Drosophila TRF2 is a preferential core promoter regulator.

Transcription of protein-coding genes is highly dependent on the RNA polymerase II core promoter. Core promoters, generally defined as the regions that direct transcription initiation, consist of functional core promoter motifs (such as the TATA-box, initiator [Inr], and downstream core promoter element [DPE]) that confer specific properties to the core promoter. The known basal transcription factors that support TATA-dependent transcription are insufficient for in vitro transcription of DPE-dependent promoters. In search of a transcription factor that supports DPE-dependent transcription, we used a biochemical complementation approach and identified the Drosophila TBP (TATA-box-binding protein)-related factor 2 (TRF2) as an enriched factor in the fractions that support DPE-dependent transcription. We demonstrate that the short TRF2 isoform preferentially activates DPE-dependent promoters. DNA microarray analysis reveals the enrichment of DPE promoters among short TRF2 up-regulated genes. Using primer extension analysis and reporter assays, we show the importance of the DPE in transcriptional regulation of TRF2 target genes. It was previously shown that, unlike TBP, TRF2 fails to bind DNA containing TATA-boxes. Using microfluidic affinity analysis, we discovered that short TRF2-bound DNA oligos are enriched for Inr and DPE motifs. Taken together, our findings highlight the role of short TRF2 as a preferential core promoter regulator.

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.

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.

Molecular assessment of the effect of light and heterotrophy in the scleractinian coral Stylophora pistillata.

Corals acquire nutrients via the transfer of photosynthates by their endosymbionts (autotrophy), or via zooplankton predation by the animal (heterotrophy). During stress events, corals lose their endosymbionts, and undergo starvation, unless they increase their heterotrophic capacities. Molecular mechanisms by which heterotrophy sustains metabolism in stressed corals remain elusive. Here for the first time, to the best of our knowledge, we identified specific genes expressed in heterotrophically fed and unfed colonies of the scleractinian coral Stylophora pistillata, maintained under normal and light-stress conditions. Physiological parameters and gene expression profiling demonstrated that fed corals better resisted stress than unfed ones by exhibiting less oxidative damage and protein degradation. Processes affected in light-stressed unfed corals (HLU), were related to energy and metabolite supply, carbohydrate biosynthesis, ion and nutrient transport, oxidative stress, Ca(2+) homeostasis, metabolism and calcification (carbonic anhydrases, calcium-transporting ATPase, bone morphogenetic proteins). Two genes (cp2u1 and cp1a2), which belong to the cytochrome P450 superfamily, were also upregulated 249 and 10 times, respectively, in HLU corals. In contrast, few of these processes were affected in light-stressed fed corals (HLF) because feeding supplied antioxidants and energetic molecules, which help repair oxidative damage. Altogether, these results show that heterotrophy helps prevent the cascade of metabolic problems downstream of oxidative stress.

Cellular transcriptome analysis reveals differential expression of pro- and antiapoptosis genes by varicella-zoster virus-infected neurons and fibroblasts.

Transcriptional changes following varicella-zoster virus (VZV) infection of cultured human neurons derived from embryonic stem cells were compared to those in VZV-infected human foreskin fibroblasts. Transcription of 340 neuronal genes significantly altered by VZV infection included 223 transcript changes unique to neurons. Strikingly, genes inhibiting apoptosis were upregulated in neurons, while proapoptotic gene transcription was increased in fibroblasts. These data are a basis for discovery of differences in virus-host interactions between these VZV targets.

Postnatal TLR2 activation impairs learning and memory in adulthood.

Neuroinflammation in the central nervous system is detrimental for learning and memory, as evident form epidemiological studies linking developmental defects and maternal exposure to harmful pathogens. Postnatal infections can also induce neuroinflammatory responses with long-term consequences. These inflammatory responses can lead to motor deficits and/or behavioral disabilities. Toll like receptors (TLRs) are a family of innate immune receptors best known as sensors of microbial-associated molecular patterns, and are the first responders to infection. TLR2 forms heterodimers with either TLR1 or TLR6, is activated in response to gram-positive bacterial infections, and is expressed in the brain during embryonic development. We hypothesized that early postnatal TLR2-mediated neuroinflammation would adversely affect cognitive behavior in the adult. Our data indicate that postnatal TLR2 activation affects learning and memory in adult mice in a heterodimer-dependent manner. TLR2/6 activation improved motor function and fear learning, while TLR2/1 activation impaired spatial learning and enhanced fear learning. Moreover, developmental TLR2 deficiency significantly impairs spatial learning and enhances fear learning, stressing the involvement of the TLR2 pathway in learning and memory. Analysis of the transcriptional effects of TLR2 activation reveals both common and unique transcriptional programs following heterodimer-specific TLR2 activation. These results imply that adult cognitive behavior could be influenced in part, by activation or alterations in the TLR2 pathway at birth.

