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Natural light cycles are important for synchronizing behavioural and physiological rhythms over varying time periods in both plants and animals. An endogenous clock, regulated by positive and negative elements, interacting in feedback loops controls these rhythms. Many corals exhibit diel cycles of polyp expansion and contraction entrained by solar light patterns and monthly cycles of spawning or planulation that correspond to nocturnal lunar light cycles. However, despite considerable interest in studies of coral reproduction, there is currently not enough molecular information about the cellular pathways involved with synchronizing spawning/planulation in broadcast spawners and brooders. To determine whether the endogenous clock is implicated in the regulation of reproductive behaviour in corals, we characterized the transcriptome of Acropora digitifera colonies at twelve time points over a 2-month period of full and new moons, starting with the day of spawning in June 2014. We identified 608 transcripts with differential expression only on the spawning night during the coral setting phase and gamete release. Our data revealed an upregulation of light-sensing molecules and rhodopsin-like receptors that initiate signalling cascades, including the glutamate, SMAD signalling and WNT signalling pathways, neuroactive ligand-receptor interactions and calcium signalling. These are all involved in cell cycling, cell movement, tissue polarity, focal adhesion and cytoskeleton reorganization and together lead to gamete release. These findings can improve the understanding of many time-based cycles and extend our knowledge of the interplay between exogenous signals and the endogenous clock in cnidarians. [ABSTRACT FROM AUTHOR]

Abstract Background Recent studies have provided extensive evidence for multitudes of non-coding RNA (ncRNA) transcripts in a wide range of eukaryotic genomes. ncRNAs are emerging as key players in multiple layers of cellular regulation. With the availability of many whole genome sequences, comparative analysis has become a powerful tool to identify ncRNA molecules. In this study, we performed a systematic genome-wide in silico screen to search for novel small ncRNAs in the genome of Trypanosoma brucei using techniques of comparative genomics. Results In this study, we identified by comparative genomics, and validated by experimental analysis several novel ncRNAs that are conserved across multiple trypanosomatid genomes. When tested on known ncRNAs, our procedure was capable of finding almost half of the known repertoire through homology over six genomes, and about two-thirds of the known sequences were found in at least four genomes. After filtering, 72 conserved unannotated sequences in at least four genomes were found, 29 of which, ranging in size from 30 to 392 nts, were conserved in all six genomes. Fifty of the 72 candidates in the final set were chosen for experimental validation. Eighteen of the 50 (36%) were shown to be expressed, and for 11 of them a distinct expression product was detected, suggesting that they are short ncRNAs. Using functional experimental assays, five of the candidates were shown to be novel H/ACA and C/D snoRNAs; these included three sequences that appear as singletons in the genome, unlike previously identified snoRNA molecules that are found in clusters. The other candidates appear to be novel ncRNA molecules, and their function is, as yet, unknown. Conclusions Using comparative genomic techniques, we predicted 72 sequences as ncRNA candidates in T. brucei. The expression of 50 candidates was tested in laboratory experiments. This resulted in the discovery of 11 novel short ncRNAs in procyclic stage T. brucei, which have homologues in the other trypansomatids. A few of these molecules are snoRNAs, but most of them are novel ncRNA molecules. Based on this study, our analysis suggests that the total number of ncRNAs in trypanosomatids is in the range of several hundred.

Abstract Background Detection of non coding RNA (ncRNA) molecules is a major bioinformatics challenge. This challenge is particularly difficult when attempting to detect H/ACA molecules which are involved in converting uridine to pseudouridine on rRNA in trypanosomes, because these organisms have unique H/ACA molecules (termed H/ACA-like) that lack several of the features that characterize H/ACA molecules in most other organisms. Results We present here a computational tool called Psiscan, which was designed to detect H/ACA-like molecules in trypanosomes. We started by analyzing known H/ACA-like molecules and characterized their crucial elements both computationally and experimentally. Next, we set up constraints based on this analysis and additional phylogenic and functional data to rapidly scan three trypanosome genomes (T. brucei, T. cruzi and L. major) for sequences that observe these constraints and are conserved among the species. In the next step, we used minimal energy calculation to select the molecules that are predicted to fold into a lowest energy structure that is consistent with the constraints. In the final computational step, we used a Support Vector Machine that was trained on known H/ACA-like molecules as positive examples and on negative examples of molecules that were identified by the computational analyses but were shown experimentally not to be H/ACA-like molecules. The leading candidate molecules predicted by the SVM model were then subjected to experimental validation. Conclusion The experimental validation showed 11 molecules to be expressed (4 out of 25 in the intermediate stage and 7 out of 19 in the final validation after the machine learning stage). Five of these 11 molecules were further shown to be bona fide H/ACA-like molecules. As snoRNA in trypanosomes are organized in clusters, the new H/ACA-like molecules could be used as starting points to manually search for additional molecules in their neighbourhood. All together this study increased our repertoire by fourteen H/ACA-like and six C/D snoRNAs molecules from T. brucei and L. Major. In addition the experimental analysis revealed that six ncRNA molecules that are expressed are not downregulated in CBF5 silenced cells, suggesting that they have structural features of H/ACA-like molecules but do not have their standard function. We termed this novel class of molecules AGA-like, and we are exploring their function. This study demonstrates the power of tight collaboration between computational and experimental approaches in a combined effort to reveal the repertoire of ncRNA molecles.

