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.

Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring.

Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5' tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Injection of sperm small RNA fractions from MTX-treated males into normal fertilized eggs generated cranial cartilage defects in the offspring. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.

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.

A different transcriptional landscape sheds light on Russian sturgeon (Acipenser gueldenstaedtii) mechanisms to cope with bacterial infection and chronic heat stress.

Sturgeons are chondrostean fish of high economic value and critically endangered due to anthropogenic activities, which has led to sturgeon aquaculture development. Russian sturgeon (Acipenser gueldenstaedtii), the second most important species reared for caviar, is successfully farmed in subtropical countries, including Uruguay. However, during the Uruguayan summer, sturgeons face intolerable warmer temperatures that weaken their defences and favour infections by opportunistic pathogens, increasing fish mortality and farm economic losses. Since innate immunity is paramount in fish, for which the liver plays a key role, we used deep RNA sequencing to analyse differentially expressed genes in the liver of Russian sturgeons exposed to chronic heat stress and challenged with Aeromonas hydrophila. We assembled 149.615 unigenes in the Russian sturgeon liver transcriptome and found that metabolism and immune defence pathways are among the top five biological processes taking place in the liver. Chronic heat stress provoked profound effects on liver biological functions, up-regulating genes related to protein folding, heat shock response and lipid and protein metabolism to meet energy demands for coping with heat stress. Besides, long-term exposure to heat stress led to cell damage triggering liver inflammation and diminishing liver ability to mount an innate response to A. hydrophila challenge. Accordingly, the reprogramming of liver metabolism over an extended period had detrimental effects on fish health, resulting in weight loss and mortality, with the latter increasing after A. hydrophila challenge. To our knowledge, this is the first transcriptomic study describing how chronic heat-stressed sturgeons respond to a bacterial challenge, suggesting that liver metabolism alterations have a negative impact on the innate anti-bacterial response.

Different response of Acipenser gueldenstaedtii CRP/SAP and SAA to bacterial challenge and chronic thermal stress sheds light on the innate immune system of sturgeons.

Sturgeons are chondrostean fish critically endangered due to anthropogenic loss and degradation of natural habitat and overfishing for meat and caviar production. Consequently, sturgeon aquaculture has extensively developed lately, being Russian sturgeon (Acipenser gueldenstaedtii) the second most important species reared for caviar production. However, Russian sturgeon aquaculture in subtropical countries, such as Uruguay, confronts difficulties because fish have to endure excessive summertime warm temperatures, which weaken their innate defences facilitating opportunistic infections. To address this problem, we look for identifying putative acute phase proteins (APPs), which might be robust serum biomarkers of both infection and chronic thermal stress, applied to monitoring Russian sturgeon health status in farms. We focused on the C-Reactive Protein/Serum Amyloid P (CRP/SAP) pentraxin since the pentraxin family includes well-known APPs, better characterised in mammals than fish. We identified A.gueldenstaedtii CRP/SAP (AgCRP/SAP), as a member of the universal CRP/SAP pentraxin sub-family, and studied AgCRP/SAP involvement in sturgeon response to bacterial challenge and chronic thermal stress, in comparison with A. gueldenstaedtii Serum Amyloid A (AgSAA), a previously described positive APP. Results showed that AgCRP/SAP is a constitutive serum component that remained constant upon Aeromonas hydrophila challenge and chronic thermal stress. Contrastingly, serum AgSAA was subjected to regulation by bacterial and thermal stress challenges, showing a 50-fold increase and 3-fold decline in serum levels, respectively. Overall, results highlight the potential value of AgSAA, but not of AgCRP/SAP, as a biomarker of bacterial infection and the need to continue searching for robust chronic thermal stress biomarkers in sturgeons.

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).

Serum amyloid A is a positive acute phase protein in Russian sturgeon challenged with Aeromonas hydrophila.

The immune system of sturgeons, one of the most ancient and economically valuable fish worldwide, is poorly understood. The lack of molecular tools and data about infection biomarkers hinders the possibility to monitor sturgeon health during farming and detect infection outbreaks. To tackle this issue, we mined publicly available transcriptomic datasets and identified putative positive acute-phase proteins (APPs) of Russian sturgeons that could be induced by a bacterial infection and monitored using non-invasive methods. Teleost literature compelled us to focus on five promising candidates: hepcidin, a warm acclimation associated hemopexin, intelectin, serum amyloid A protein (SAA) and serotransferrin. Among them, SAA was the most upregulated protein at the mRNA level in the liver of sturgeons challenged with heat-inactivated or live Aeromonas hydrophila. To assess whether this upregulation yielded increasing SAA levels in circulation, we developed an in-house ELISA to quantify SAA levels in sturgeon serum. Circulating SAA rose upon bacterial challenge and positively correlated with hepatic saa expression. This is the first time serum SAA has been quantified in an Actinopterygii fish. Since APPs vary across different fish species, our work sheds light on sturgeon acute-phase response, revealing that SAA is a positive APP with potential value as infection biomarker.

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.

Russian sturgeon cultured in a subtropical climate shows weaken innate defences and a chronic stress response.

Russian sturgeon (Acipenser gueldenstaedtii) has been successfully farmed in Uruguay for the past ten years. However, during the Uruguayan summer fish endure high water temperatures and increased bacterial infections that threaten aquaculture. Our understanding of sturgeon's immune system and its interplay with environmental factors like temperature is almost unknown. This study analysed the way in which seasonal variations affect enzymatic blood components of Russian sturgeon's innate defences, including the serum alternative complement pathway (ACP), ceruloplasmin (Cp) and lysozyme activities. Results showed that summertime conditions in the farm altered these defences in different ways, inducing a significant decrease in ACP and Cp, and an increase in lysozyme. In addition, serum levels of total protein and cortisol decreased in summer, suggesting a chronic stress response was induced in parallel. Subsequently, we analysed whether the increase in water river temperature during summer could account for the observed results. To that end, we acclimated juvenile sturgeons to mild (18 °C) or warm (24 °C) temperatures for 37 days. Like in summer, sturgeons exposed to 24 °C showed lower levels of serum ACP, Cp and total proteins, together with a progressive decrease in body weight and increased fish mortality. Administration of an immunostimulant containing Se and Zn slightly reverted the temperature-induced effects on sturgeon's defences. Altogether, our study provides novel data on various physiological parameters of the Russian sturgeon and highlights the impact warm temperature has on stress and innate immunity in this chondrostean fish.