Profiling of extracellular small RNAs highlights a strong bias towards non-vesicular secretion

ABSTRACTExtracellular environment consists of a plethora of different molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, it is generally accepted that most of the biological activity is attributed to EV-associated miRNAs. The capability of EVs to transport cargoes has attracted much interest towards developing EVs as therapeutic short RNA carriers by using endogenous loading strategies for miRNA enrichment. Here, by overexpressing miRNA and shRNA sequences of interest in source cells and using size exclusion liquid chromatography (SEC) to separate the cellular secretome into EV and non-EV fractions, we saw that strikingly, <2% of all secreted overexpressed miRNA were found in association with EVs. To see whether the prominent non-EV miRNA secretion also holds true at the basal expression level of native miRNA transcripts, both fractions were further analysed by small RNA sequencing. This revealed a global correlation of EV and non-EV miRNA abundance to that of their parent cells and showed an enrichment only for miRNAs with a relatively low cellular expression level. Further quantification showed that similarly to the transient overexpression context, an outstanding 96.2-99.9% of total secreted miRNA at its basal level was secreted to the non-EV fraction. Yet, even though EVs contain only a fraction of secreted miRNAs, these molecules were found stable at 37°C in serum-containing environment, indicating that if sufficient miRNA loading to EVs is achieved, EVs can remain miRNA delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy can be a relatively wasteful way of loading miRNA to EVs and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.

Application of single nuclei RNA sequencing to assess the hepatic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Cell-specific transcriptional responses are lost in the averages of bulk RNA sequencing. We performed single nuclei RNA sequencing (snSeq) on frozen liver samples from male C57BL/6 mice in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Approximately 19,907 hepatic genes were detected across 16,015 sequenced nuclei from control and treated samples. Eleven cell-(sub)types were identified including distinct hepatocyte sub-populations, consistent with the cell diversity of the liver. TCDD increased macrophages from 0.5% to 24.7%, while neutrophils were only present in treated samples. The number of differentially expressed genes correlated with the basal expression level of Ahr. In addition to expected functional enrichments within each cell-(sub)type, RAS signaling was enriched in nonparenchymal cells. snSeq also identified a Kupffer cell subtype highly expressing Gpnmb, consistent with a dietary NASH model. Overall, snSeq distinguished cell-specific transcriptional changes and population shifts consistent with the hepatotoxicity of TCDD.

Functional divergence and potential mechanisms of the duplicate recA genes in Myxococcus xanthus

RecA is a ubiquitous multifunctional protein for bacterial homologous recombination and SOS response activation. Myxococcus xanthus DK1622 possesses two recA genes, and their functions and mechanisms are almost unclear. Here, we showed that the transcription of recA1 (MXAN_1441) was less than one-tenth of recA2 (MXAN_1388). Expressions of the two recA genes were both induced by ultraviolet (UV) irradiation, but in different periods. Deletion of recA1 did not affect the growth, but significantly decreased the UV-irradiation survival, the homologous recombination ability, and the induction of the LexA-dependent SOS genes. Comparably, the deletion of recA2 markedly prolonged the lag phase for cellular growth and antioxidation of hydrogen peroxide, but did not change the UV-irradiation resistance and the SOS-gene inducibility. The two RecA proteins are both DNA-dependent ATPase enzymes. We demonstrated that RecA1, but not RecA2, had in vitro DNA recombination capacity and LexA-autolysis promotion activity. Transcriptomic analysis indicated that the duplicate RecA2 has evolved to mainly regulate the gene expressions for cellular transportation and antioxidation. We discuss the potential mechanisms for the functional divergence. This is the first time to clearly determine the divergent functions of duplicated recA genes in bacterial cells. The present results highlight that the functional divergence of RecA duplicates facilitates the exertion of multiple RecA functions.Author summaryMyxobacteria has a large-size genome, contains many DNA repeats that are rare in the prokaryotic genome. It encodes bacterial RecA that could promote recombination between homologous DNA sequences. How myxobacteria avoid the undesired recombination between DNA repeats in its genome is an interesting question. M. xanthus encodes two RecA proteins, RecA1 (MXAN_1441) and RecA2 (MXAN_1388). Both RecA1 and RecA2 shows more than 60% sequence consistency with E. coli RecA (EcRecA) and can partly restore the UV resistance of E. coli recA mutant. Here, our results proved their divergent functions of the two RecAs. RecA1 retains the ability to catalyze DNA recombination, but its basal expression level is very low. RecA2 basal expression level is high, but no recombination activity is detected in vitro. This may be a strategy for M. xanthus to adapt to more repetitive sequences in its genome and avoid incorrect recombination.HighlightsM. xanthus has two recAs, which are expressed with significantly different levels. Both recAs are inducible by UV irradiation, but in different stages.The absence of recA1 reduces bacterial UV-irradiation resistance, while the absence of recA2 delays bacterial growth and antioxidant capacity.RecA1 retains the DNA recombination and SOS induction abilities, while RecA2 has evolved to regulate the expression of genes for cellular transport and antioxidation.