Abstract Background In recent years, RNA molecules that are not translated into proteins (ncRNAs) have drawn a great deal of attention, as they were shown to be involved in many cellular functions. One of the most important computational problems regarding ncRNA is to predict the secondary structure of a molecule from its sequence. In particular, we attempted to predict the secondary structure for a set of unaligned ncRNA molecules that are taken from the same family, and thus presumably have a similar structure. Results We developed the RNAspa program, which comparatively predicts the secondary structure for a set of ncRNA molecules in linear time in the number of molecules. We observed that in a list of several hundred suboptimal minimal free energy (MFE) predictions, as provided by the RNAsubopt program of the Vienna package, it is likely that at least one suggested structure would be similar to the true, correct one. The suboptimal solutions of each molecule are represented as a layer of vertices in a graph. The shortest path in this graph is the basis for structural predictions for the molecule. We also show that RNA secondary structures can be compared very rapidly by a simple string Edit-Distance algorithm with a minimal loss of accuracy. We show that this approach allows us to more deeply explore the suboptimal structure space. Conclusion The algorithm was tested on three datasets which include several ncRNA families taken from the Rfam database. These datasets allowed for comparison of the algorithm with other methods. In these tests, RNAspa performed better than four other programs.

Background: The hypothalamic-pituitary-adrenal (HPA) and -gonadal (HPG) axes are two major pathways that connect the neural and endocrine systems in vertebrates. Factors such as prenatal stress and maternal exposure to exogenous steroids have been shown to affect these pathways during fetal development. Another less studied factor is the transfer of hormones across fetuses in multifetal pregnancies. This form of transfer has been shown to influence the morphology, anatomy, physiology, and behavior of the offspring in litter-bearing mammals, an influence termed the intrauterine position (IUP) effect. In this study, we sought to delineate how the IUP effects HPA and HPG brain receptors, peptides, and enzymes (hereafter components) in utero and how these influences may differ between males and females., Methods: We utilized the unconventional model of culled free-ranging nutria (Myocastor coypus), with its large natural variation. We collected brain tissues from nutria fetuses and quantified the expression of key HPA and HPG components in three brain regions: prefrontal cortex, hypothalamus, and striatum., Results: We found an interaction between sex and IUP in the mineralocorticoid receptor (MR), gonadotropin-releasing hormone receptor (GNRHR), androgen receptor (AR), and estrogen receptor alpha (ESR1). IUP was significant in both gonadotropin-releasing hormone (GnRH) and its receptor GNRHR, but in different ways. In the hypothalamus, fetuses adjacent to same-sex neighbors had higher expression of GnRH than fetuses neighboring the opposite sex. Conversely, in the cortex, GNRHR exhibited the inverse pattern, and fetuses that were neighboring the opposite sex had higher expression levels than those neighboring the same sex. Regardless of IUP, in most components that showed significant sex differences, female fetuses had higher mRNA expression levels than male fetuses. We also found that HPA and HPG components were highly related in the early stages of gestation, and that there was an interaction between sex and developmental stage. In the early stages of pregnancy, female component expression levels were more correlated than males', but in the last trimester of pregnancy, male components were more related to each other than female's., Conclusions: This study suggests that there are sexually different mechanisms to regulate the HPA and HPG axes during fetal development. Higher mRNA expression levels of endocrine axes components may be a mechanism to help females cope with prolonged androgen exposure over a long gestational period. Additionally, these findings suggest different coordination requirements of male and female endocrine axes during stages of fetal development., (© 2024. The Author(s).)

