Ribonuclease activity undermines immune sensing of naked extracellular RNA.

The plasma membrane and the membrane of endosomal vesicles are considered physical barriers preventing extracellular RNA uptake. While naked RNA can be spontaneously internalized by certain cells types, functional delivery of naked RNA into the cytosol has been rarely observed. Here we show that extracellular ribonucleases, mainly derived from cell culture supplements, have so far hindered the study of extracellular RNA functionality. In the presence of active ribonuclease inhibitors (RI), naked bacterial RNA is pro-inflammatory when spiked in the media of dendritic cells and macrophages. In murine cells, this response mainly depends on the action of endosomal Toll-like receptors. However, we also show that naked RNA can perform endosomal escape and engage with cytosolic RNA sensors and ribosomes. For example, naked mRNAs encoding reporter proteins can be spontaneously internalized and translated by a variety of cell types, in an RI-dependent manner. In vivo, RI co-injection enhances the activation induced by naked extracellular RNA on splenic lymphocytes and myeloid-derived leukocytes. Furthermore, naked extracellular RNA is inherently pro-inflammatory in ribonuclease-poor compartments such as the peritoneal cavity. Overall, these results demonstrate that naked RNA is bioactive and does not need encapsulation inside synthetic or biological lipid vesicles for functional uptake, making a case for nonvesicular extracellular RNA-mediated intercellular communication.

Small RNA structural biochemistry in a post-sequencing era.

High-throughput sequencing has had an enormous impact on small RNA research during the past decade. However, sequencing only offers a one-dimensional view of the transcriptome and is often highly biased. Additionally, the 'sequence, map and annotate' approach, used widely in small RNA research, can lead to flawed interpretations of the data, lacking biological plausibility, due in part to database issues. Even in the absence of technical biases, the loss of three-dimensional information is a major limitation to understanding RNA stability, turnover and function. For example, noncoding RNA-derived fragments seem to exist mainly as dimers, tetramers or as nicked forms of their parental RNAs, contrary to widespread assumptions. In this perspective, we will discuss main sources of bias during small RNA-sequencing, present several useful bias-reducing strategies and provide guidance on the interpretation of small RNA-sequencing results, with emphasis on RNA fragmentomics. As sequencing offers a one-dimensional projection of a four-dimensional reality, prior structure-level knowledge is often needed to make sense of the data. Consequently, while less-biased sequencing methods are welcomed, integration of orthologous experimental techniques is also strongly recommended.

Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids.

Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function, and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine, and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of several hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.

Exomeres and Supermeres: monolithic or diverse?

Extracellular vesicles (EVs), including exosomes and microvesicles, are far from being the only RNA-containing extracellular particles (EPs). Recently, new 35 nm-sized EPs were discovered by asymmetric-flow field-flow fractionation and termed "exomeres". Purification of exomeres was later performed by differential ultracentrifugation as well. More recently, the supernatant of the high-speed ultracentrifugation used to collect exomeres was further centrifuged to collect a new class of EP, termed "supermeres". Supermeres contain high quantities of extracellular RNA and are enriched in miR-1246. They are also replete in disease biomarkers and can induce metabolic and adaptive changes in recipient cells. Here, we reanalyzed proteomic and transcriptomic data obtained in this exciting study to obtain further insights into the molecular composition of exomeres and supermeres. We found that the top-ranking RNAs in supermeres correspond to the footprints of extracellular protein complexes. These complexes protect fragments of the small nuclear RNA U2 and the 28S rRNA from extracellular ribonucleases (exRNases). We suggest that intracellular nanoparticles such as the U2 ribonucleoprotein, ribosomes and LGALS3BP ring-like decamers are released into the extracellular space. These heterogeneous EPs might be further processed by exRNases and co-isolate by ultracentrifugation with other components of exomeres and supermeres. We look forward to continuing progress in defining exRNA carriers, bridging process definitions with molecular composition and function.

Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research.

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.

Socioeconomic, Clinical, and Molecular Features of Breast Cancer Influence Overall Survival of Latin American Women.