The hnRNP F/H homologue of Trypanosoma brucei is differentially expressed in the two life cycle stages of the parasite and regulates splicing and mRNA stability.

Trypanosomes are protozoan parasites that cycle between a mammalian host (bloodstream form) and an insect host, the Tsetse fly (procyclic stage). In trypanosomes, all mRNAs are trans-spliced as part of their maturation. Genome-wide analysis of trans-splicing indicates the existence of alternative trans-splicing, but little is known regarding RNA-binding proteins that participate in such regulation. In this study, we performed functional analysis of the Trypanosoma brucei heterogeneous nuclear ribonucleoproteins (hnRNP) F/H homologue, a protein known to regulate alternative splicing in metazoa. The hnRNP F/H is highly expressed in the bloodstream form of the parasite, but is also functional in the procyclic form. Transcriptome analyses of RNAi-silenced cells were used to deduce the RNA motif recognized by this protein. A purine rich motif, AAGAA, was enriched in both the regulatory regions flanking the 3' splice site and poly (A) sites of the regulated genes. The motif was further validated using mini-genes carrying wild-type and mutated sequences in the 3' and 5' UTRs, demonstrating the role of hnRNP F/H in mRNA stability and splicing. Biochemical studies confirmed the binding of the protein to this proposed site. The differential expression of the protein and its inverse effects on mRNA level in the two lifecycle stages demonstrate the role of hnRNP F/H in developmental regulation.

Basal splicing factors regulate the stability of mature mRNAs in trypanosomes.

Gene expression in trypanosomes is mainly regulated post-transcriptionally. Genes are transcribed as polycistronic mRNAs that are dissected by the concerted action of trans-splicing and polyadenylation. In trans-splicing, a common exon, the spliced leader, is added to all mRNAs from a small RNA. In this study, we examined by microarray analysis the transcriptome following RNAi silencing of the basal splicing factors U2AF65, SF1, and U2AF35. The transcriptome data revealed correlations between the affected genes and their splicing and polyadenylation signaling properties, suggesting that differential binding of these factors to pre-mRNA regulates trans-splicing and hence expression of specific genes. Surprisingly, all these factors were shown to affect not only splicing but also mRNA stability. Affinity purification of SF1 and U2AF35 complexes supported their role in mRNA stability. U2AF35 but not SF1 was shown to bind to ribosomes. To examine the role of splicing factors in mRNA stability, mutations were introduced into the polypyrimidine tract located in the 3' UTR of a mini-gene, and the results demonstrate that U2AF65 binds to such a site and controls the mRNA stability. We propose that transcripts carrying splicing signals in their 3' UTR bind the splicing factors and control their stability.

Gene expression profiles during short-term heat stress in the red sea coral Stylophora pistillata.

During the past several decades, corals worldwide have been affected by severe bleaching events leading to wide-spread coral mortality triggered by global warming. The symbiotic Red Sea coral Stylophora pistillata from the Gulf of Eilat is considered an opportunistic 'r' strategist. It can thrive in relatively unstable environments and is considered a stress-tolerant species. Here, we used a S. pistillata custom microarray to examine gene expression patterns and cellular pathways during short-term (13-day) heat stress. The results allowed us to identify a two-step reaction to heat stress, which intensified significantly as the temperature was raised to a 32 °C threshold, beyond which, coping strategies failed at 34 °C. We identified potential 'early warning genes' and 'severe heat-related genes'. Our findings suggest that during short-term heat stress, S. pistillata may divert cellular energy into mechanisms such as the ER-unfolded protein response (UPR) and ER-associated degradation (ERAD) at the expense of growth and biomineralization processes in an effort to survive and subsequently recover from the stress. We suggest a mechanistic theory for the heat stress responses that may explain the success of some species which can thrive under a wider range of temperatures relative to others.