Transcription initiates at the core promoter, which contains distinct core promoter elements. Here, we highlight the complexity of transcriptional regulation by outlining the effect of core promoter-dependent regulation on embryonic development and the proper function of an organism. We demonstrate in vivo the importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach using both CRISPR and nascent transcriptomics, we show the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7 bp substitution mutation results in massive perturbation of the Tinman regulatory network that orchestrates dorsal musculature, which is manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features, and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Natural killer (NK) cells are critical to the innate immune system, as they recognize antigens without prior sensitization, and contribute to the control and clearance of viral infections and cancer. However, a significant proportion of NK cells in mice and humans do not express classical inhibitory receptors during their education process and are rendered naturally "anergic", i.e., exhibiting reduced effector functions. The molecular events leading to NK cell anergy as well as their relation to those underlying NK cell exhaustion that arises from overstimulation in chronic conditions, remain unknown. Here, we characterize the "anergic" phenotype and demonstrate functional, transcriptional, and phenotypic similarities to the "exhausted" state in tumor-infiltrating NK cells. Furthermore, we identify zinc finger transcription factor Egr2 and diacylglycerol kinase DGKα as common negative regulators controlling NK cell dysfunction. Finally, experiments in a 3D organotypic spheroid culture model and an in vivo tumor model suggest that a nanoparticle-based delivery platform can reprogram these dysfunctional natural killer cell populations in their native microenvironment. This approach may become clinically relevant for the development of novel anti-tumor immunotherapeutic strategies., (© 2024. The Author(s).)

Leishmania is the causative agent of cutaneous and visceral diseases affecting millions of individuals worldwide. Pseudouridine (Ψ), the most abundant modification on rRNA, changes during the parasite life cycle. Alterations in the level of a specific Ψ in helix 69 (H69) affected ribosome function. To decipher the molecular mechanism of this phenotype, we determine the structure of ribosomes lacking the single Ψ and its parental strain at ∼2.4-3 Å resolution using cryo-EM. Our findings demonstrate the significance of a single Ψ on H69 to its structure and the importance for its interactions with helix 44 and specific tRNAs. Our study suggests that rRNA modification affects translation of mRNAs carrying codon bias due to selective accommodation of tRNAs by the ribosome. Based on the high-resolution structures, we propose a mechanism explaining how the ribosome selects specific tRNAs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCK's functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis , pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant ( NvClk Δ ), revealing circadian behavior loss under constant dark (DD) or light (LL), while maintaining a 24 hr rhythm under light-dark condition (LD). Transcriptomics analysis revealed distinct rhythmic genes in wild-type (WT) polypsunder LD compared to DD conditions. In LD, NvClk Δ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClk Δ/Δ polyps showed alterations in temporal pacemaker gene expression, impacting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption, and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior with the diel rhythm of the earth's biosphere., Competing Interests: RA, MR, NS, TD, LA, OL No competing interests declared, (© 2023, Aguillon et al.)

Changes in land use strongly affect soil biological and physico-chemical structure and characteristics, which are strongly related to agricultural conversion of natural habitats to man-made usage. These are among the most important and not always beneficial changes, affecting loss of habitats. In Golan Heights basaltic soils, vineyards are currently a driving force in land-use change. Such changes could have an important effect on soil microbial community that play an important role in maintaining stable functioning of soil ecosystems. This study investigated the microbial communities in five different agro-managements using molecular tools that can clarify the differences in microbial community structure and function. Significant differences in soil microbial community composition were found. However, no differences in alpha diversity or functionality were found between the treatments. To the best of our knowledge, this is the first report indicating that the bacterial community in different agro-managements provide an insight into the potential function of a vineyard system., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal. A single knock-out of a snoRNA guiding Ψ530 on H69 altered the composition of the 80S monosome. These changes specifically affected the translation of only a subset of proteins. This study correlates a single site Ψ modification with changes in ribosomal protein stoichiometry, supported by a high-resolution cryo-EM structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins., (© 2023. The Author(s).)

In a recent study, we found a distinct soil bacterial community associated with male and female plants of the desert gymnosperm Welwitschia mirabilis. In this subsequent study, we also found that the soil fungal community associated with Welwitschia differs between male and female plants, and between unvegetated areas and the soil under plants. Site location, pH, and soil moisture also had an important influence on the composition of the fungal community. A number of Ascomycota and Chytrid species were found to be distinct indicators of male and female plants, respectively, but there was no overall difference at the phylum level or in terms of diversity. The unvegetated areas between plants also differed in terms of several Ascomycota OTUs. Network connectivity of the fungal communities was found to be higher under both male and female Welwitschia plants than in unvegetated control areas. As with the bacterial community, it is unclear what processes produce the gender-distinct fungal community, and also the more general plant-associated community, and also what the effects on the biology of the plants are. One possibility behind the gender-related difference in fungal community is that there are differences in the production of pollen or nectar between the two plant genders, affecting the below-ground soil community., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Quantifying coral reef biodiversity is challenging for cryptofauna and organisms in early life stages. We demonstrate the utility of eDNA metabarcoding as a tool for comprehensively evaluating invertebrate communities on complex 3D structures for reef reformation, and the role these structures play in provisioning habitat for organisms. 3D design and printing were used to create 18 complex tiles, which were used to form artificial reef structures. eDNA was collected from scraping tile surfaces for organismal biomass and from seawater samples around the artificial reefs in the Gulf of Eilat/Aqaba, Red Sea. Metabarcoding targeted the mitochondrial COI gene with specific primers for marine biodiversity. We provide the first eDNA biodiversity baseline for the Gulf of Eilat/Aqaba, capturing extensive information on species abundance, richness, and diversity. Tile tops had higher phylogenetic diversity and richness, despite a higher abundance of organisms on tile bottoms, highlighting the detection of cryptic organisms with eDNA. We recommend eDNA metabarcoding for reef restoration initiatives, especially for complex marine structures, to improve success and evaluation of biodiversity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Prof. Oren Levy, Bar Ilan University. Published by Elsevier B.V. All rights reserved.)