Molecular profile of breast cancer in Latin-American women was studied in five countries: Argentina, Brazil, Chile, Mexico, and Uruguay. Data about socioeconomic characteristics, risk factors, prognostic factors, and molecular subtypes were described, and the 60-month overall cumulative survival probabilities (OS) were estimated. From 2011 to 2013, 1,300 eligible Latin-American women 18 years or older, with a diagnosis of breast cancer in clinical stage II or III, and performance status ≦̸1 were invited to participate in a prospective cohort study. Face-to-face interviews were conducted, and clinical and outcome data, including death, were extracted from medical records. Unadjusted associations were evaluated by Chi-squared and Fisher's exact tests and the OS by Kaplan-Meier method. Log-rank test was used to determine differences between cumulative probability curves. Multivariable adjustment was carried out by entering potential confounders in the Cox regression model. The OS at 60 months was 83.9%. Multivariable-adjusted death hazard differences were found for women living in Argentina (2.27), Chile (1.95), and Uruguay (2.42) compared with Mexican women, for older (≥60 years) (1.84) compared with younger (≤40 years) women, for basal-like subtype (5.8), luminal B (2.43), and HER2-enriched (2.52) compared with luminal A subtype, and for tumor clinical stages IIB (1.91), IIIA (3.54), and IIIB (3.94) compared with stage IIA women. OS was associated with country of residence, PAM50 intrinsic subtype, age, and tumor stage at diagnosis. While the latter is known to be influenced by access to care, including cancer screening, timely diagnosis and treatment, including access to more effective treatment protocols, it may also influence epigenetic changes that, potentially, impact molecular subtypes. Data derived from heretofore understudied populations with unique geographic ancestry and sociocultural experiences are critical to furthering our understanding of this complexity.

The Transcriptomic Portrait of Locally Advanced Breast Cancer and Its Prognostic Value in a Multi-Country Cohort of Latin American Patients.

Purposes: Most molecular-based published studies on breast cancer do not adequately represent the unique and diverse genetic admixture of the Latin American population. Searching for similarities and differences in molecular pathways associated with these tumors and evaluating its impact on prognosis may help to select better therapeutic approaches. Patients and Methods: We collected clinical, pathological, and transcriptomic data of a multi-country Latin American cohort of 1,071 stage II-III breast cancer patients of the Molecular Profile of Breast Cancer Study (MPBCS) cohort. The 5-year prognostic ability of intrinsic (transcriptomic-based) PAM50 and immunohistochemical classifications, both at the cancer-specific (OSC) and disease-free survival (DFS) stages, was compared. Pathway analyses (GSEA, GSVA and MetaCore) were performed to explore differences among intrinsic subtypes. Results: PAM50 classification of the MPBCS cohort defined 42·6% of tumors as LumA, 21·3% as LumB, 13·3% as HER2E and 16·6% as Basal. Both OSC and DFS for LumA tumors were significantly better than for other subtypes, while Basal tumors had the worst prognosis. While the prognostic power of traditional subtypes calculated with hormone receptors (HR), HER2 and Ki67 determinations showed an acceptable performance, PAM50-derived risk of recurrence best discriminated low, intermediate and high-risk groups. Transcriptomic pathway analysis showed high proliferation (i.e. cell cycle control and DNA damage repair) associated with LumB, HER2E and Basal tumors, and a strong dependency on the estrogen pathway for LumA. Terms related to both innate and adaptive immune responses were seen predominantly upregulated in Basal tumors, and, to a lesser extent, in HER2E, with respect to LumA and B tumors. Conclusions: This is the first study that assesses molecular features at the transcriptomic level in a multicountry Latin American breast cancer patient cohort. Hormone-related and proliferation pathways that predominate in PAM50 and other breast cancer molecular classifications are also the main tumor-driving mechanisms in this cohort and have prognostic power. The immune-related features seen in the most aggressive subtypes may pave the way for therapeutic approaches not yet disseminated in Latin America. Clinical Trial Registration: ClinicalTrials.gov (Identifier: NCT02326857).

Population-based screening of Uruguayan Ashkenazi Jews for recurrent BRCA1 and BRCA2 pathogenic sequence variants.

