Dror Eliaz

Two splicing factors carrying serine-arginine motifs, TSR1 and TSR1IP, regulate splicing, mRNA stability, and rRNA processing in Trypanosoma brucei

In trypanosomes, mRNAs are processed by trans-splicing; in this process, a common exon, the spliced leader, is added to all mRNAs from a small RNA donor, the spliced leader RNA (SL RNA). However, little is known regarding how this process is regulated. In this study, we investigated the function of two serine-arginine-rich proteins, TSR1 and TSR1IP, implicated in trans-splicing in Trypanosoma brucei. Depletion of these factors by RNAi suggested their role in both cis- and trans-splicing. Microarray was used to examine the transcriptome of the silenced cells. The level of hundreds of mRNAs was changed, suggesting that these proteins have a role in regulating only a subset of T. brucei mRNAs. Mass-spectrometry analyses of complexes associated with these proteins suggest that these factors function in mRNA stability, translation, and rRNA processing. We further demonstrate changes in the stability of mRNA as a result of depletion of the two TSR proteins. In addition, rRNA defects were observed under the depletion of U2AF35, TSR1, and TSR1IP, but not SF1, suggesting involvement of SR proteins in rRNA processing.

Stabilizing RNA by the Sonochemical Formation of RNA Nanospheres

Biological macromolecules, including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. Unlike DNA, RNA is a more versatile molecule whose range in the cell is from 21 to thousands of nucleotides and is usually folded into stem and loop structures. RNA is unique in nanoscale fabrication due to its diversity in size, function, and structure. Because gene expression analysis is becoming a clinical reality and there is a need to collect RNA in minute amounts from clinical samples, keeping the RNA intact is a growing challenge. RNA samples are notoriously difficult to handle because of their highly labile nature and tendency to degrade even under controlled RNase-free conditions and maintenance in the cold. Silencing the RNA that induces the RNA interference is viewed as the next generation of therapeutics. The stabilization and delivery of RNA to cells are the major concerns in making siRNAs usable drugs. For the first time, ultrasonic waves are shown to convert native RNA molecules to RNA nanospheres. The creation of the nanobubbles is performed by a one-step reaction. The RNA nanospheres are stable at room temperature for at least one month. Additionally, the nanospheres can be inserted into mammalian cancer cells (U2OS). This research achieves: 1) a solution to RNA storage; and 2) a way to convert RNA molecules to RNA particles. RNA nanosphere formation is a reversible process, and by using denaturing conditions, the RNA can be refolded into intact molecules.