The demand for reliable indicators to quantify soil health has increased recently. We propose and test the use of soil microbial functional diversity as an indicator of multifunctional performance in agriculturally important areas. Agricultural fields in the Mediterranean and semiarid regions of Israel were selected as test sites and measured in Spring and Autumn seasons. Measurements included microbial parameters, basic soil abiotic properties and biological responses to agricultural management relative to measures of a natural ecosystem. Using a canonical correlation analysis we found that soil moisture was the most important basic soil property with different responses in Spring and Autumn. In Spring, it had a strongly negative relation with microbial biomass (MB), community level physiological profiling (CLPP) and the Shannon-Weaver index H', while in Autumn it had a strong relation with CLPP. We further show a significant interaction between CLPP and climate for land-use type "orchards". CLPP measured in the autumn season was thus identified as a useful and rapid biological soil health indicator, recommended for application in semiarid and Mediterranean agricultural regions. Apart from obtaining a better understanding of CLPP as the soil indicator, the study concludes that CLPP is well suited to differentiate between soils in different climates, seasons and land use types. The study shows a promising direction for further research on characterizing soil health under a larger variety of conditions., (© 2022. The Author(s).)

The nucleolus is a subnuclear 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 the lytic reactivation of KSHV. This was manifested by the redistribution of key nucleolar proteins, including the rRNA transcription factor UBF. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Significantly, the redistribution of UBF was dependent on viral DNA replication or late viral gene expression. No significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found by RT-qPCR and Northern blot analysis. Furthermore, fluorescent in situ hybridization (FISH), combined with immunofluorescence, revealed an overlap between Fibrillarin and internal transcribed spacer 1 (ITS1), which represents the primary product of the pre-rRNA, suggesting that the processing of rRNA proceeds during lytic reactivation. Finally, small changes in the levels of pseudouridylation (Ψ) and 2'- O -methylation (Nm) were documented across the rRNA; however, none were localized to the functional domain. Taken together, our results suggest that despite dramatic changes in the nucleolar organization, rRNA transcription and processing persist during lytic reactivation of KSHV. Whether the observed nucleolar alterations favor productive infection or signify cellular anti-viral responses remains to be determined.

Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (Ψs) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T. brucei. We show using HydraPsiSeq and RiboMeth-seq that the Ψ on C/D snoRNA guiding 2'-O-methylation increased the efficiency of the guided modification on its target, rRNA. We found differential levels of Ψs on these noncoding RNAs in the two life stages (insect host and mammalian host) of the parasite. Furthermore, tRNA isoform abundance and Ψ modifications were characterized in these two life stages demonstrating stage-specific regulation. We conclude that the differential Ψ modifications identified here may contribute to modulating the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of the parasite between its two hosts., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The parasite Trypanosoma brucei causes African sleeping sickness that is fatal to patients if untreated. Parasite differentiation from a replicative slender form into a quiescent stumpy form promotes host survival and parasite transmission. Long noncoding RNAs (lncRNAs) are known to regulate cell differentiation in other eukaryotes. To determine whether lncRNAs are also involved in parasite differentiation, we used RNA sequencing to survey the T. brucei genome, identifying 1428 previously uncharacterized lncRNA genes. We find that grumpy lncRNA is a key regulator that promotes parasite differentiation into the quiescent stumpy form. This function is promoted by a small nucleolar RNA encoded within the grumpy lncRNA. snoGRUMPY binds to messenger RNAs of at least two stumpy regulatory genes, promoting their expression. grumpy overexpression reduces parasitemia in infected mice. Our analyses suggest that T. brucei lncRNAs modulate parasite-host interactions and provide a mechanism by which grumpy regulates cell differentiation in trypanosomes.

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings., Competing Interests: The authors declare that they have no conflict of interest.