Evidence for Phosphate Starvation of Rhizobia without Terminal Differentiation in Legume Nodules

Phosphate homeostasis is tightly modulated in all organisms, including bacteria, which harbor both high- and low-affinity transporters acting under conditions of fluctuating phosphate levels. It was thought that nitrogen-fixing rhizobia, named bacteroids, inhabiting root nodules of legumes are not phosphate limited. Here, we show that the high-affinity phosphate transporter PstSCAB, rather than the low-affinity phosphate transporter Pit, is essential for effective nitrogen fixation of Sinorhizobium fredii in soybean nodules. Symbiotic and growth defects of the pst mutant can be effectively restored by knocking out PhoB, the transcriptional repressor of pit. The pst homologs of representative rhizobia were actively transcribed in bacteroids without terminal differentiation in nodules of diverse legumes (soybean, pigeonpea, cowpea, common bean, and Sophora flavescens) but exhibited a basal expression level in terminally differentiated bacteroids (alfalfa, pea, and peanut). Rhizobium leguminosarum bv. viciae Rlv3841 undergoes characteristic nonterminal and terminal differentiations in nodules of S. flavescens and pea, respectively. The pst mutant of Rlv3841 showed impaired adaptation to the nodule environment of S. flavescens but was indistinguishable from the wild-type strain in pea nodules. Taken together, root nodule rhizobia can be either phosphate limited or nonlimited regarding the rhizobial differentiation fate, which is a host-dependent feature.

Abstract 238: ABCA1 and HDL3 are Required to Modulate Smooth Muscle Cells Trandifferentiation in a Myocardin-miR143/145-dependent Process

Cells of the artery wall may accumulate free cholesterol and cholesteryl esters becoming foam cells. Up to 50% of foam cells in human lesions originates from smooth muscle cells (SMCs). Arterial SMCs express the ATP binding cassette (ABC) transporter ABCA1 and, upon cholesterol loading, express macrophage markers and a phagocytic activity. To characterize the role of ABCA1 and HDL3 in this transdifferentiation process, we evaluated the phenotypic changes in SMCs isolated from wild type (WT) and ABCA1 knock out (KO) mice and how HDL3 affects these changes. Cholesterol loading causes the downregulation of the expression of SMC markers including ACTA2, alpha-tropomyosin and myosin heavy chain and increases the expression of macrophage-related genes such as CD68, Mac-2, SRB1, MMPs, ABCG1 and ABCA1. HDL3 treatment in WT cells is able to normalize the expression of ACTA2, while the expression of macrophage-related genes is reduced. On the contrary, the preventive effect of HDL3 is completely lost in ABCA1 KO cells. Interestingly, the presence of HDL3 does not differently affect neutral lipid accumulation in WT or ABCA1 KO cells but stimulates phospholipids removal only in WT cells. ApoAI addition does not reverse the phenotypic changes induced by cholesterol not only in KO but also in WT cells. Moreover, cholesterol loading reduces the expression of myocardin, the master SMC specific-transcriptional coactivator involved in SMC differentiation, by up to 55% (p<0.01 vs respective control) in both cell types. HDL3 normalizes myocardin levels in WT cells while it does not have any effect in ABCA1 KO cells. Similar results are obtained evaluating the levels of miR-143/145, which positively regulate myocardin. The basal expression level of KLF4, a myocardin repressor, is almost double in ABCA1 KO cells compared to WT. After cholesterol loading, KLF4 is slightly reduced in WT cells, while its expression is halved in ABCA1 KO cells. HDL3 restores KLF4 to basal levels in KO cells, but it further reduces them in WT cells. These results indicate that HDL3, modulating the miR143/145-myocardin axis in SMC, prevents the cholesterol-induced gene expression modification regardless of its cholesterol unloading capacity and the presence of ABCA1 is required.

Limited GADD45α expression and function in IL-1β toxicity towards insulin-producing cells.

Growth arrest and DNA damage-inducible (GADD) 45 proteins are regulators of cell death and survival. The proinflammatory cytokine IL-1β strongly increases the level of the transcript encoding GADD45α in rat insulin-producing INS-1E cells. The activation of Gadd45α gene is clearly dependent on JNK and NF-κB activation and the synthesis of the secondary mediator nitric oxide (NO). Interestingly, the observed twelve-fold increase in the GADD45α-coding transcript level is not followed by increased expression of GADD45α at the protein level. An analysis of IL-1β toxicity in INS-1E cells overexpressing GADD45α revealed no correlation between the GADD45α protein level and the sensitivity to IL-1β toxicity. These findings suggest that the potential engagement of GADD45α in IL-1β toxicity towards beta cells is limited to the effects induced by the basal expression level of this protein.