Double-stranded RNA uptake in Caenorhabditis elegans

dsRNA uptake from the environment is a common phenomenon in many invertebrates and can be harnessed for acquiring pest control. In the nematode Caenorhabditis elegans, a transmembrane protein named systemic RNAi deficient-2 (sid-2) is required for dsRNA uptake from the environment through the intestine. Furthermore, the tyrosine-protein kinase sid-3 promotes import dsRNA. However, the molecular mechanism underlying environmental RNA uptake and its natural phenotypic roles are largely unknown. Here I characterized the intracellular properties of SID-2 and its role during the nematode development. I determined SID-2 localization at the intestinal apical membrane and the trans-Golgi-network (TGN). This localization is dependent on sid-3 and its potential substrate viro-2, hence demonstrating a molecular interplay between the different SID proteins. The spatial organisation of SID-2 is conserved across species, suggesting a role for the TGN in environmental RNA uptake. By combing small RNA and transcriptome profiling with developmental studies I identified a novel role for systemic RNAi in worm morphology. A detailed phenotypical analysis ruled out a nutritional role for dsRNA uptake, implying that environmental RNA uptake affects morphology through gene regulation. These results uncover the first phenotypic role for the Sid pathway and identifies the TGN as a central cellular compartment for environmental dsRNA uptake

RNA Control of HIV-1 Particle Size Polydispersity

HIV-1, an enveloped RNA virus, produces viral particles that are known to be much more heterogeneous in size than is typical of non-enveloped viruses. We present here a novel strategy to study HIV-1 Viral Like Particles (VLP) assembly by measuring the size distribution of these purified VLPs and subsequent viral cores thanks to Atomic Force Microscopy imaging and statistical analysis. This strategy allowed us to identify whether the presence of viral RNA acts as a modulator for VLPs and cores size heterogeneity in a large population of particles. These results are analyzed in the light of a recently proposed statistical physics model for the self-assembly process. In particular, our results reveal that the modulation of size distribution by the presence of viral RNA is qualitatively reproduced, suggesting therefore an entropic origin for the modulation of RNA uptake by the nascent VLP

Management of pest insects and plant diseases by non-transformative RNAi

Since the discovery of RNA interference (RNAi), scientists have made significant progress towards the development of this unique technology for crop protection. The RNAi mechanism works at the mRNA level by exploiting a sequence-dependent mode of action with high target specificity due to the design of complementary dsRNA molecules, allowing growers to target pests more precisely compared to conventional agrochemicals. The delivery of RNAi through transgenic plants is now a reality with some products currently in the market. Conversely, it is also expected that more RNA-based products reach the market as non-transformative alternatives. For instance, topically applied dsRNA/siRNA (SIGS - Spray Induced Gene Silencing) has attracted attention due to its feasibility and low cost compared to transgenic plants. Once on the leaf surface, dsRNAs can move directly to target pest cells (e.g., insects or pathogens) or can be taken up indirectly by plant cells to then be transferred into the pest cells. Water-soluble formulations containing pesticidal dsRNA provide alternatives, especially in some cases where plant transformation is not possible or takes years and cost millions to be developed (e.g., perennial crops). The ever-growing understanding of the RNAi mechanism and its limitations has allowed scientists to develop non-transgenic approaches such as trunk injection, soaking, and irrigation. While the technology has been considered promising for pest management, some issues such as RNAi efficiency, dsRNA degradation, environmental risk assessments, and resistance evolution still need to be addressed. Here, our main goal is to review some possible strategies for non-transgenic delivery systems, addressing important issues related to the use of this technology

Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages

<p>Abstract</p> <p>Background</p> <p>Exosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages.</p> <p>Method</p> <p>Exosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy.</p> <p>Results</p> <p>RNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages.</p> <p>Conclusions</p> <p>Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.</p

Platelet Transcriptome Heterogeneity: A Role for RNA Uptake in Vascular Health and Disease

As our understanding of the platelet’s systemic role continues to expand beyond hemostasis and thrombosis, interrogation of the platelet’s ability to affect diverse biological processes is required. Studies of the platelet’s non-traditional roles have focused on developing our understanding of the platelet’s relation to specific disease phenotypes as well as elucidation of platelet characteristics, content, and function. The generic content, traditional function and heterogeneity of platelets have long been accepted; more ambiguous and controversial has been how these factors are interrelated. Investigation of platelet content revealed the presence of biologically functional RNA in anucleated platelets, the correlation of platelet RNA to distinct phenotypes, and the ability of platelets to transfer RNA to other vascular cells; however how these processes occur is unclear. To further interrogate platelet RNA processes, we utilized sorting and RNA sequencing to develop platelet subpopulation transcriptome profiles. We found that platelet heterogeneity extends to the platelet transcriptome: distinct RNA profiles exist dependent on platelet size. We hypothesized that this RNA heterogeneity is the result of RNA transfer between platelets and vascular cells. Using in vitro and in vivo modeling, we were able to show the novel ability of platelets to take up RNA from vascular cells, correlating to the unique functional profile associated with small platelet transcriptomes. These findings reveal a role for platelet RNA transfer in platelet RNA heterogeneity, with potential correlation to platelet functional diversity previously proposed. The ability of the platelet to bidirectionally transfer RNA within circulation has implications for vascular health and beyond