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania , which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2'- O -methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The parasite Trypanosoma brucei cycles between an insect and a mammalian host and is the causative agent of sleeping sickness. Here, we performed high-throughput mapping of pseudouridines (Ψs) on mRNA from two life stages of the parasite. The analysis revealed ~273 Ψs, including developmentally regulated Ψs that are guided by homologs of pseudouridine synthases (PUS1, 3, 5, and 7). Mutating the U that undergoes pseudouridylation in the 3' UTR of valyl-tRNA synthetase destabilized the mRNA level. To investigate the mechanism by which Ψ affects the stability of this mRNA, proteins that bind to the 3' UTR were identified, including the RNA binding protein RBSR1. The binding of RBSR1 protein to the 3' UTR was stronger when lacking Ψ compared to transcripts carrying the modification, suggesting that Ψ can inhibit the binding of proteins to their target and thus affect the stability of mRNAs. Consequently, Ψ modification on mRNA adds an additional level of regulation to the dominant post-transcriptional control in these parasites., (© 2021 John Wiley & Sons Ltd.)

Key Message: Arabidopsis pollen transcriptome analysis revealed new intergenic transcripts of unknown function, many of which are long non-coding RNAs, that may function in pollen-specific processes, including the heat stress response. The male gametophyte is the most heat sensitive of all plant tissues. In recent years, long noncoding RNAs (lncRNAs) have emerged as important components of cellular regulatory networks involved in most biological processes, including response to stress. While examining RNAseq datasets of developing and germinating Arabidopsis thaliana pollen exposed to heat stress (HS), we identified 66 novel and 246 recently annotated intergenic expressed loci (XLOCs) of unknown function, with the majority encoding lncRNAs. Comparison with HS in cauline leaves and other RNAseq experiments indicated that 74% of the 312 XLOCs are pollen-specific, and at least 42% are HS-responsive. Phylogenetic analysis revealed that 96% of the genes evolved recently in Brassicaceae. We found that 50 genes are putative targets of microRNAs and that 30% of the XLOCs contain small open reading frames (ORFs) with homology to protein sequences. Finally, RNAseq of ribosome-protected RNA fragments together with predictions of periodic footprint of the ribosome P-sites indicated that 23 of these ORFs are likely to be translated. Our findings indicate that many of the 312 unknown genes might be functional and play a significant role in pollen biology, including the HS response.

The parasite Trypanosoma brucei is the causative agent of sleeping sickness and cycles between insect and mammalian hosts. The parasite appears to lack conventional transcriptional regulation of protein coding genes, and mRNAs are processed from polycistronic transcripts by the concerted action of trans- splicing and polyadenylation. Regulation of mRNA function is mediated mainly by RNA binding proteins affecting mRNA stability and translation. In this study, we describe the identification of 62 non-coding (nc) RNAs that are developmentally regulated and/or respond to stress. We characterized two novel anti-sense RNA regulators (TBsRNA-33 and 37) that originate from the rRNA loci, associate with ribosomes and polyribosomes, and interact in vivo with distinct mRNA species to regulate translation. Thus, this study suggests for the first-time anti-sense RNA regulators as an additional layer for controlling gene expression in these parasites., Competing Interests: The authors declare no competing financial interests., (© 2020 The Authors.)

Desert and semi-desert plants are often associated with a distinct soil biota under the plants and close to the root system. We aimed to understand if similar effects could be found in the taxonomically isolated desert gymnosperm Welwitschia mirabilis in the Namib Desert, and whether this island effect varied with climate and with gender of plants. We took soil cores adjacent to the plants in environments ranging from extreme desert to arid shrubland, and in nearby control sites between the plants. Soil chemistry was analysed, and deoxyribonucleic acid was extracted and sequenced for the bacterial 16s region. Soil under the plants was richer in organic C, N and moisture. Despite the range of climates, the soil around Welwitschia plants was consistently associated with a particular bacterial community composition that was distinct from samples further away. Compared to unvegetated control patches, bacterial diversity close to the plants was reduced. In the plant-associated soil community, there was a clear gender effect across all sites with a distinct community composition and greater diversity under male plants. It is unclear what differences in the soil environment might be producing these gender-associated differences, which provide an additional dimension to the fertile island effect., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

The parasite Trypanosoma brucei cycles between insect and mammalian hosts, and is the causative agent of sleeping sickness. Here, we performed genome-wide mapping of 2'- O -methylations (Nms) on trypanosome rRNA using three high-throughput sequencing methods; RibOxi-seq, RiboMeth-seq and 2'- O Me-seq. This is the first study using three genome-wide mapping approaches on rRNA from the same species showing the discrepancy among the methods. RibOxi-seq detects all the sites, but RiboMeth-seq is the only method to evaluate the level of a single Nm site. The sequencing revealed at least ninety-nine Nms guided by eighty-five snoRNAs among these thirty-eight Nms are trypanosome specific sites. We present the sequence and target of the C/D snoRNAs guiding on rRNA. This is the highest number of Nms detected to date on rRNA of a single cell parasite. Based on RiboMeth-seq, several Nm sites were found to be differentially regulated at the two stages of the parasite life cycle, the insect procyclic form (PCF) versus the bloodstream form (BSF) in the mammalian host.

