Structural and mechanistic insights into the function of Leishmania ribosome lacking a single pseudouridine modification.

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.

CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues.

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 (NvClkΔ), 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, NvClkΔ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClkΔ/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.

Effects of Vineyard Agro-management Practices on Soil Bacterial Community Composition, and Diversity.

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.

A single pseudouridine on rRNA regulates ribosome structure and function in the mammalian parasite Trypanosoma brucei.

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.

A single DPE core promoter motif contributes to in vivo transcriptional regulation and affects cardiac function.

Transcription is initiated at the core promoter, which confers specific functions depending on the unique combination of core promoter elements. The downstream core promoter element (DPE) is found in many genes related to heart and mesodermal development. However, the function of these core promoter elements has thus far been studied primarily in isolated, in vitro or reporter gene settings. tinman (tin) encodes a key transcription factor that regulates the formation of the dorsal musculature and heart. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, we show that a substitution mutation of the functional tin DPE motif within the natural context of the core promoter results in a massive perturbation of Tinman's regulatory network orchestrating dorsal musculature and heart formation. Mutation of endogenous tin DPE reduced the expression of tin and distinct target genes, resulting in significantly reduced viability and an overall decrease in adult heart function. We demonstrate the feasibility and importance of characterizing DNA sequence elements in vivo in their natural context, and accentuate the critical impact a single DPE motif has during Drosophila embryogenesis and functional heart formation.

Evaluating biodiversity for coral reef reformation and monitoring on complex 3D structures using environmental DNA (eDNA) metabarcoding.

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.

Plant Gender Affects Soil Fungal Microbiota Associated with Welwitschia mirabilis, an Unusual Desert Gymnosperm.

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.

A sensitive soil biological indicator to changes in land-use in regions with Mediterranean climate.

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.

Lytic Reactivation of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Is Accompanied by Major Nucleolar Alterations.

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.

A long noncoding RNA promotes parasite differentiation in African trypanosomes.

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.

Identification and functional implications of pseudouridine RNA modification on small noncoding RNAs in the mammalian pathogen Trypanosoma brucei.

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.

Experimental evolution links post-transcriptional regulation to Leishmania fitness gain.

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.

Genome instability drives epistatic adaptation in the human pathogen Leishmania.

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.

Pseudouridines on Trypanosoma brucei mRNAs are developmentally regulated: Implications to mRNA stability and protein binding.

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.

Characterization of novel pollen-expressed transcripts reveals their potential roles in pollen heat stress response in Arabidopsis thaliana.

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.

Developmentally Regulated Novel Non-coding Anti-sense Regulators of mRNA Translation in Trypanosoma b rucei.

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.

The 'fertile island effect' of Welwitschia plants on soil microbiota is influenced by plant gender.

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 large repertoire of 2'-O-methylation guided by C/D snoRNAs on Trypanosoma brucei rRNA.

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'-OMe-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.

Sex differences in testosterone reactivity and sensitivity in a non-model gerbil.

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 ß-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.