In Ashkenazi Jews (AJ) three recurring pathogenic sequence variants (PSVs) are detected in ~2.5% of the general population in the BRCA1 (c.68_69del = 185delAG, c.5266dup = 5382insC), and BRCA2 (c.5946del = 6174delT). Population-based screening for these PSVs in AJ women is part of the health basket in Israel. To assess the feasibility and outcome of BRCA genotyping in the Jewish population of Uruguay, AJ in the greater Montevideo area were recruited using ethically approved protocol and without pretest counseling were genotyped for the three predominant AJ PSVs in the BRCA genes. Independently confirmed PSV carriers were counseled, and genetic testing was offered to additional family members. Overall, 327 participants were enrolled: 312 (95%) female, 261 (80%) had all four grandparents AJ, and 14 (4%) women were breast cancer survivors with a mean age ± standard deviation (SD) 50 ± 11.5 years. The BRCA1 c.68_69del PSV was detected in three cancer free participants (0.92%, CI 95% 0.31-2.6), all with a suggestive family history. No carriers of the other two recurrent PSVs were detected. Online oncogenetic counseling was provided for all carriers. In conclusion, the rate of the BRCA1 c.68_69del PSV was similar with the rate in other AJ communities. AJ population BRCA genotyping screens in Uruguay seem feasible and should be promoted.

RI-SEC-seq: Comprehensive Profiling of Nonvesicular Extracellular RNAs with Different Stabilities.

Exosomes and other extracellular vesicles (EVs) are considered the main vehicles transporting RNAs in extracellular samples, including human bodily fluids. However, a major proportion of extracellular RNAs (exRNAs) do not copurify with EVs and remain in ultracentrifugation supernatants of cell-conditioned medium or blood serum. We have observed that nonvesicular exRNA profiles are highly biased toward those RNAs with intrinsic resistance to extracellular ribonucleases. These highly resistant exRNAs are interesting from a biomarker point of view, but are not representative of the actual bulk of RNAs released to the extracellular space. In order to understand exRNA dynamics and capture both stable and unstable RNAs, we developed a method based on size-exclusion chromatography (SEC) fractionation of RNase inhibitor (RI)-treated cell-conditioned medium (RI-SEC-seq). This method has allowed us to identify and study extracellular ribosomes and tRNAs, and offers a dynamical view of the extracellular RNAome which can impact biomarker discovery in the near future. Graphical abstract: Overview of the RI-SEC-seq protocol: sequencing of size-exclusion chromatography fractions from nonvesicular extracellular samples treated or not with RNase inhibitors (+/- RI).

Revisiting Extracellular RNA Release, Processing, and Function.

It is assumed that RNAs enriched in extracellular samples were selected for release by their parental cells. However, recent descriptions of extracellular RNA (exRNA) biogenesis and their differential stabilities question this assumption, as they could produce identical outcomes. Here, we share our opinion about the importance of considering both selective and nonselective mechanisms for RNA release into the extracellular environment. In doing so, we provide new perspectives on RNA-mediated intercellular communication, including an analogy to communication through social media. We also argue that technical limitations have restricted the study of some of the most abundant exRNAs, both inside and outside extracellular vesicles (EVs). These RNAs may be better positioned to induce a response in recipient cells compared with low abundance miRNAs.

Circulating SNORD57 rather than piR-54265 is a promising biomarker for colorectal cancer: common pitfalls in the study of somatic piRNAs in cancer.

There is increasing interest among cancer researchers in the study of Piwi-interacting RNAs (piRNAs), a group of small RNAs important for maintaining genome stability in the germline. Aberrant expression of piRNAs in cancer could imply an involvement of these regulatory RNAs in neoplastic transformation. On top of that, it could enable early cancer diagnosis based on RNA analysis in liquid biopsies, as piRNAs are not expected to widely circulate in the bloodstream of healthy individuals. Indeed, it has recently been shown that serum piR-54265 allows for excellent discrimination between colorectal cancer patients and healthy controls. However, we have also shown that most somatic piRNAs reported to date in mammals are actually fragments of other noncoding RNAs. Herein, we show that reports positioning piR-54265 as a noninvasive biomarker for colorectal cancer were actually measuring variations in the levels of a full-length (72 nt) small nucleolar RNA in serum. This should place a cautionary note for future research in somatic and cancer-specific piRNAs. We deeply encourage this line of research but discuss proper ways to identify somatic piRNAs without the interference of erroneous entries contained in piRNA databases. We also introduce the concept of miscellaneous-piRNAs (m-piRNAs) to distinguish between canonical piRNAs and other small RNAs circumstantially associated with PIWI proteins in somatic cells.