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.

A Cyanobacterial Component Required for Pilus Biogenesis Affects the Exoproteome.

Protein secretion as well as the assembly of bacterial motility appendages are central processes that substantially contribute to fitness and survival. This study highlights distinctive features of the mechanism that serves these functions in cyanobacteria, which are globally prevalent photosynthetic prokaryotes that significantly contribute to primary production. Our studies of biofilm development in the cyanobacterium Synechococcus elongatus uncovered a novel component required for the biofilm self-suppression mechanism that operates in this organism. This protein, which is annotated as "hypothetical," is denoted EbsA (essential for biofilm self-suppression A) here. EbsA homologs are highly conserved and widespread in diverse cyanobacteria but are not found outside this clade. We revealed a tripartite complex of EbsA, Hfq, and the ATPase homolog PilB (formerly called T2SE) and demonstrated that each of these components is required for the assembly of the hairlike type IV pili (T4P) appendages, for DNA competence, and affects the exoproteome in addition to its role in biofilm self-suppression. These data are consistent with bioinformatics analyses that reveal only a single set of genes in S. elongatus to serve pilus assembly or protein secretion; we suggest that a single complex is involved in both processes. A phenotype resulting from the impairment of the EbsA homolog in the cyanobacterium Synechocystis sp. strain PCC 6803 implies that this feature is a general cyanobacterial trait. Moreover, comparative exoproteome analyses of wild-type and mutant strains of S. elongatus suggest that EbsA and Hfq affect the exoproteome via a process that is independent of PilB, in addition to their involvement in a T4P/secretion machinery.IMPORTANCE Cyanobacteria, environmentally prevalent photosynthetic prokaryotes, contribute ∼25% of global primary production. Cyanobacterial biofilms elicit biofouling, thus leading to substantial economic losses; however, these microbial assemblages can also be beneficial, e.g., in wastewater purification processes and for biofuel production. Mechanistic aspects of cyanobacterial biofilm development were long overlooked, and genetic and molecular information emerged only in recent years. The importance of this study is 2-fold. First, it identifies novel components of cyanobacterial biofilm regulation, thus contributing to the knowledge of these processes and paving the way for inhibiting detrimental biofilms or promoting beneficial ones. Second, the data suggest that cyanobacteria may employ the same complex for the assembly of the motility appendages, type 4 pili, and protein secretion. A shared pathway was previously shown in only a few cases of heterotrophic bacteria, whereas numerous studies demonstrated distinct systems for these functions. Thus, our study broadens the understanding of pilus assembly/secretion in diverse bacteria and furthers the aim of controlling the formation of cyanobacterial biofilms.

Expression and chromosomal organization of mouse meiotic genes.

Microarray technology which enables large scale analysis of gene expression and thus comparison between transcriptomes of different cell types, cells undergoing different treatments or cells at different developmental stages has also been used to study the transcriptome involved with spermatogenesis. Many new germ cell-specific genes were determined, and the resulting genes were classified according to different criteria. However, the biological significance of these classifications and their clustering according to developmental transcriptional patterns during spermatogenesis have not yet been addressed. In this study we utilized mouse testicular transcriptome analysis at five distinct post-natal ages (Days 7, 10, 12, 14, and 17), representing distinct meiotic stages, in an attempt to better understand the biological significance of genes clustered into similar expression patterns during this process. Among 790 sequences that showed an expression level change of twofold or more in any of the five key stages that were monitored, relative to the geometric average of all stages, about 40% peaked and about 30% were specifically suppressed at post-natal day 14 (representing the early pachytene stage of spermatocytes), reflecting tight transcriptional regulation at this stage. We also found that each of the six main transcription clusters that were determined was characterized by statistically significant representation of genes related to specific biological processes. Finally, our results indicated that genes important for meiosis are not randomly distributed along the mouse genome but rather preferentially located on specific chromosomes, suggesting for the first time that chromosomal location might be a regulating factor of meiotic gene expression.

Human Pluripotent Stem Cell Fate Regulation by SMARCB1.