Although testosterone (T) is a key regulator in vertebrate development, physiology, and behaviour in both sexes, studies suggest that its regulation may be sex-specific. We measured circulating T levels in Baluchistan gerbils (Gerbillus nanus) in the field and in the lab all year round and found no significant sex differences. However, we observed sex differences in circulating T levels following gonadotropin-releasing hormone (GnRH) challenge and T implants in this non-model species. Whereas only males elevated T following a GnRH challenge, females had higher serum T concentrations following T implant insertion. These differences may be a result of different points of regulation along the hypothalamic-pituitary-gonadal (HPG) axis. Consequently, we examined sex differences in the mRNA expression of the androgen receptor (AR) in multiple brain regions. We identified AR and β-actin sequences in assembled genomic sequences of members of the Gerbillinae, which were analogous to rat sequences, and designed primers for them. The distribution of the AR in G. nanus brain regions was similar to documented expression profiles in rodents. We found lower AR mRNA levels in females in the striatum. Additionally, G. nanus that experienced housing in mixed-sex pairs had higher adrenal AR expression than G. nanus that were housed alone. Regulation of the gerbil HPG axis may reflect evolutionary sex differences in life-history strategies, with males ready to reproduce when receptive females are available, while the possible reproductive costs associated with female T direct its regulation upstream., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Life on earth has evolved under constant environmental changes; in response to these changes, most organisms have developed an endogenous clock that allows them to anticipate daily and seasonal changes and adapt their biology accordingly. Light cycles synchronize biological rhythms and are controlled by an endogenous clock that is entrained by environmental cues. Light is known to play a key role in the biology of symbiotic corals as they exhibit many biological processes entrained by daily light patterns. In this study, we aimed at determining the effect of constant dim light on coral's perception of diel and monthly cycles. Our results show that under constant dim light corals display a loss of rhythmic processes and constant stimuli by light, which initiates signal transduction that results in an abnormal cell cycle, cell proliferation, and protein synthesis. The results emphasize how constant dim light can mask the biological clock of Acropora digitifera., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The pro-longevity enzyme SIRT6 regulates various metabolic pathways. Gene expression analyses in SIRT6 heterozygotic mice identify significant decreases in PPARα signaling, known to regulate multiple metabolic pathways. SIRT6 binds PPARα and its response element within promoter regions and activates gene transcription. Sirt6 +/- results in significantly reduced PPARα-induced β-oxidation and its metabolites and reduced alanine and lactate levels, while inducing pyruvate oxidation. Reciprocally, starved SIRT6 transgenic mice show increased pyruvate, acetylcarnitine, and glycerol levels and significantly induce β-oxidation genes in a PPARα-dependent manner. Furthermore, SIRT6 mediates PPARα inhibition of SREBP-dependent cholesterol and triglyceride synthesis. Mechanistically, SIRT6 binds PPARα coactivator NCOA2 and decreases liver NCOA2 K780 acetylation, which stimulates its activation of PPARα in a SIRT6-dependent manner. These coordinated SIRT6 activities lead to regulation of whole-body respiratory exchange ratio and liver fat content, revealing the interactions whereby SIRT6 synchronizes various metabolic pathways, and suggest a mechanism by which SIRT6 maintains healthy liver., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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 at elevated temperatures. We further demonstrate that Ψ strengthens U4/U6 RNA-RNA and U2B"/U2A' proteins-U2 snRNA interaction at elevated temperatures. The existence of single hairpin RNAs that modify both the spliceosome and ribosome RNAs is unique for these parasites, and may be related to their ability to cycle between their two hosts that differ in temperature., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

As human populations grow and lighting technologies improve, artificial light gradually alters natural cycles of light and dark that have been consistent over long periods of geological and evolutionary time. While considerable ecological implications of artificial light have been identified in both terrestrial and aquatic habitats, knowledge about the physiological and molecular effects of light pollution is vague. To determine if ecological light pollution (ELP) impacts coral biological processes, we characterized the transcriptome of the coral Acropora eurystoma under two different light regimes: control conditions and treatment with light at night. Here we show that corals exposed to ELP have approximately 25 times more differentially expressed genes that regulate cell cycle, cell proliferation, cell growth, protein synthesis and display changes in photo physiology. The finding of this work confirms that ELP acts as a chronic disturbance that may impact the future of coral reefs., Competing Interests: Competing interestsThe authors declare no competing interests.