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome.

A major proportion of extracellular RNAs (exRNAs) do not copurify with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes are probably not silent from an immunological perspective and could possibly be decoded as damage-associated molecular patterns.

Extracellular tRNAs and tRNA-derived fragments.

Fragmentation of tRNAs generates a family of small RNAs collectively known as tRNA-derived fragments. These fragments vary in sequence and size but have been shown to regulate many processes involved in cell homoeostasis and adaptations to stress. Additionally, the field of extracellular RNAs (exRNAs) is rapidly growing because exRNAs are a promising source of biomarkers in liquid biopsies, and because exRNAs seem to play key roles in intercellular and interspecies communication. Herein, we review recent descriptions of tRNA-derived fragments in the extracellular space in all domains of life, both in biofluids and in cell culture. The purpose of this review is to find consensus on which tRNA-derived fragments are more prominent in each extracellular fraction (including extracellular vesicles, lipoproteins and ribonucleoprotein complexes). We highlight what is becoming clear and what is still controversial in this field, in order to stimulate future hypothesis-driven studies which could clarify the role of full-length tRNAs and tRNA-derived fragments in the extracellular space.

Stable tRNA halves can be sorted into extracellular vesicles and delivered to recipient cells in a concentration-dependent manner.

Extracellular vesicles (EVs) are cell-derived nanoparticles that act as natural carriers of nucleic acids between cells. They offer advantages as delivery vehicles for therapeutic nucleic acids such as small RNAs. Loading of desired nucleic acids into EVs can be achieved by electroporation or transfection once purified. An attractive alternative is to transfect cells with the desired small RNAs and harness the cellular machinery for RNA sorting into the EVs. This possibility has been less explored because cells are believed to secrete only specific RNAs. However, we hypothesized that, even in the presence of selective secretion, concentration-driven RNA sorting to EVs would still be feasible. To show this, we transfected cells with glycine 5' tRNA halves, which we have previously shown to better resist RNases. We then measured their levels in EVs and in recipient cells and found that, in contrast to unstable RNAs of random sequence, these tRNA halves were present in vesicles and in recipient cells in amounts proportional to the concentration of RNA used for transfection. Similar efficiencies were obtained with other stable oligonucleotides of random sequence. Our results demonstrate that RNA stability is a key factor needed to maintain high intracellular concentrations, a prerequisite for efficient non-selective RNA sorting to EVs and delivery to cells. Given that glycine 5' tRNA halves belong to the group of stress-induced tRNA fragments frequently detected in extracellular space and biofluids, we propose that upregulation of extracellular tRNA fragments is consequential to cellular stress and might be involved in intercellular signalling.

Non-coding RNA fragments account for the majority of annotated piRNAs expressed in somatic non-gonadal tissues.

PIWI-interacting RNAs (piRNAs) are regarded as the guardians of the genome because they tackle genome stability-threatening transposable elements in the germline. Recently, piRNAs were also reported in other types of cells, including mouse brain, malignant and non-malignant somatic tissues, and human plasma. This suggests that piRNA function might be broader than previously expected. Here, we show that different piRNA databases contain a subset of sequences that correspond to piRNA-sized fragments of ncRNAs (rRNAs, tRNAs, YRNAs, snRNAs, and snoRNAs) and intermediates of miRNA biogenesis. We discuss that the biogenesis of these sequences is probably independent of the PIWI pathway, and can therefore be considered contaminants in piRNA databases. Although a minority of annotated piRNAs falls in this category, they account for the vast majority of piRNA expression in somatic non-gonadal tissues. Since ncRNA fragments are ubiquitous and abundant, their confusion with piRNAs strongly impacts the estimation of piRNA expression outside of mammalian gonads.

Dimerization confers increased stability to nucleases in 5' halves from glycine and glutamic acid tRNAs.

