Search for: All records

Abstract Extracellular vesicles (EVs) are small membrane-bound vesicles that are released by most cells. EVs have been shown to transport molecules including proteins and various types of RNAs between cells of even different types. Furthermore, EV RNAs are shown to modulate gene expression in physiological and pathological conditions in recipient cells which can be utilized in therapeutics by engineering cells to enrich RNA of interest in EVs. However, how specific RNA species are enriched in EVs is a long-standing question in the field. Here, we used sequence features of RNAs to predict its enrichment in EVs. These features include length, nucleotide and dinucleotide frequencies, secondary structure information, number of exons, coding probability for non-coding RNAs as well as RNA binding protein (RBP) motifs. The model achieved a performance (AU-ROC: 90%, 77%) for circRNAs and mRNAs, respectively. Here, we present a web tool called, EV RNA Cargo Enrichment Prediction Tool (EVRCEPT), that allows users to predict likelihood of input RNA to be enriched into EVs. This tool will also provide the list of RBPs that are likely to interact with the input RNA and works with both linear and circular RNAs. This webtool, which is freely accessible athttps://euler.dbi.udel.edu/evrcept, will help understand extracellular RNA transport and guide the design of therapeutic RNAs to maximize their incorporation in EVs towards targeted personalized medicine. 

Background: Ewing’s sarcoma (EwS) is a pediatric bone and soft tissue cancer driven by the oncogenic fusion protein EWS::FLI1. Currently, EwS lacks targeted therapies, necessitating the identification of novel regulatory mechanisms. While the role of microRNAs and long non-coding RNAs has been explored in EwS, the presence and functional significance of circular RNAs (circRNAs) in EwS is not reported. This is the first study to report the presence and role of oncogenic circRNA, circZNF609 in EwS tumor progression. Methods: Expression of circZNF609 was validated in 5 different EwS cell lines using qPCR. Cellular localization of circZNF609 was identified using circFISH. Functional assays for proliferation, migration and apoptosis were performed in wild type and circZNF609 knocked down (KD) cell lines to confirm its oncogenic role. The impact of circZNF609 on EWS::FLI1 protein levels was confirmed using western blots, immunofluorescence, and polysome fractionation. Mechanistic insights were gained utilizing bioinformatic, dual-luciferase reporter assays, rescue experiments, and microscopy to identify and validate the circRNA-miRNA-mRNA regulatory axis. Results: We report the first identification of circZNF609 in EwS, demonstrating that its expression is EWS::FLI1-dependent. Functional analysis reveals that circZNF609 promotes cell proliferation and metastasis while inhibiting apoptosis. Mechanistically, circZNF609 acts as a molecular sponge for miR-145-5p. By sequestering this miRNA, circZNF609 prevents the translational repression of EWS::FLI1, thereby sustaining oncogenic signaling. Conclusions: These findings identify circZNF609 as a novel post-transcriptional regulator of EWS::FLI1 and establish its critical role in EwS pathogenesis. Our results suggest that targeting the circZNF609/miR-145-5p/EWS::FLI1 axis may offer a promising therapeutic strategy for EwS. 

U6 small nuclear RNA (U6 snRNA), a critical spliceosome component primarily found in the nucleus, plays a vital role in RNA splicing. Our previous study, using the simian immunodeficiency virus (SIV) macaque model, revealed an increase of U6 snRNA in plasma extracellular vesicles (EVs) in acute retroviral infection. Given the limited understanding of U6 snRNA dynamics across cells and EVs, particularly in SIV infection, this research explores U6 snRNA trafficking and its association with splicing proteins in the nucleus, cytoplasm, and EVs. We observed a redistribution of U6 snRNA from the nucleus to EVs post-infection, accompanied by distinct protein profile changes and alterations in nucleic acid metabolism and spliceosome pathways. In addition, U6 machinery proteins changed in cells and EVs in a contrasting manner. The redistribution of U6 and related proteins we observed could be part of a viral strategy to redirect host splicing machinery, suggesting that U6 may have regulatory roles and be part of retroviral infection signature. 

CircRNAs are a category of regulatory RNAs that have garnered significant attention in the field of regulatory RNA research due to their structural stability and tissue-specific expression. Their circular configuration, formed via back-splicing, results in a covalently closed structure that exhibits greater resistance to exonucleases compared to linear RNAs. The distinctive regulation of circRNAs is closely associated with several physiological processes, as well as the advancement of pathophysiological processes in several human diseases. Despite a good understanding of the biogenesis of circular RNA, details of their biological roles are still being explored. With the steady rise in the number of investigations being carried out regarding the involvement of circRNAs in various regulatory pathways, understanding the biological and clinical relevance of circRNA-mediated regulation has become challenging. Given the vast landscape of circRNA research in the development of the heart and vasculature, we evaluated cardiovascular system research as a model to critically review the state-of-the-art understanding of the biologically relevant functions of circRNAs. We conclude the review with a discussion of the limitations of current functional studies and provide potential solutions by which these limitations can be addressed to identify and validate the meaningful and impactful functions of circRNAs in different physiological processes and diseases. 

We present an efficient, effective, and economical approach, named E3technology, for proteomics sample preparation. By immobilizing silica microparticles into the polytetrafluoroethylene matrix, we develop a robust membrane medium, which could serve as a reliable platform to generate proteomics-friendly samples in a rapid and low-cost fashion. We benchmark its performance using different formats and demonstrate them with a variety of sample types of varied complexity, quantity, and volume. Our data suggest that E3technology provides proteome-wide identification and quantitation performance equivalent or superior to many existing methods. We further propose an enhanced single-vessel approach, named E4technology, which performs on-filter in-cell digestion with minimal sample loss and high sensitivity, enabling low-input and low-cell proteomics. Lastly, we utilized the above technologies to investigate RNA-binding proteins and profile the intact bacterial cell proteome.