The sedimentary Marl mudstone soil is composed primarily of CaCO 3 , and is an important pedologic and geomorphologic element known as Marl, extensively dispersed in slopes and ridges in the northern Negev Desert, Israel. The wide Marl soil-layer areas are barren, with well-developed streamsides and no perennial vegetation cover. Soil systems in the Negev Desert have been widely studied, yet very little information was collected on Marl soils, and even less on the microbiome present in the Negev. Thus, an evaluation of the microbial-community inhabitants in a Marl soil layer was conducted in an attempt to distinguish between Marl with surface green mat and bare Marl soil layer. Our objective was to investigate the microbiome and abiotic components of the upper layer (0-5 cm) of Marl and Marl-with-green-mat soil collected in the Negev Desert. Plate-counting enabled the estimation of fungal and bacterial population size, while nested polymerase chain reaction (nPCR) and Ion Torrent sequencing were used to analyze biological diversity. The results indicate significant differences in microbial biomass and microbial-community diversity between Marl and Marl-green mat, despite similar pH levels. Further study is needed to enhance understanding of the activity of the biotic components and their contribution to slope stabilization.

In trypanosomes, in contrast to most eukaryotes, the large subunit (LSU) ribosomal RNA is fragmented into two large and four small ribosomal RNAs (srRNAs) pieces, and this additional processing likely requires trypanosome-specific factors. Here, we examined the role of 10 abundant small nucleolar RNAs (snoRNAs) involved in rRNA processing. We show that each snoRNA involved in LSU processing associates with factors engaged in either early or late biogenesis steps. Five of these snoRNAs interact with the intervening sequences of rRNA precursor, whereas the others only guide rRNA modifications. The function of the snoRNAs was explored by silencing snoRNAs. The data suggest that the LSU rRNA processing events do not correspond to the order of rRNA transcription, and that srRNAs 2, 4 and 6 which are part of LSU are processed before srRNA1. Interestingly, the 6 snoRNAs that affect srRNA1 processing guide modifications on rRNA positions that span locations from the protein exit tunnel to the srRNA1, suggesting that these modifications may serve as check-points preceding the liberation of srRNA1. This study identifies the highest number of snoRNAs so far described that are involved in rRNA processing and/or rRNA folding and highlights their function in the unique trypanosome rRNA maturation events., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cucurbits represent an attractive model to explore the dynamics of fruit set, whose regulation is not fully understood, despite its importance for yield determination. A fertilized ovary must integrate signals from distant plant parts and 'decide' whether to set fruit, or remain inhibited and later senesce. Here, we set out to characterize first-fruit inhibition (FFI), that is, the inhibitory effect of the first fruit on subsequent development of younger ovaries during pollination-induced and parthenocarpic fruit set. After the first fertilized ovaries set fruit, younger fertilized ovaries remained in a temporary state of inhibition. Such ovaries preserved their size and green color, and if the older fruit were removed within a 1-week reversibility window, they set fruit. The FFI effect was documented in both fertilized and parthenocarpic ovaries. We compared the gene expression profiles of pollinated ovaries (committed to set fruit) with respect to those affected by FFI, and to non-pollinated ovaries (undergoing senescence). The three fates of the ovaries were characterized by wide changes in gene expression, with several specific transcripts being up- or down-regulated in response to pollination, and to the presence of inhibitory fruit. Metabolic profiling was undertaken and integrated with the transcriptomic data in order to characterize early physiological changes that occur in post-anthesis ovaries in parthenocarpic and non-parthenocarpic genotypes. The combined results are discussed with respect to current models of fruit set and specifically with regard to FFI. Moreover, these metabolome and transcriptome data provide a valuable resource for studying ovary development and fruit set., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

The parasite Trypanosoma brucei is the causative agent of African sleeping sickness and is known for its unique RNA processing mechanisms that are common to all the kinetoplastidea including Leishmania and Trypanosoma cruzi. Trypanosomes possess two canonical RNA poly (A) polymerases (PAPs) termed PAP1 and PAP2. PAP1 is encoded by one of the only two genes harboring cis-spliced introns in this organism, and its function is currently unknown. In trypanosomes, all mRNAs, and non-coding RNAs such as small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs), undergo trans-splicing and polyadenylation. Here, we show that the function of PAP1, which is located in the nucleus, is to polyadenylate non-coding RNAs, which undergo trans-splicing and polyadenylation. Major substrates of PAP1 are the snoRNAs and lncRNAs. Under the silencing of either PAP1 or PAP2, the level of snoRNAs is reduced. The dual polyadenylation of snoRNA intermediates is carried out by both PAP2 and PAP1 and requires the factors essential for the polyadenylation of mRNAs. The dual polyadenylation of the precursor snoRNAs by PAPs may function to recruit the machinery essential for snoRNA processing., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Microbial function, composition, and distribution play a fundamental role in ecosystem ecology. The interaction between desert plants and their associated microbes is expected to greatly affect their response to changes in this harsh environment. Using comparative analyses, we studied the impact of three desert shrubs, Atriplex halimus (A), Artemisia herba-alba (AHA), and Hammada scoparia (HS), on soil- and leaf-associated microbial communities. DNA extracted from the leaf surface and soil samples collected beneath the shrubs were used to study associated microbial diversity using a sequencing survey of variable regions of bacterial 16S rRNA and fungal ribosomal internal transcribed spacer (ITS1). We found that the composition of bacterial and fungal orders is plant-type-specific, indicating that each plant type provides a suitable and unique microenvironment. The different adaptive ecophysiological properties of the three plant species and the differential effect on their associated microbial composition point to the role of adaptation in the shaping of microbial diversity. Overall, our findings suggest a link between plant ecophysiological adaptation as a "temporary host" and the biotic-community parameters in extreme xeric environments.

