Keap1 recognizes EIAV early accessory protein Rev to promote antiviral defense

The Nrf2/Keap1 axis plays a complex role in viral susceptibility, virus-associated inflammation and immune regulation. However, whether or how the Nrf2/Keap1 axis is involved in the interactions between equine lentiviruses and their hosts remains unclear. Here, we demonstrate that the Nrf2/Keap1 axis was activated during EIAV infection. Mechanistically, EIAV-Rev competitively binds to Keap1 and releases Nrf2 from Keap1-mediated repression, leading to the accumulation of Nrf2 in the nucleus and promoting Nrf2 responsive genes transcription. Subsequently, we demonstrated that the Nrf2/Keap1 axis represses EIAV replication via two independent molecular mechanisms: directly increasing antioxidant enzymes to promote effective cellular resistance against EIAV infection, and repression of Rev-mediated RNA transport through direct interaction between Keap1 and Rev. Together, these data suggest that activation of the Nrf2/Keap1 axis mediates a passive defensive response to combat EIAV infection. The Nrf2/Keap1 axis could be a potential target for developing the strategies for combating EIAV infection.

The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions

RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two color imaging directly revealed single mRNAs transported by single KIF1C motors, with the 3’UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal a widespread RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role in transporting RNAs and clustering them within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA suggesting a possible feedback loop acting at the level of mRNA transport.

Abstract P347: Transcriptional Features Of Biological Age Maintained In Cultured Cardiac Interstitial Cells

Introduction: Ex vivo expansion of cells is necessary in regenerative medicine to generate large populations for therapeutic use. Adaptation to culture conditions prompt an increase in transcriptome diversity and decreased population heterogeneity in cKit+ cardiac interstitial cells (cCICs). The “transcriptional memory” influenced by cellular origin remains unexplored and is likely to differ between neonatal versus senescent input cells undergoing culture expansion. Approach: cCICs isolated from neonatal and adult cardiac tissue (Left Ventricular Assist Device; LVAD). Single cell libraries from in vitro expanded cells were prepared following five passages as previously demonstrated by our group to promote transcriptional homogeneity. “Transcriptional memory” was surveyed via bioinformatic analysis including unsupervised clustering, differential expression analysis, gene ontology and pathway analysis. Transitional states were assessed through pseudotime analysis. Results: Cell cycle imprint associated with biological age after culture was observed in Neonatal cCICs via upregulation of G2M genes. LVAD derived cCICs retained a widespread senescent profile, in particular high expression of interleukins and elements of the senescence associated secretory phenotype (SASP). Nuclear pore complex TPR and UBC9 and translation initiation factors, displayed age-associated downregulation of elements in the RNA transport and processing pathway. Pathway and co-expression analysis of fibroblast markers Ddr2, Tcf21, Vimentin, Periostin and Collagen deposition markers indicated a primed fibrotic phenotype in senescent cells. A small subset of cCICs exist in a transcriptional continuum between “youthful” phenotype and the damaged microenvironment of adult tissue in which they were embedded. Conclusion: The influence of age, pathology and the cellular stress associated to the in vivo tissue microenvironment persist after culture adaptation, influencing targets of 1) cell cycle, 2) senescence associated secretory phenotype (SASP), 3) RNA transport, and 4) ECM-receptor/fibrosis. The connate transcriptional phenotypes offer fundamental biological insight and highlights cellular input as a consideration in culture expansion and adoptive transfer protocols.

RNAs and extracellular vesicles - Keeping up the appearances

Since the advent of extracellular vesicle (EV) research in the last decade, these particles have been associated with RNAs. Traded as promising new biomarkers, RNA transport vehicles, or ultimately as potential therapeutic RNA delivery vehicles. However, this view is currently undergoing a change in which RNA may no longer be a major component of EVs. In this short opinion paper, we would like to encourage a reconsideration of our view on EVs and RNAs and open it up to new thoughts.

Concatemeric Broccoli reduces mRNA stability and induces aggregates

Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.

An active RNA transport mechanism into plant vacuoles

RNA degradation inside the plant vacuole by the ribonuclease RNS2 is essential for maintaining nucleotide concentrations and cellular homeostasis via the nucleotide salvage pathway. However, the mechanisms by which RNA is transported into the vacuole are not well understood. While selective macroautophagy may contribute to this transport, macroautophagy-independent transport pathways also exist. Here we demonstrate a mechanism for direct RNA transport into vacuoles that is active in purified vacuoles and is ATP hydrolysis-dependent. We identify the RNA helicase SKI2 as a factor required for this transport pathway, as ski2 mutant vacuoles are defective in transport. ski2 mutants have an increased autophagy phenotype that can be rescued by exogenous addition of nucleosides, consistent with a function in nucleotide salvage. This newly-described transport mechanism is therefore critical for RNA degradation, recycling and cytoplasmic nucleotide homeostasis.

