Papers by Shulamit Michaeli
Journal of Bacteriology, 1984
Transcription of the metA gene of Escherichia coli K-12 is from a promoter which is under methion... more Transcription of the metA gene of Escherichia coli K-12 is from a promoter which is under methionine control and is located next to a region which has an extensive sequence homology with the operator regions of the metBL and metF genes. However, in the metA gene there is a second transcription start point which is located 74 base pairs upstream and which is independent of the intracellular methionine concentration.
Nano-Structures & Nano-Objects
Journal of Nanomedicine & Nanotechnology, 2016
Nucleic Acids Research, 2019
The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an inse... more The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an insect and a mammalian host. Here, we investigated the presence of pseudouridines (Ψs) on the spliceosomal small nuclear RNAs (snRNAs), which may enable growth at the very different temperatures characterizing the two hosts. To this end, we performed the first high-throughput mapping of spliceosomal snRNA Ψs by small RNA Ψ-seq. The analysis revealed 42 Ψs on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding Ψ on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA Ψs are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth ...
The nucleolus is a sub-nuclear compartment whose primary function is the biogenesis of ribosomal ... more The nucleolus is a sub-nuclear compartment whose primary function is the biogenesis of ribosomal subunits. Certain viral infections affect the morphology and composition of the nucleolar compartment and influence ribosomal RNA (rRNA) transcription and maturation. However, no description of nucleolar morphology and function during infection with Kaposi’s sarcoma-associated herpesvirus (KSHV) is available to date. Using immunofluorescence microscopy, we documented extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV. This was manifested by redistribution of key nucleolar proteins, including the rRNA transcription factor, UBF, the essential pre-rRNA processing factor Fibrillarin, and the nucleolar multifunctional phosphoproteins Nucleophosmin (NPM1) and Nucleolin. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programme...
Acinetobacter species, Acinetobacter calcoaceticus RA57,was isolated bystandard enrichment cultur... more Acinetobacter species, Acinetobacter calcoaceticus RA57,was isolated bystandard enrichment culture techniques on thebasis ofitsability toutilize theoily sludge foundinthevicinity ofalocal gasstation. Strain RA57was foundtocontain fourplasmids: pSR1(5.1 kilobases [kb]), pSR2(5.4 kb),pSR3 (10.5 kb),andpSR4(20kb).Bothsupercoiled andopen circular formsofthefirst threeplasmids were identified bytwo-dimensional gelelectrophoresis. Restriction endonuclease analysis ofpSR4demonstrated that theplasmid contained acircular map. Colonies wereisolated atrandomafter growth inthepresenceofacridine orangeandfound tofall into twocategories: (i) those whichhadlost theability togrow on anddisperse crude oilinliquid culture andconcurrently were cured ofpSR4and(ii) those whichretained theability tobothgrow on anddisperse crudeoilandwhichcontained pSR4.Strains fromthefirst class continued togrow on hydrocarbon vapors,indicating thatthedefect associated withthecuring ofpSR4was related tothephysical intera...
Bioconjugate Chemistry, 2021
Leishmaniasis is among the five parasitic diseases that still require the development of new drug... more Leishmaniasis is among the five parasitic diseases that still require the development of new drugs. Ultrasmall cerium (Ce3/4+) cation-doped maghemite (γ-Fe2O3) nanoparticles (NPs) were tested as a potential drug to treat visceral leishmaniasis, a disease affecting millions of people worldwide. The NPs were engineered for binding a polycationic branched polyethylenimine (PEI) polymer, thereby rupturing the single lysosome of these parasites and enabling entry of the anti-Leishmania drug, pentamidine. Exploiting the known lanthanide cation/complex-based coordinative chemical reactivity enabled the binding of both active agents onto the surface of the NPs. To optimize the fabrication of the cytotoxic NPs, optimization via a DoE (Design of Experiments) process was used to identify the optimal NP with toxicity against the two stages of the parasite, promastigotes, which propagate in the insect, and amastigotes, which infect the mammalian host. The screen identified a single optimized NP (DoE Opt) that was further examined in a mouse model of visceral leishmaniasis. Intravenous injection of the NPs had no adverse effects on the cellular composition or biochemical parameters of the blood, demonstrating no signs of systemic toxicity. The optimized NP was able to eradicate visceral disease caused by Leishmania donovani infection. The study demonstrates the versatile ability of the cerium-doped NPs to bind at least two cytotoxic ligands. This approach could be used for optimizing the binding of different drugs for the treatment of other diseases, including cancer. Since resistance to treatment with nanocarriers was not reported to date, such an approach could potentially overcome drug resistance that emerges when using soluble small molecule drugs.