Epigenetic regulation by the SWI/SNF complex is essential for normal self-renewal capacity and pluripotency of human pluripotent stem cells (hPSCs). It has been shown that different subunits of the complex have a distinct role in this regulation. Specifically, the SMARCB1 subunit has been shown to regulate the activity of enhancers in diverse types of cells, including hPSCs. Here, we report the establishment of conditional hPSC lines, enabling control of SMARCB1 expression from complete loss of function to significant overexpression. Using this system, we show that any deviation from normal SMARCB1 expression leads to cell differentiation. We further found that SMARCB1 expression is not required for differentiation of hPSCs into progenitor cells, but rather for later stages of differentiation. Finally, we identify SMARCB1 as a critical player in regulation of cell-cell and cell-ECM interactions in hPSCs and show that this regulation is mediated at least in part by the WNT pathway.

Endogenous siRNAs promote proteostasis and longevity in germline-less Caenorhabditis elegans.

How lifespan and the rate of aging are set is a key problem in biology. Small RNAs are conserved molecules that impact diverse biological processes through the control of gene expression. However, in contrast to miRNAs, the role of endo-siRNAs in aging remains unexplored. Here, by combining deep sequencing and genomic and genetic approaches in Caenorhabditis elegans, we reveal an unprecedented role for endo-siRNA molecules in the maintenance of proteostasis and lifespan extension in germline-less animals. Furthermore, we identify an endo-siRNA-regulated tyrosine phosphatase, which limits the longevity of germline-less animals by restricting the activity of the heat shock transcription factor HSF-1. Altogether, our findings point to endo-siRNAs as a link between germline removal and the HSF-1 proteostasis and longevity-promoting somatic pathway. This establishes a role for endo siRNAs in the aging process and identifies downstream genes and physiological processes that are regulated by the endo siRNAs to affect longevity.

Unique Aspects of rRNA Biogenesis in Trypanosomatids.

Trypanosomatids are protozoan parasites that cycle between an insect and a mammalian host. The large-subunit rRNA of these organisms undergoes unique processing events absent in other eukaryotes. Recently, small nucleolar RNAs (snoRNAs) that mediate these specific cleavages were identified. Trypanosomatid rRNA is rich in RNA modifications such as 2'-O-methylation (Nm) and pseudouridylation (Ψ) that are also guided by these snoRNAs. A subset of these modifications is developmentally regulated and increased in the parasite form that propagates in the mammalian host. Such hypermodification contributes the temperature adaptation and hence infectivity during cycling of the parasite. rRNA processing and modification should be considered promising drug targets for fighting the diseases caused by these parasites.

The Algal Symbiont Modifies the Transcriptome of the Scleractinian Coral Euphyllia paradivisa During Heat Stress.

The profound mutualistic symbiosis between corals and their endosymbiotic counterparts, Symbiodiniaceae algae, has been threatened by the increase in seawater temperatures, leading to breakdown of the symbiotic relationship-coral bleaching. To characterize the heat-stress response of the holobiont, we generated vital apo-symbiotic Euphylliaparadivisa corals that lacked the endosymbiotic algae. Using RNA sequencing, we analyzed the gene expression of these apo-symbionts vs. symbiotic ones, to test the effect of the algal presence on the tolerance of the coral. We utilized literature-derived lists of "symbiosis differentially expressed genes" and "coral heat-stress genes" in order to compare between the treatments. The symbiotic and apo-symbiotic samples were segregated into two separate groups with several different enriched gene ontologies. Our findings suggest that the presence of endosymbionts has a greater negative impact on the host than the environmental temperature conditions experienced by the holobiont. The peak of the stress reaction was identified as 28 °C, with the highest number of differentially expressed genes. We suggest that the algal symbionts increase coral holobiont susceptibility to elevated temperatures. Currently, we can only speculate whether coral species, such as E.paradivisa, with the plasticity to also flourish as apo-symbionts, may have a greater chance to withstand the upcoming global climate change challenge.

A microcin processing peptidase-like protein of the cyanobacterium Synechococcus elongatus is essential for secretion of biofilm-promoting proteins.