Sonochemical Synthesis of DNA Nanospheres

Biological macromolecules, including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. DNA nanotechnology is a subfield of nanotechnology that seeks to use the unique molecular-recognition properties of DNA and other nucleic acids to create novel, controllable structures of DNA. Chemically, DNA consists of two long polymers. DNA is normally a linear molecule, in that its axis is unbranched. Different results are obtained when DNA in aqueous solution and DNA in biological tissue are exposed to ultrasound. The influence of ultrasonic waves on native DNA molecules has been previously reported. In those studies, it was shown that 2 min of ultrasonication of an aqueous solution of DNA splits the DNA helix into fragments; this makes ultrasonication a useful and convenient tool for obtaining DNA fragments on a preparative scale. Here, we show, for the first time, that ultrasonic waves can be used to convert native DNA molecules to extremely stable DNA nanoparticles (DNA nanospheres, DNs). In addition, the genetic information that was encoded in the DNA nanospheres was successfully delivered to competent cells and to human U2OS cancer cells, and expressed in competent (E. coli) cells. Our fundamental research on the synthesis and characterization of sonochemically produced DNA nanospheres provides an estimate of the efficiency of the sonochemical process in converting the native DNA molecules to biologically active DNA nanospheres. Ultrasonic emulsification is a well-known process that occurs in biphasic systems. Emulsification is necessary for microcapsule formation. Micrometer-sized gasor liquid-filled micro/ nanospheres can be produced from various kinds of proteins such as bovine serum albumin (BSA), human serum albumin (HSA), hemoglobin (Hb), and from combination of proteins. The mechanism of the sonochemical formation of protein microspheres (PMs) has been discussed previously. The microspheres are formed by chemically crosslinking cysteine residues, which undergo oxidation by HO2 radicals formed around a micron-sized gas bubble or a nonaqueous droplet. The formation of S!S bonds is a direct result of the chemical effects of the ultrasound radiation on an aqueous medium. Our reaction involved the sonication of an aqueous solution of DNA and dodecane (or soya oil) in a 50 mL sonication cell for three minutes. Five kinds of DNA were used in this work: 1) genomic DNA extracted from cells, 2) genomic DNA extracted from leaves, 3) DNA plasmid, 4) linear DNA extracted from DNA plasmid, and 5) single-stranded DNA. For all five DNAs and for both organic solvents we obtained DNA nanospheres. No difference was found between DNA nanospheres filled with dodecane and those filled with soya oil. We further demonstrate that the denaturing conditions as well as denaturing agents, which are commonly used in DNA isolation, cannot destroy the dsDNA (double-stranded DNA) nanospheres of the four DNA’s, while the nanospheres obtained from ssDNA could disintegrate to re-form the individual starting molecules. The efficiency of the sonochemical method in converting native DNA to DNA nanospheres was analyzed by spectrophotometry (NanoDrop 1000 spectrophotometer). It was found that 73.6% of DNA was converted into nanospheres under air and 96% under argon. It is worth underlining that unlike PMs, which are formed only under air and not under argon, DNA nanospheres are formed under both. In order to be sure that the nanobubbles in the solution after sonication are DNA nanospheres and not a combination of fragmented DNA, EtBr dye solution (commonly used for DNA and RNA detection) was added to the product solution. Nanospheres were colored red; this means that the walls of the nanobubbles consisted of DNA molecules (see the Supporting Information). The morphology of the nanospheres in the solution was determined by using scanning environmental-electron microscopy (E-SEM) and light microscopy (Apo-Tome Zeiss1 microscope). For all five types of DNA, the same spherical structure was observed. In Figure 1A, an E-SEM image of the DNA nanospheres that were produced from DNA type I is presented. The spherical morphology of the nanospheres made of DNA is very similar to that of proteinaceous microspheres, but the diameter of the DNA nanospheres (280 nm) was much smaller than the 2500 nm seen for PMs. A large number of dsDNA (DNA type I) are presented in Figure 1B. The size distribution of three kinds of DN (from DNA types I, III, and V) was examined on a DLS apparatus. When a solution of native DNA molecules was sonicated, the DLS results yielded spheres with an average size varying from 290 to 486 nm. A table summarizing average sizes and electrical charges of DNA nanospheres versus DNA type is presented in the Supporting Information. The DNA nanospheres were found to have an electrical charge of !40.7 mV. The electrical charge (z-potential) of [a] Dr. U. Shimanovich, I. Vayman, Prof. Dr. A. Gedanken Department of Chemistry and Kanbar Laboratory for Nanomaterials Bar-Ilan University Center for Advanced Materials and Nanotechnology Bar-Ilan University Ramat-Gan 52900 (Israel) E-mail : gedanken@mail.biu.ac.il [b] D. Eliaz, A. Aizer, Prof. Dr. S. Michaeli, Dr. Y. Shav-Tal Mina and Everard Goodman Faculty of Life Sciences Bar-Ilan University Center for Advanced Materials and Nanotechnology Bar-Ilan University Ramat-Gan, 52900 (Israel) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201100009: Experimental preparation of DNs and the details of the instrumentation used in the current experiment are available in this section.

Tetracycline Nanoparticles as Antibacterial and Gene-Silencing Agents

The spread of antibiotic-resistant bacteria and parasites calls for the development of new therapeutic strategies with could potentially reverse this trend. Here, a proposal is presented to exploit a sonochemical method to restore the antibiotic activity of tetracycline (TTCL) against resistant bacteria by converting the antibiotic into a nanoparticulate form. The demonstrated sonochemical method allows nanoscale TTCL assembly to be driven by supramolecular hydrogen bond formation, with no further modification to the antibiotics chemical structure. It is shown that tetracycline nanoparticles (TTCL NPs) can act as antibacterial agents, both against TTCL sensitive and against resistant bacterial strains. Moreover, the synthesized antibiotic nanoparticles (NPs) can act as effective gene-silencing agents through the use of a TTCL repressor in Trypanosome brucei parasites. It is demonstrated that the NPs are nontoxic to human cells and T. brucei parasites and are able to release their monomer components in an active form in a manner that results in enhanced antimicrobial activity relative to a homogeneous solution of the precursor monomer. As the TTCL NPs are biocompatible and biodegradable, sonochemical formation of TTCL NPs represents a new promising approach for generation of pharmaceutically active nanomaterials.