Biomaterials Science, 2021
Exosomes are promising vectors for anti-tumor therapy. In this research, both in-vivo CT tracking... more Exosomes are promising vectors for anti-tumor therapy. In this research, both in-vivo CT tracking and ex-vivo measurements revealed better tumor targeting, accumulation and penetration of MSC-derived exosomes as compared to A431-derived exosomes.
Diamond and Related Materials, 2020
We present a novel method for aqueous effective disaggregation, dispersion, and stabilization of ... more We present a novel method for aqueous effective disaggregation, dispersion, and stabilization of detonation nanodiamonds (NDs) that also allows easy further second-step nanodiamond (ND) functionalization/surface engineering through lanthanide-based coordination chemistry. This method includes ultrasonic irradiation of NDs in the presence of a strong mono-electronic ceric ammonium nitrate (CAN, [Ce(IV)(NH 4) 2 (NO 3) 6 ]) oxidant. The resulting CAN-treated NDs are positively charged with lanthanide [CeL n ] 3/4+ complexes/cations, enabling an anti-aggregation effect together with the ability to be further surface-modified through [CeL n ] 3/4+ ligand exchange (lanthanide coordinative chemistry). Therefore, this method produces~10 nm-sized CAN-modified nanoparticles (NDs-CAN NPs) that are highly positively charged (ξ potential maximal value: +45.7 mV & average zeta potential: +34.6 mV). The obtained ND surface modification by [CeL n ] 3/4+ complexes/cations enabled an organic-type coordination attachment of various different organic molecules. This innovative way of dealing with the well-known ND aggregation phenomenon enables a novel way for the development of a wide range of biomedical, imaging, and diagnostic-related ND-based applications.
Journal of Biological Chemistry, 2019
The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein... more The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein 1 (TEP1), is found in many eukaryotes. However, previous studies of vtRNAs, for example in mammalian cells, have failed to reach a definitive conclusion about their function. vtRNAs are related to Y RNAs, which are complexed with Ro protein and influence Ro's function in noncoding RNA (ncRNA) quality control and processing. In Trypanosoma brucei, the small noncoding TBsRNA-10 was first described in a survey of the ncRNA repertoire in this organism. Here, we report that TBsRNA-10 in T. brucei is a vtRNA, based on its association with TEP1 and sequence similarity to those of other known and predicted vtRNAs. We observed that like vtRNAs in other species, TBsRNA-10 is transcribed by RNA polymerase III, which in trypanosomes also generates the spliceosomal U-rich small nuclear RNAs. In T. brucei, spliced leader (SL)mediated trans-splicing of pre-mRNAs is an obligatory step in gene expression, and we found here that T. brucei's vtRNA is highly enriched in a non-nucleolar locus in the cell nucleus implicated in SL RNP biogenesis. Using a newly developed permeabilized cell system for the bloodstream form of T. brucei, we show that down-regulated vtRNA levels impair transspliced mRNA production, consistent with a role of vtRNA in trypanosome mRNA metabolism. Our results suggest a common theme for the functions of vtRNAs and Y RNAs. We conclude that by complexing with their protein-binding partners TEP1 and Ro, respectively, these two RNA species modulate the metabolism of various RNA classes.
