New Research Hot Spot: Extracellular RNA

What is Extracellular RNA?

Extracellular RNA, abbreviated as exRNA, refers to RNA located outside of cells, including RNA in the environment as well as RNA found in the intercellular spaces or bodily fluids of multicellular organisms. ExRNA was initially discovered in plasma and serum, but due to the presence of RNAases in the extracellular space and bodily fluids, its discovery did not attract much attention from the scientific community at that time, as it was generally believed that exRNA could not exist stably. It wasn't until 2006 and 2007 when two groundbreaking studies respectively demonstrated that RNA exists within microvesicles and exosomes, which can be secreted into the extracellular environment, acting as signaling molecules that influence the behavior of recipient cells. This process can occur between adjacent cells as well as over long distances. These two studies opened the door to exRNA research and provided a new perspective for exploring intercellular signaling. Subsequently, exRNA was found to exist in almost all bodily fluids, including blood, saliva, urine, breast milk, cerebrospinal fluid, amniotic fluid, ascites, bile, and pleural effusions. Results from high-throughput sequencing also indicate that, apart from mRNA, exRNA includes various types of non-coding RNA, such as miRNA, piRNA, tRNA, lncRNA, nucleolar small RNA, and others. The widespread presence and diversity of exRNA suggest that it likely participates in important biological processes, regulating normal growth and development as well as the occurrence of cancer and diseases.

Schematic diagram of processing, release and delivery of extracellular RNA. (J, P, Tosar.; et al, 2021)

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Functions of Extracellular RNA

ExRNA was first reported in the culture medium of cultured mouse and human cells 50 years ago. These exRNAs circulate in various carriers, potentially serving as mediators for intercellular communication and playing a role in immune regulation. RNAseq analysis of plasma RNA indicates that microRNAs (miRNAs) are the most abundant RNA species in plasma, accounting for over 80% of total RNA reads in both mice and healthy humans. Other detectable plasma RNA species include rRNA, piRNA, mRNA, and tRNA, but in much lower quantities. miRNAs are a group of small (20–22 nt), single-stranded (ss), non-coding RNAs. The conventional role of intracellular miRNAs is to regulate gene expression by binding to the 3' untranslated region (3'-UTR) of target genes, leading to mRNA degradation and translation suppression. Thus, by affecting gene translation in the cytoplasm, miRNAs act as regulators of widespread biological processes such as inflammation and play a dominant role in both health and disease. In miRNA biogenesis, primary miRNAs (pri-miRNAs) are transcribed from gene loci and processed into precursor miRNAs (pre-miRNAs) by the nuclear RNase III enzyme Drosha. Pre-miRNAs are then exported to the cytoplasm and further cleaved by another RNase III, Dicer, to generate double-stranded (ds) miRNA duplexes. Once formed, ds miRNA duplexes are loaded onto Argonaute proteins (Agos). Subsequently, the passenger strand of the duplex is ejected and degraded, while the guide strand forms a RNA-induced silencing complex (RISC) with Ago2, which is the effector protein of RISC, and then targets genes by binding to their 3'-UTR.

As Plasma Carriers

Circulating RNAs such as miRNAs are packaged into various plasma carriers and carried by them, such as extracellular vesicles (EVs), high-density lipoproteins (HDL), and Ago-2. These large molecular complexes protect miRNAs from plasma RNase digestion and exhibit various biological activities by delivering their cargo miRNAs to target cells.

As Innate Immune Modulators

During critical illnesses such as sepsis, trauma, and ischemic injury in animals and humans, host cell nucleic acids such as DNA and RNA are released into the bloodstream. These extracellular endogenous nucleic acids can be easily recognized by various innate immune sensors located inside cells. Intracellular sensors such as TLR3 and MDA5 for dsRNA, RIG-I for blunt-ended short dsRNA, TLR7/8 for ssRNA, and TLR9 for DNA are present in the endosomal and cytosolic compartments. Studies in both in vitro and in vivo systems have shown that RNA extracts from organs (potentially containing single-stranded and double-stranded RNA as well as miRNA mimics) can act as innate immune activators, promoting the production of complement factor B, MAP kinases, cytokines, peritonitis, cardiac inflammation, and pro-coagulation tissue factors. Although endogenous exRNA may induce innate immune responses through TLR3, TLR7, and MyD88, exogenous miRNA mimics seem to signal only through TLR7 in vitro and in vivo.

Applications of Extracellular RNA

Potential of ncRNA Carried by EVs in Liquid Biopsy and Cancer Therapy

Extracellular vesicles (EVs) serve as crucial mediators of intercellular communication, closely associated with information exchange between tumor cells. Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) associated with EVs hold potential applications as cancer biomarkers and in cancer therapy. Researchers have observed significant phenotypic changes upon transferring lncRNAs and circRNAs via EVs to recipient tumor cells. These EV-carried ncRNAs exhibit tissue-specific expression, stability, and targeted delivery capabilities, showing promising potential in liquid biopsy and cancer therapy.

Novel Roles of Non-coding RNA in Small Extracellular Vesicles

Extracellular vesicles (EVs) play an important role in intercellular communication by transferring non-coding RNAs (ncRNAs) between cells. For instance, long non-coding RNAs can serve as potential diagnostic tools but can also mediate resistance to certain cancer-specific chemotherapy drugs. Additionally, piRNAs play a crucial role in maintaining genome integrity.

RNA Delivery by Extracellular Vesicles in Mammalian Cells and Its Applications

Extracellular vesicles, derived from intracellular compartments (exosomes) or plasma membrane shedding (microvesicles, apoptotic bodies), carry various biomolecules including RNA, proteins, lipids, and DNA, serving as reservoirs of biomarkers for various diseases. The RNA cargo of extracellular vesicles can alter gene expression and function in recipient cells.

Plasma Extracellular RNA as a Potential Predictor for Alzheimer's Disease During the Latent Period

Alzheimer's disease (AD) has a latent period of up to 15 years before symptomatic onset. Using SILVER-seq biopsy technology, a study analyzing the extracellular RNA (exRNA) profile in human plasma samples discovered that exRNA in blood could serve as a novel potential biomarker for AD. This research provides new insights into early prediction and diagnosis studies for AD patients.

RNA Associated with Neutrophil Extracellular Traps and LL37 Aggravates Psoriasis

Psoriasis is an inflammatory skin disease often characterized by intense neutrophil (PMN) infiltration and high levels of antimicrobial peptide LL37. The antimicrobial peptide LL37, complexed with DNA and RNA, exacerbates disease by acting on plasmacytoid dendritic cells. Research indicates that PMNs induce neutrophil extracellular traps (NETs) and cytokine responses, contributing to disease exacerbation.

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Reference

1. J, P, Tosar.; et al. Revisiting Extracellular RNA Release, Processing, and Function. Frontiers in Cardiovascular Medicine. 2021, 46(6): 438-445.