We have previously shown that 5' halves from tRNAGlyGCC and tRNAGluCUC are the most enriched small RNAs in the extracellular space of human cell lines, and especially in the non-vesicular fraction. Extracellular RNAs are believed to require protection by either encapsulation in vesicles or ribonucleoprotein complex formation. However, deproteinization of non-vesicular tRNA halves does not affect their retention in size-exclusion chromatography. Thus, we considered alternative explanations for their extracellular stability. In-silico analysis of the sequence of these tRNA-derived fragments showed that tRNAGly 5' halves can form homodimers or heterodimers with tRNAGlu 5' halves. This capacity is virtually unique to glycine tRNAs. By analyzing synthetic oligonucleotides by size exclusion chromatography, we provide evidence that dimerization is possible in vitro. tRNA halves with single point substitutions preventing dimerization are degraded faster both in controlled nuclease digestion assays and after transfection in cells, showing that dimerization can stabilize tRNA halves against the action of cellular nucleases. Finally, we give evidence supporting dimerization of endogenous tRNAGlyGCC 5' halves inside cells. Considering recent reports have shown that 5' tRNA halves from Ala and Cys can form tetramers, our results highlight RNA intermolecular structures as a new layer of complexity in the biology of tRNA-derived fragments.

Detection and Analysis of Non-vesicular Extracellular RNA.

Although extracellular vesicles (EVs) are by far the most studied carriers of extracellular small RNAs involved in cell-to-cell communication, most extracellular small RNAs are actually present as soluble vesicle-free supramolecular complexes. Several proteins have been described as the counterparts of these RNase-protected complexes. Here we describe a method for the purification and analysis of non-vesicular extracellular RNA derived from the conditioned media of mammalian cell culture. Focus on this fraction will increase our understanding on extracellular RNA biology, while serving as a source for biomarker discovery complementary to EVs.

Ribonucleic artefacts: are some extracellular RNA discoveries driven by cell culture medium components?

In a recently published study, Anna Krichevsky and colleagues raise the important question of whether results of in vitro extracellular RNA (exRNA) studies, including extracellular vesicle (EV) investigations, are confounded by the presence of RNA in cell culture medium components such as foetal bovine serum (FBS). The answer, according to their data, is a resounding "yes". Even after lengthy ultracentrifugation to remove bovine EVs from FBS, the majority of exRNA in FBS remained. Although technical factors may affect the degree of depletion, residual EVs and exRNA in FBS could influence the conclusions of in vitro studies: certainly, for secreted RNA, and possibly also for cell-associated RNA. In this commentary, we critically examine some of the literature in this field, including a recent study from some of the authors of this piece, in light of the Wei et al. study and explore how cell culture-derived RNAs may affect what we think we know about EV RNAs. These findings hold particular consequence as the field moves towards a deeper understanding of EV-RNA associations and potential functions.

Electrochemical Sandwich Immunosensor for Determination of Exosomes Based on Surface Marker-Mediated Signal Amplification.

Extracellular vesicles (EVs), namely, exosomes and microvesicles, are important mediators of intercellular communication pathways. Since EVs can be detected in a variety of biofluids and contain a specific set of biomarkers which are reminiscent of their parental cells, they show great promise in clinical diagnostics as EV analysis can be performed in minimally invasive liquid biopsies. However, reliable, fast and cost-effective methods for their determination are still needed, especially if decentralized analysis is intended. In this study, we developed an electrochemical biosensor which works with 1.5 µL sample volume and can detect as low as 200 exosomes per microliter, with a linear range spanning almost 4 orders of magnitude. The sensor is specific and readily differentiates exosomes from microvesicles in samples containing 1000-fold excess of the latter. Capability of detecting exosomes in real samples (diluted serum) was shown. This was achieved by immobilizing rabbit antihuman CD9 antibodies on gold substrates and using monoclonal antibodies against CD9 for detection of captured exosomes. Signal amplification is presumably obtained from the fact that multiple detector antibodies bind to the surface of each captured vesicle. Detection is performed based on electrochemical reduction of 3,3',5,5'-tetramethyl benzidine (TMB) after addition of horseradish peroxidase (HRP)-conjugated anti-IgG antibodies. This amperometric biosensor can be easily incorporated into future miniaturized and semiautomatic devices for EV determination.