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.

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.

Core promoter elements play a pivotal role in the transcriptional output, yet they are often detected manually within sequences of interest. Here, we present 2 contributions to the detection and curation of core promoter elements within given sequences. First, the Elements Navigation Tool (ElemeNT) is a user-friendly web-based, interactive tool for prediction and display of putative core promoter elements and their biologically-relevant combinations. Second, the CORE database summarizes ElemeNT-predicted core promoter elements near CAGE and RNA-seq-defined Drosophila melanogaster transcription start sites (TSSs). ElemeNT's predictions are based on biologically-functional core promoter elements, and can be used to infer core promoter compositions. ElemeNT does not assume prior knowledge of the actual TSS position, and can therefore assist in annotation of any given sequence. These resources, freely accessible at http://lifefaculty.biu.ac.il/gershon-tamar/index.php/resources, facilitate the identification of core promoter elements as active contributors to gene expression.

We present the draft genome sequence of Pseudoalteromonas sp. strain PLSV, isolated from the feces of an Aplysia sea slug. The addition of the PLSV genome to the existing genomes of three other ulvan-degrading bacterial species will enhance our understanding of ulvan utilization., (Copyright © 2014 Kopel et al.)

Here, we report the draft genome sequence of two ulvan-degrading Alteromonas spp. isolated from the feces of the sea slug, Aplysia. These sequenced genomes display a unique ulvan degradation machinery compared with ulvanolytic enzymes previously identified in Nonlabens ulvanivorans., (Copyright © 2014 Kopel et al.)

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., (© 2014 Kedmi et al.; Published by Cold Spring Harbor Laboratory Press.)

Here we report the draft genome sequence of the bacterium Nonlabens ulvanivorans, which was recently isolated. To our knowledge, this is the first published genome of a characterized ulvan-degrading bacterium. Revealing the ulvan utilization pathways may provide access to a vast marine biomass source that has yet to be exploited., (Copyright © 2014 Kopel et al.)

Background: The human genome encodes thousands of unique long non-coding RNAs (lncRNAs), and these transcripts are emerging as critical regulators of gene expression and cell fate. However, the transcriptional regulation of their expression is not fully understood. The pivotal transcription factor E2F1 which can induce both proliferation and cell death, is a critical downstream target of the tumor suppressor, RB. The retinoblastoma pathway is often inactivated in human tumors resulting in deregulated E2F activity., Results: Here, we report that lncRNA XLOC 006942, which we named ERIC, is regulated by E2F1 and, most probably, also E2F3. We show that expression levels of ERIC were elevated upon activation of exogenous E2F1, E2F3 or endogenous E2Fs. Moreover, knockdown of either E2F1 or E2F3 reduced ERIC levels and endogenous E2F1 binds ERIC's promoter. Expression of ERIC was cell cycle regulated and peaked in G1 in an E2F1-dependent manner. Inhibition of ERIC expression increased E2F1-mediated apoptosis, suggesting that E2F1 and ERIC constitute a negative feedback loop that modulates E2F1 activity. Furthermore, ERIC levels were increased following DNA damage by the chemotherapeutic drug Etoposide, and inhibition of ERIC expression enhanced Etoposide -induced apoptosis., Conclusions: Our data identify ERIC as a novel lncRNA that is transcriptionally regulated by E2Fs, and restricts apoptosis induced by E2F1, as well as by DNA damage.

Trypanosome protozoan parasites are the causative agents of devastating diseases. Trypanosome telomeres grow in an uncontrolled manner and the variant surface glycoprotein (VSG) genes are located in subtelomeric domains. The gene encoding telomerase reverse transcriptase (TERT) was identified and in this study, we describe the Trypanosoma brucei telomerase RNA (TER). TER RNA is bound by the core proteins of the C/D small nucleolar RNA (snoRNA) family and associates with the methyltransferase-associated protein (MTAP), whose homologue binds to mammalian TER. Silencing of TbTER resulted in telomere shortening. This is the first report of a TER that binds the C/D snoRNA core proteins., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)