Small secreted compounds, e.g. microcins, are characterized by a double-glycine (GG) secretion motif that is cleaved off upon maturation. Genomic analysis suggests that small proteins that possess a GG motif are widespread in cyanobacteria; however, the roles of these proteins are largely unknown. Using a biofilm-proficient mutant of the cyanobacterium Synechococcus elongatus PCC 7942 in which the constitutive biofilm self-suppression mechanism is inactivated, we previously demonstrated that four small proteins, Enable biofilm formation with a GG motif (EbfG1-4), each with a GG motif, enable biofilm formation. Furthermore, a peptidase belonging to the C39 family, Peptidase transporter enabling Biofilm (PteB), is required for secretion of these proteins. Here, we show that the microcin processing peptidase-like protein encoded by gene Synpcc7942_1127 is also required for biofilm development - inactivation of this gene in the biofilm-proficient mutant abrogates biofilm development. Additionally, this peptidase-like protein (denoted EbfE - enables biofilm formation peptidase) is required for secretion of the EbfG biofilm-promoting small proteins. Given their protein-domain characteristics, we suggest that PteB and EbfE take part in a maturation-secretion system, with PteB being located to the cell membrane while EbfE is directed to the periplasmic space via its secretion signal.

Setting the pace: host rhythmic behaviour and gene expression patterns in the facultatively symbiotic cnidarian Aiptasia are determined largely by Symbiodinium.

BACKGROUND: All organisms employ biological clocks to anticipate physical changes in the environment; however, the integration of biological clocks in symbiotic systems has received limited attention. In corals, the interpretation of rhythmic behaviours is complicated by the daily oscillations in tissue oxygen tension resulting from the photosynthetic and respiratory activities of the associated algal endosymbiont Symbiodinium. In order to better understand the integration of biological clocks in cnidarian hosts of Symbiodinium, daily rhythms of behaviour and gene expression were studied in symbiotic and aposymbiotic morphs of the sea-anemone Aiptasia diaphana. RESULTS: The results showed that whereas circatidal (approx. 12-h) cycles of activity and gene expression predominated in aposymbiotic morphs, circadian (approx. 24-h) patterns were the more common in symbiotic morphs, where the expression of a significant number of genes shifted from a 12- to 24-h rhythm. The behavioural experiments on symbiotic A. diaphana displayed diel (24-h) rhythmicity in body and tentacle contraction under the light/dark cycles, whereas aposymbiotic morphs showed approximately 12-h (circatidal) rhythmicity. Reinfection experiments represent an important step in understanding the hierarchy of endogenous clocks in symbiotic associations, where the aposymbiotic Aiptasia morphs returned to a 24-h behavioural rhythm after repopulation with algae. CONCLUSION: Whilst some modification of host metabolism is to be expected, the extent to which the presence of the algae modified host endogenous behavioural and transcriptional rhythms implies that it is the symbionts that influence the pace. Our results clearly demonstrate the importance of the endosymbiotic algae in determining the timing and the duration of the extension and contraction of the body and tentacles and temporal gene expression.

Identifying genes and regulatory pathways associated with the scleractinian coral calcification process.

Reef building corals precipitate calcium carbonate as an exo-skeleton and provide substratum for prosperous marine life. Biomineralization of the coral's skeleton is a developmental process that occurs concurrently with other proliferation processes that control the animal extension and growth. The development of the animal body is regulated by large gene regulatory networks, which control the expression of gene sets that progressively generate developmental patterns in the animal body. In this study we have explored the gene expression profile and signaling pathways followed by the calcification process of a basal metazoan, the Red Sea scleractinian (stony) coral, Stylophora pistillata. When treated by seawater with high calcium concentrations (addition of 100 gm/L, added as CaCl2.2H2O), the coral increases its calcification rates and associated genes were up-regulated as a result, which were then identified. Gene expression was compared between corals treated with elevated and normal calcium concentrations. Calcification rate measurements and gene expression analysis by microarray RNA transcriptional profiling at two time-points (midday and night-time) revealed several genes common within mammalian gene regulatory networks. This study indicates that core genes of the Wnt and TGF-ß/BMP signaling pathways may also play roles in development, growth, and biomineralization in early-diverging organisms such as corals.

Draft Genome Sequence of a New Bacterium Named Rappaport israeli, gen. nov., sp. nov., Isolated from a Blood Culture.

Here, we report the draft genome sequence of a Gram-negative microbe found in a blood culture (B08008) from a patient. The organism was proposed to be from a new unknown genus and species. This publication will increase worldwide microbial knowledge and may improve microbial identification and antibiotic treatment for patients.