Construction and Analysis of Coexpression Network to Understand Biological Responses in Chickens Infected by Eimeria tenella

Coccidiosis, caused by various Eimeria species, is a major parasitic disease in chickens. Our understanding of how chickens respond to coccidian infections is highly limited at both the molecular and cellular levels. In this study, coexpression modules were identified by weighted gene coexpression network analysis in chickens infected with Eimeria tenella. A total of 15 correlation modules were identified using 5,175 genes with 24 chicken samples, 12 with primary and 12 with secondary E. tenella infection. The analysis of the interactions between these modules showed a high degree of scale independence. Gene Ontology and Kyoto Encyclopedia of Gene and Genomes enrichment analyses revealed that genes in these functional modules were involved in a broad categories of functions, such as immune response, amino acid metabolism, cellular responses to lipids, sterol biosynthetic processes, and RNA transport. Two modules viz yellow and magenta were identified significantly associating with infection status. Preservation analysis showed that most of the modules identified in E. tenella infections were highly or moderately preserved in chickens infected with either Eimeria acervulina or Eimeria maxima. These analyses outline a biological responses landscape for chickens infected by E. tenella, and also indicates that infections with these three Eimeria species elicit similar biological responses in chickens at the system level. These findings provide new clues and ideas for investigating the relationship between parasites and host, and the control of parasitic diseases.

Opposing roles for Egalitarian and Staufen in transport, anchoring and localization of oskar mRNA in the Drosophila oocyte

Localization of oskar mRNA includes two distinct phases: transport from nurse cells to the oocyte, a process typically accompanied by cortical anchoring in the oocyte, followed by posterior localization within the oocyte. Signals within the oskar 3’ UTR directing transport are individually weak, a feature previously hypothesized to facilitate exchange between the different localization machineries. We show that alteration of the SL2a stem-loop structure containing the oskar transport and anchoring signal (TAS) removes an inhibitory effect such that in vitro binding by the RNA transport factor, Egalitarian, is elevated as is in vivo transport from the nurse cells into the oocyte. Cortical anchoring within the oocyte is also enhanced, interfering with posterior localization. We also show that mutation of Staufen recognized structures (SRSs), predicted binding sites for Staufen, disrupts posterior localization of oskar mRNA just as in staufen mutants. Two SRSs in SL2a, one overlapping the Egalitarian binding site, are inferred to mediate Staufen-dependent inhibition of TAS anchoring activity, thereby promoting posterior localization. The other three SRSs in the oskar 3’ UTR are also required for posterior localization, including two located distant from any known transport signal. Staufen, thus, plays multiple roles in localization of oskar mRNA.

RNA Motifs and Modification Involve in RNA Long-Distance Transport in Plants

A large number of RNA molecules have been found in the phloem of higher plants, and they can be transported to distant organelles through the phloem. RNA signals are important cues to be evolving in fortification strategies by long-distance transportation when suffering from various physiological challenges. So far, the mechanism of RNA selectively transportation through phloem cells is still in progress. Up to now, evidence have shown that several RNA motifs including Polypyrimidine (poly-CU) sequence, transfer RNA (tRNA)-related sequence, Single Nucleotide Mutation bound with specific RNA binding proteins to form Ribonucleotide protein (RNP) complexes could facilitate RNA mobility in plants. Furthermore, some RNA secondary structure such as tRNA-like structure (TLS), untranslation region (UTR) of mRNA, stem-loop structure of pre-miRNA also contributed to the mobility of RNAs. Latest researchs found that RNA methylation such as methylated 5′ cytosine (m5C) played an important role in RNA transport and function. These studies lay a theoretical foundation to uncover the mechanism of RNA transport. We aim to provide ideas and clues to inspire future research on the function of RNA motifs in RNA long-distance transport, furthermore to explore the underlying mechanism of RNA systematic signaling.

Intertissue small RNA communication mediates the acquisition and inheritance of hormesis in Caenorhabditis elegans

Environmental conditions can cause phenotypic changes, part of which can be inherited by subsequent generations via soma-to-germline communication. However, the signaling molecules or pathways that mediate intertissue communication remain unclear. Here, we show that intertissue small RNA communication systems play a key role in the acquisition and inheritance of hormesis effects – stress-induced stress resistance – in Caenorhabditis elegans. The miRNA-processing enzyme DRSH-1 is involved in both the acquisition and the inheritance of hormesis, whereas worm-specific Argonaute (WAGO) proteins, which function with endo-siRNAs, are involved only in its inheritance. Further analyses demonstrate that the miRNA production system in the neuron and the small RNA transport machinery in the intestine are both essential for its acquisition and that both the transport of small RNAs in the germline and the germline Argonaute HRDE-1 complex are required for its inheritance. Our results thus demonstrate that overlapping and distinct roles of small RNA systems in the acquisition and inheritance of hormesis effects.