<p><b>(A) Identification of proteins purified by affinity-selection</b>. Cells ... more <p><b>(A) Identification of proteins purified by affinity-selection</b>. Cells containing the <i>SmD1</i> silencing construct were induced for 48h, as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006245#ppat.1006245.ref009" target="_blank">9</a>]. An extract was prepared from 2×10<sup>9</sup> cells. The extract was separated on a Superdex 200 column, and SL RNA containing fractions were subjected to affinity selection as described in Materials and Methods. Proteins obtained from the control experiment lacking the selecting oligonucleotide (-Oligo) and proteins from the affinity selected particles (+Oligo) were extracted from the streptavidin beads, separated on a 12% acrylamide SDS gel, and stained with silver. <b>(B) <i>ZC3H41</i>silencing.</b> Cell lines expressing the <i>ZC3H41</i> stem-loop silencing construct were induced for 48 hrs. Cells (~10<sup>6</sup> cells/ lane) were subjected to western analysis using ZC3H41 and PTB1 antibodies. <b>(C) <i>ZC3H41</i> is an essential gene for trypanosome survival.</b> Cells were either induced (+Tet) or un-induced (-Tet), and growth was monitored. The arrow indicates the time of tetracycline addition. The number of un-induced cells is designated by triangles, and of induced cells by squares. <b>(D) ZC3H41 binds loosely to SL RNA.</b> Cells expressing TAP-Myc-His- ZC3H41 fusion protein and the <i>SmD1</i>-silencing construct were silenced for 48 hrs. Cells (1.5×10<sup>9</sup>) were UV irradiated, as described in Materials and Methods. Extracts prepared from control (-UV) cells, and cells following UV irradiation were affinity selected on IgG beads. The RNA was extracted from the beads and analyzed by primer extension with SL and U3 RNAs specific primers. T, Total extract (5%); S, supernatant after removing the IgG beads (5%); P, the entire RNA sample bound to beads. The position of the cap-4 modification is indicated. <b>(E) Depletion of ZC3H41 in <i>SmE/ ZC3H41</i> silenced cells.</b> Western analysis was performed, as described in panel B. <b>(F) Levels of SL RNA under <i>SmE</i> and <i>SmE/ ZC3H41</i> silencing.</b> 10 μg of total RNA was subjected to primer extension with primers specific to SL RNA, U4, and U3 snoRNAs (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006245#ppat.1006245.s011" target="_blank">S1 Table</a>). The extension products were separated on a 6% denaturing gel. The identity of the cell line and the position of the modified cap nts are indicated. The statistical analysis represents the mean ± s.e.m of quantification from three independent experiments. **<i>P</i> <0.01, and ***<i>P</i> <0.005 compared to–Tet, using Student's <i>t</i>-test. <b>(G) Changes in localization of ZC3H41 and SL RNA during <i>SmD1</i> silencing.</b> Cells carrying the <i>SmD1</i> silencing construct were induced for the time indicated and subjected to <i>in situ</i> hybridization with SL RNA (red) and IFA with ZC3H41 antibodies (green). The nucleus was stained with DAPI. The merge was performed on DAPI staining and SL RNA hybridization and the time points of silencing are indicated.</p
Extracellular vesicles (EVs) isolated from pathogens mediate communication between parasites and ... more Extracellular vesicles (EVs) isolated from pathogens mediate communication between parasites and their hosts under a variety of physiological and pathological conditions. EVs deliver cell-free messages via a transfer of RNA, proteins, and even DNA to modulate and induce inflammation and to control the host infection process. EVs can provide valuable information on how a pathogen sends messages to other pathogens and hosts (Torrecilhas et al., 2012; Campos et al., 2015; OfirBirin and Regev-Rudzki, 2019; Torrecilhas et al. 2020). This Research Topic provides an overview of the mechanism of EV-mediated communication between hosts and viruses, parasites, and fungi. This Research Topic consists of 17 papers, including 8 reviews and 9 original papers. Several studies on this topic assessed the effects of EVs on the interactions between pathogens and hosts, and the mechanisms of EV-mediated communication between hosts and pathogens were also addressed. The first original article published ...
Molecular and Biochemical Parasitology, 2009
Early in the assembly of eukaryotes the branch-point binding protein (BBP, also called SF1) recog... more Early in the assembly of eukaryotes the branch-point binding protein (BBP, also called SF1) recognizes the branch point sequence, whereas the heterodimer U2AF, consisting of a 65 and a 35 kDa subunit, contacts the polypyrimidine tract and the AG splice site, respectively. Herein, we identified, cloned and expressed the Trypanosoma cruzi and Trypanosoma brucei U2AF35, U2AF65 and SF1. Trypanosomatid U2AF65 strongly diverged from yeast and human homologues. On the contrary, trypanosomatid SF1 was conserved but lacked the C-terminal sequence present in the mammalian protein. Yeast two hybrid approaches were used to assess their interactions. The interaction between U2AF35 and U2AF65 was very weak or not detectable. However, as in other eukaryotes, the interaction between U2AF65 and SF1 was strong. At the cellular level, these results were confirmed by fractionation and affinity-selection experiments in which SF1 and U2AF65 were affinity-selected with TAP tagged SF1, but not with TAP tagged U2AF35. Silencing one of the three factors affected growth and trans-splicing in the first step of this reaction. Trypanosomes are the first described example of eukaryotic cells in which the interaction of two expressed U2AF factors seemed to be very weak, or not detectable.
