin modification. ARGONAUTE proteins sit at the nexus of this process, linking the small RNA information to the programming of chromatin. ARGONAUTE proteins physically incorporate the small RNAs as guides to target specific regions of the genome. In this issue of Genes & Development, Wang and colleagues (pp. 103-118) add substantial new detail to the processes of ARGONAUTE RNA loading, preference, cleavage, and retention, which together accomplish RNA-directed chromatin modification. They show that after catalytic cleavage by the plant ARGONAUTE protein AGO4, the cleaved fragment remains bound. This happens during two distinct RNA cleavage reactions performed by AGO4: first for a passenger RNA strand of the siRNA duplex, and second for a nascent transcript at the target DNA locus. Cleaved fragment retention of the nascent transcript explains how the protein complex accumulates to high levels at the target locus, amplifying chromatin modification.

Construction of Fusion Protein for Enhanced Small RNA Loading to Extracellular Vesicles.

Extracellular vesicles (EVs) naturally carry cargo from producer cells, such as RNA and protein, and can transfer these messengers to other cells and tissue. This ability provides an interesting opportunity for using EVs as delivery vehicles for therapeutic agents, such as for gene therapy. However, endogenous loading of cargo, such as microRNAs (miRNAs), is not very efficient as the copy number of miRNAs per EV is quite low. Therefore, new methods and tools to enhance the loading of small RNAs is required. In the current study, we developed fusion protein of EV membrane protein CD9 and RNA-binding protein AGO2 (hCD9.hAGO2). We show that the EVs engineered with hCD9.hAGO2 contain significantly higher levels of miRNA or shRNA (miR-466c or shRNA-451, respectively) compared to EVs that are isolated from cells that only overexpress the desired miRNA or shRNA. These hCD9.hAGO2 engineered EVs also transfer their RNA cargo to recipient cells more efficiently. We were not able to detect changes in gene expression levels in recipient cells after the EV treatments, but we show that the cell viability of HUVECs was increased after hCD9.hAGO2 EV treatments. This technical study characterizes the hCD9.hAGO2 fusion protein for the future development of enhanced RNA loading to EVs.

Abstract 1537: Understanding role of uniquely enriched RNAs carried in non-small cell lung cancer derived extracellular vesicles and dynamics of their selective export

Abstract The dynamic interaction mediated by extracellular vesicles (EVs) between cancer cells and microenvironment has been shown to regulate cancer progression. Specifically, tumor derived EVs alter the phenotypes of recipient cells by delivering functional biomolecules including RNAs (EV-RNA). Studies from our lab reveal the impact of NSCLC derived EVs (Calu6 and H358) in promoting invasion of non-tumorigenic cells (BEAS-2B) and disruption of an epithelial barrier. We further determined that of all the major macromolecules contained in EVs, EV-RNA is a significant contributor to the observed phenotypes. Additionally, our data supports dysregulation of RNA subsets enclosed in NSCLC cell-derived EVs in comparison with non-tumorigenic EVs. Interestingly, RNA sequencing analysis revealed preferential enrichment of several small RNAs (<200nt), especially miRNAs, in EVs isolated from NSCLC cell lines which suggest the presence of precise mechanisms involved in loading and export of RNAs into EVs. We shortlisted a cohort of uniquely enriched miRNAs in EVs derived from Calu6 and H358 cell lines to understand their combinatorial effects on non-tumorigenic recipient cells. Amongst the shortlisted candidates miR-100, miR-10b, miR-21, miR-155 and miR-486 stand out as potential contributors to NSCLC-EV mediated function. To understand the dynamics of export of miRNAs into EVs, we transfected Calu6 cells with one of the uniquely enriched fluorescently labelled candidate miRNA (miR-451) and tracked its export into EVs. We first verified that inclusion of a fluorophore to the RNA does not impair RNA loading into EVs. To set the experiment up, one EV-enriched RNA and one Cell-enriched RNA (as a control) conjugated with different fluorophores were transfected into cells, and cellular and EV fluorescence were monitored. Interestingly, cellular fluorescence corresponding to the EV-enriched RNA diminished, while fluorescence corresponding to the Cell-enriched RNA was retained. Fluorescence signal for EV-enriched RNA showed retention in EVs isolated 48 hours after transfection. Validation of release of candidate miRNA into EVs was performed by inhibiting biosynthesis of EVs after treatment with nSMase inhibitor Gw4869. Inhibition of EV release caused EV-enriched RNA to be retained by the cells which was confirmed following flow cytometry analysis of treated cells. Despite clear evidence that dysregulated EV-RNA subsets enclosed in cancer cell-derived EVs can modulate the cellular microenvironment, research showing dynamics and mechanisms of their export is limiting. Hence, there is a critical need to reveal mechanistic details of RNA export into EVs. Resulting data from this study is expected to not only reveal functional EV-RNA subsets and dynamics of their loading but will ultimately point to novel targets for future therapeutic intervention. Citation Format: Humna Hasan, Nadia A. Lanman, Sagar Utturkar, Andrea L. Kasinski. Understanding role of uniquely enriched RNAs carried in non-small cell lung cancer derived extracellular vesicles and dynamics of their selective export [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1537.

AtRsmD Is Required for Chloroplast Development and Chloroplast Function in Arabidopsis thaliana.