Exosome secretion affects social motility in Trypanosoma brucei

Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite Trypanosoma brucei, exosome secretion is induced by stress that affects trans-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites.

Genome-wide analysis of small nucleolar RNAs of Leishmania major reveals a rich repertoire of RNAs involved in modification and processing of rRNA

Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. These organisms regulate their gene expression mainly at the post-transcriptional level and possess characteristic RNA processing mechanisms. In this study, we analyzed the complete repertoire of Leishmania major small nucleolar (snoRNA) RNAs by performing RNA-seq analysis on RNAs that were affinity-purified using the C/D snoRNA core protein, SNU13, and the H/ACA core protein, NHP2. This study revealed a large collection of C/D and H/ACA snoRNAs, organized in gene clusters generally containing both snoRNA types. Abundant snoRNAs were identified and predicted to guide trypanosome-specific rRNA cleavages. The repertoire of snoRNAs was compared to that of the closely related Trypanosoma brucei, and 80% of both C/D and H/ACA molecules were found to have functional homologues. The comparative analyses elucidated how snoRNAs evolved to generate molecules with analogous functions in both species. Interestingly, H/ACA RNAs have great flexibility in their ability to guide modifications, and several of the RNA species can guide more than one modification, compensating for the presence of single hairpin H/ACA snoRNA in these organisms. Placing the predicted modifications on the rRNA secondary structure revealed hypermodification regions mostly in domains which are modified in other eukaryotes, in addition to trypanosome-specific modifications.

A pseudouridylation switch in rRNA is implicated in ribosome function during the life cycle of Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa, undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector. However, little is known about how the parasite performs most molecular functions in such different environments. Here, we provide evidence for the intriguing possibility that pseudouridylation of rRNA plays an important role in the capacity of the parasite to transit between the insect midgut and the mammalian bloodstream. Briefly, we mapped pseudouridines (Ψ) on rRNA by Ψ-seq in procyclic form (PCF) and bloodstream form (BSF) trypanosomes. We detected 68 Ψs on rRNA, which are guided by H/ACA small nucleolar RNAs (snoRNA). The small RNome of both life cycle stages was determined by HiSeq and 83 H/ACAs were identified. We observed an elevation of 21 Ψs modifications in BSF as a result of increased levels of the guiding snoRNAs. Overexpression of snoRNAs guiding modification on H69 provided a slight growth advantage to PCF parasites at 30 °C. Interestingly, these modifications are predicted to significantly alter the secondary structure of the large subunit (LSU) rRNA suggesting that hypermodified positions may contribute to the adaption of ribosome function during cycling between the two hosts.

Transcriptome and proteome analyses and the role of atypical calpain protein and autophagy in the spliced leader silencing pathway in Trypanosoma brucei.

Under persistent ER stress, Trypanosoma brucei parasites induce the spliced leader silencing (SLS) pathway. In SLS, transcription of the SL RNA gene, the SL donor to all mRNAs, is extinguished, arresting trans‐splicing and leading to programmed cell death (PCD). In this study, we investigated the transcriptome following silencing of SEC63, a factor essential for protein translocation across the ER membrane, and whose silencing induces SLS. The proteome of SEC63‐silenced cells was analyzed with an emphasis on SLS‐specific alterations in protein expression, and modifications that do not directly result from perturbations in trans‐splicing. One such protein identified is an atypical calpain SKCRP7.1/7.2. Co‐silencing of SKCRP7.1/7.2 and SEC63 eliminated SLS induction due its role in translocating the PK3 kinase. This kinase initiates SLS by migrating to the nucleus and phosphorylating TRF4 leading to shut‐off of SL RNA transcription. Thus, SKCRP7.1 is involved in SLS signaling and the accompanying PCD. The role of autophagy in SLS was also investigated; eliminating autophagy through VPS34 or ATG7 silencing demonstrated that autophagy is not essential for SLS induction, but is associated with PCD. Thus, this study identified factors that are used by the parasite to cope with ER stress and to induce SLS and PCD.