RNA, 2005
Small nucleolar RNAs (snoRNAs) constitute newly discovered noncoding small RNAs, most of which fu... more Small nucleolar RNAs (snoRNAs) constitute newly discovered noncoding small RNAs, most of which function in guiding modifications such as 2′-O-ribose methylation and pseudouridylation on rRNAs and snRNAs. To investigate the genome organization of Trypanosoma brucei snoRNAs and the pattern of rRNA modifications, we used a whole-genome approach to identify the repertoire of these guide RNAs. Twenty-one clusters encoding for 57 C/D snoRNAs and 34 H/ACA-like RNAs, which have the potential to direct 84 methylations and 32 pseudouridines, respectively, were identified. The number of 2′-O-methyls (Nms) identified on rRNA represent 80% of the expected modifications. The modifications guided by these RNAs suggest that trypanosomes contain many modifications and guide RNAs relative to their genome size. Interestingly, ~40% of the Nms are species-specific modifications that do not exist in yeast, humans, or plants, and 40% of the species-specific predicted modifications are located in unique po...
RNA, 2009
Trypanosomatid genomes encode for numerous proteins containing an RNA recognition motif (RRM), bu... more Trypanosomatid genomes encode for numerous proteins containing an RNA recognition motif (RRM), but the function of most of these proteins in mRNA metabolism is currently unknown. Here, we report the function of two such proteins that we have named PTB1 and PTB2, which resemble the mammalian polypyrimidine tract binding proteins (PTB). RNAi silencing of these factors indicates that both are essential for life. PTB1 and PTB2 reside mostly in the nucleus, but are found in the cytoplasm, as well. Microarray analysis performed on PTB1 and PTB2 RNAi silenced cells indicates that each of these factors differentially affects the transcriptome, thus regulating a different subset of mRNAs. PTB1 and PTB2 substrates were categorized bioinformatically, based on the presence of PTB binding sites in their 5′ and 3′ flanking sequences. Both proteins were shown to regulate mRNA stability. Interestingly, PTB proteins are essential for trans-splicing of genes containing C-rich polypyrimidine tracts. P...
Prostaglandins & Other Lipid Mediators, 2003
The present study was conducted to utilize a double-stranded RNA (dsRNA) specific for cyclooxygen... more The present study was conducted to utilize a double-stranded RNA (dsRNA) specific for cyclooxygenase (COX) II and demonstrate inhibition of the expression of COX II protein and its product PGE. A 21-dsRNA specific for COX II was introduced by lipofectamine into a primary cell culture of bovine aortic coronary endothelial cells (BAECs). BAECs basally express COX I but not COX II, and COX II expression is only apparent after stimulation with phorbol 12-myristate acetate (PMA). We first demonstrated that the lipofected fluorescent dsRNA-COX II is accumulated and localized within the cultured cells. We then demonstrated gene silencing of PMA-induced COX II protein expression by dsRNA-COX II using immuno-histochemistry. Western blot analysis and radioimmunoassay were used to quantitate the percent of inhibition. It was found that lipofected dsRNA-COX II reduced the percent of PMA-induced COX II enzyme by 36% and PGE production by 40%. There was no demonstrable effect of dsRNA-COX II or PMA on COX I expression.
Nucleic Acids Research, 2012
The discovery of a plethora of small non-coding RNAs (ncRNAs) has fundamentally changed our under... more The discovery of a plethora of small non-coding RNAs (ncRNAs) has fundamentally changed our understanding of how genes are regulated. In this study, we employed the power of deep sequencing of RNA (RNA-seq) to examine the repertoire of ncRNAs present in small ribonucleoprotein particles (RNPs) of Trypanosoma brucei, an important protozoan parasite. We identified new C/D and H/ACA small nucleolar RNAs (snoRNAs), as well as tens of putative novel non-coding RNAs; several of these are processed from trans-spliced and polyadenylated transcripts. The RNA-seq analysis provided information on the relative abundance of the RNAs, and their 5 0-and 3 0-termini. The study demonstrated that three highly abundant snoRNAs are involved in rRNA processing and highlight the unique trypanosome-specific repertoire of these RNAs. Novel RNAs were studied using in situ hybridization, association in RNP complexes, and 'RNA walk' to detect interaction with their target RNAs. Finally, we showed that the abundance of certain ncRNAs varies between the two stages of the parasite, suggesting that ncRNAs may contribute to gene regulation during the complex parasite's life cycle. This is the first study to provide a whole-genome analysis of the large repertoire of small RNPs in trypanosomes.
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Papers by Shulamit Michaeli