AtRsmD was recently demonstrated to be a chloroplast 16S rRNA methyltransferase (MTase) for the m2G915 modification in Arabidopsis. Here, its function of AtRsmD for chloroplast development and photosynthesis was further analyzed. The AtRsmD gene is highly expressed in green photosynthetic tissues. AtRsmD is associated with the thylakoid in chloroplasts. The atrsmd-2 mutant exhibited impaired photosynthetic efficiency in emerging leaves under normal growth conditions. A few thylakoid lamellas could be observed in the chloroplast from the atrsmd-2 mutant, and these thylakoids were loosely organized. Knockout of the AtRsmD gene had minor effects on chloroplast ribosome biogenesis and RNA loading on chloroplast ribosomes, but it reduced the amounts of chloroplast-encoded photosynthesis-related proteins in the emerging leaves, for example, D1, D2, CP43, and CP47, which reduced the accumulation of the photosynthetic complex. Nevertheless, knockout of the AtRsmD gene did not cause a general reduction in chloroplast-encoded proteins in Arabidopsis grown under normal growth conditions. Additionally, the atrsmd-2 mutant exhibited more sensitivity to lincomycin, which specifically inhibits the elongation of nascent polypeptide chains. Cold stress exacerbated the effect on chloroplast ribosome biogenesis in the atrsmd-2 mutant. All these data suggest that the AtRsmD protein plays distinct regulatory roles in chloroplast translation, which is required for chloroplast development and chloroplast function.

Robust delivery of RIG-I agonists using extracellular vesicles for anti-cancer immunotherapy.

The RIG‐I pathway can be activated by RNA containing 5′ triphosphate, leading to type I interferon release and immune activation. Hence, RIG‐I agonists have been used to induce immune responses against cancer as potential immunotherapy. However, delivery of 5′ triphosphorylated RNA molecules as RIG‐I agonists to tumour cells in vivo is challenging due to the susceptibility of these molecules to degradation. In this study, we demonstrate the use of extracellular vesicles (EVs) from red blood cells (RBCs), which are highly amenable for RNA loading and taken up robustly by cancer cells, for RIG‐I agonist delivery. We evaluate the anti‐cancer activity of two novel RIG‐I agonists, the immunomodulatory RNA (immRNA) with a unique secondary structure for efficient RIG‐I activation, and a 5′ triphosphorylated antisense oligonucleotide with dual function of RIG‐I activation and miR‐125b inhibition (3p‐125b‐ASO). We find that RBCEV‐delivered immRNA and 3p‐125b‐ASO trigger the RIG‐I pathway, and induce cell death in both mouse and human breast cancer cells. Furthermore, we observe a significant suppression of tumour growth coupled with increased immune cell infiltration mediated by the activation of RIG‐I cascade after multiple intratumoral injections of RBCEVs loaded with immRNA or 3p‐125b‐ASO. Targeted delivery of immRNA using RBCEVs with EGFR‐binding nanobody administrated via intrapulmonary delivery facilitates the accumulation of RBCEVs in metastatic cancer cells, leading to potent tumour‐specific CD8+ T cells immune response. This contributes to prominent suppression of breast cancer metastasis in the lung. Hence, this study provides a new strategy for efficient RIG‐I agonist delivery using RBCEVs for immunotherapy against cancer and cancer metastasis.

ER membrane contact sites: key platforms for biogenesis of RNA-containing extracellular vesicles.

The mechanisms by which cytoplasmic cargoes such as RNAs are incorporated into extracellular vesicles (EVs) are poorly understood. In a recent article published in Developmental Cell, we describe a novel function of endoplasmic reticulum membrane contact sites (ER MCS) in regulating biogenesis of RNA-containing EVs (Barman et al., 2022). We identified the ER MCS tether protein VAP-A and the ceramide transporter CERT as key drivers of this process. VAP-A depletion and overexpression produced corresponding changes in the overall number and RNA content of secreted EVs. Further sub-fractionation of small EVs from VAP-A depleted cells revealed a distinct loss in a specific subset of dense, RNA-loaded small EVs that are critical for the transfer of miR-100 to recipient cells. Cell imaging data confirmed the loss of RNA and RNA binding proteins (RBPs) in VAP-A-knockdown multivesicular bodies. Lipid analysis of VAP-A-knockdown EVs revealed decreases in ceramides, which are known to affect EV biogenesis. Depletion of the ceramide transfer protein CERT, which interacts with its binding partner VAP-A at ER MCS, leads to similar defects in EV number and RNA content as VAP-A-knockdown. These data suggest a model for ER MCS as platforms for biogenesis of a key EV population via ceramide transfer and RNA loading.

PiRNAs initiate transcriptional silencing of spermatogenic genes during C.elegans germline development.

SummaryEukaryotic genomes harbor invading transposable elements that are silenced by PIWI-interacting RNAs (piRNAs) to maintain genome integrity in animal germ cells. However, whether piRNAs also regulate endogenous gene expression programs remains unclear. Here, we show that C. elegans piRNAs trigger the transcriptional silencing of hundreds of spermatogenic genes during spermatogenesis, promoting sperm differentiation and function. This silencing signal requires piRNA-dependent small RNA biogenesis and loading into downstream nuclear effectors, which correlates with the dynamic reorganization of two distinct perinuclear biomolecular condensates present in germ cells. In addition, the silencing capacity of piRNAs is temporally counteracted by the Argonaute CSR-1, which targets and licenses spermatogenic gene transcription. The spatial and temporal overlap between these opposing small RNA pathways contributes to setting up the timing of the spermatogenic differentiation program. Thus, our work identifies a prominent role for piRNAs as direct regulators of endogenous transcriptional programs during germline development and gamete differentiation.