Entropic control of particle sizes during viral self-assembly

Morphologic diversity is observed across all families of viruses. Yet these supra-molecular assemblies are produced most of the time in a spontaneous way through complex molecular self-assembly scenarios. The modeling of these phenomena remains a challenging problem within the emerging field of Physical Virology. We present in this work a theoretical analysis aiming at highlighting the particular role of configuration entropy in the control of viral particle size distribution. Specializing this model to retroviruses like HIV-1, we predict a new mechanism of entropic control of both RNA uptake into the viral particle, and of the particle's size distribution. Evidence of this peculiar behavior has been recently reported experimentally

A phagocytic route for uptake of double-stranded RNA in RNAi.

RNA interference (RNAi) has a range of physiological functions including as a defence mechanism against viruses. To protect uninfected cells in a multicellular organism, not only a cell-autonomous RNAi response is required but also a systemic one. However, the route of RNA spread in systemic RNAi remains unclear. Here we show that phagocytosis can be a route for double-stranded RNA uptake. Double-stranded RNA expressed in Escherichia coli induces robust RNAi in Drosophila S2 cells, with effectiveness comparable to that of naked dsRNA. We could separate this phagocytic uptake route from that for RNAi induced by naked dsRNA. Therefore, phagocytic uptake of dsRNA offers a potential route for systemic spread of RNAi

Exosomes are natural carriers of exogenous siRNA to human cells in vitro

© 2013 Shtam et al. Background: Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication including shuttle RNA, mainly mRNA and microRNA. As exosomes naturally carry RNA between cells, these particles might be useful in gene cancer therapy to deliver therapeutic short interfering RNA (siRNA) to the target cells. Despite the promise of RNA interference (RNAi) for use in therapy, several technical obstacles must be overcome. Exogenous siRNA is prone to degradation, has a limited ability to cross cell membranes and may induce an immune response. Naturally occurring RNA carriers, such as exosomes, might provide an untapped source of effective delivery strategies. Results: This study demonstrates that exosomes can deliver siRNA to recipient cells in vitro. The different strategies were used to introduce siRNAs into human exosomes of various origins. The delivery of fluorescently labeled siRNA via exosomes to cells was confirmed using confocal microscopy and flow cytometry. Two different siRNAs against RAD51 and RAD52 were used to transfect into the exosomes for therapeutic delivery into target cells. The exosome-delivered siRNAs were effective at causing post-transcriptional gene silencing in recipient cells. Moreover, the exosome-delivered siRNA against RAD51 was functional and caused the massive reproductive cell death of recipient cancer cells. Conclusions: The results strongly suggest that exosomes effectively delivered the siRNA into the target cells. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated in vitro by the strong knockdown of RAD51, a prospective therapeutic target for cancer cells. The results give an additional evidence of the ability to use human exosomes as vectors in cancer therapy, including RNAi-based gene therapy. © 2013 Shtam et al.; licensee BioMed Central Ltd.Russian Federal Program “Scientific and Scientific-Pedagogical Personnel of Innovative Russia”, contract 14.740.11.0754 and Fellowship from the Administration of Leningrad region to Shtam T

Doctor of Philosophy

dissertationThe formed elements of blood perform essential functions and are necessary for survival. Red blood cells transport oxygen to tissues, platelets maintain hemostasis and leukocytes provide host defense. In addition to these autonomous roles, blood cells are able to communicate with one another to execute complex physiological processes. Platelet interactions with leukocytes and endothelium regulate diverse inflammatory responses. Platelet-monocyte interactions result in robust cytokine production in monocytes. Neutrophils receive signals from platelets which can result in transendothelial migration or neutrophil extracellular trap (NET) formation. Many of the mechanisms by which these processes are regulated have only been recently discovered. Here we report that platelets are able to sequester exogenous ribonucleic acid (eRNA). This uptake is time and dose dependent and is unique to platelets of the cells examined so far. Uptake of eRNA does not require activation of platelets or alter the ability of platelets to respond to thrombin. While the fate of this sequestered eRNA has yet to be determined, our observations represent the first report of this novel capacity of platelets. Our interest in platelet and leukocyte biology also led us to develop a genetic screen to identify genes required for NET formation. The nearly haploid human cell line, KBM-7, was determined to be competent to form NETs, and a retroviral gene-trap mutagenesis strategy was employed to generate a pool of mutants to assay for defects in NET formation. Due to technical limitations, the screen was unable to be executed as designed, but advances in bioinformatics may allow a modified version of the screen to be performed in the near future. Throughout the body of this work, we have used traditional techniques combined with modern innovations to interrogate the functions of platelets and leukocytes. The knowledge gained in these investigations will improve our understanding of these essential cells and provide new avenues for research and therapeutic interventions in the future