Temporal dynamics and metagenomics of phosphorothioate epigenomes in the human gut microbiome.

Background: Epigenetic regulation of gene expression and host defense is well established in microbial communities, with dozens of DNA modifications comprising the epigenomes of prokaryotes and bacteriophage. Phosphorothioation (PT) of DNA, in which a chemically-reactive sulfur atom replaces a non-bridging oxygen in the sugar-phosphate backbone, is catalyzed by dnd and ssp gene families widespread in bacteria and archaea. However, little is known about the role of PTs or other microbial epigenetic modifications in the human microbiome. Here we optimized and applied fecal DNA extraction, mass spectrometric, and metagenomics technologies to characterize the landscape and temporal dynamics of gut microbes possessing PT modifications. Results: Exploiting the nuclease-resistance of PTs, mass spectrometric analysis of limit digests of PT-containing DNA reveals PT dinucleotides as part of genomic consensus sequences, with 16 possible dinucleotide combinations. Analysis of mouse fecal DNA revealed a highly uniform spectrum of 11 PT dinucleotides in all littermates, with PTs estimated to occur in 5-10% of gut microbes. Though at similar levels, PT dinucleotides in fecal DNA from 11 healthy humans possessed signature combinations and levels of individual PTs. Comparison with a widely distributed microbial epigenetic mark, m6dA, suggested temporal dynamics consistent with expectations for gut microbial communities based on Taylor's Power Law. Application of PT-seq for site-specific metagenomic analysis of PT-containing bacteria in one fecal donor revealed the larger consensus sequences for the PT dinucleotides in Bacteroidota, Firmicutes, Actinobacteria, and Proteobacteria, which differed from unbiased metagenomics and suggested that the abundance of PT-containing bacteria did not simply mirror the spectrum of gut bacteria. PT-seq further revealed low abundance PT sites not detected as dinucleotides by mass spectrometry, attesting to the complementarity of the technologies. Conclusions: The results of our studies provide a benchmark for understanding the behavior of an abundant and chemically-reactive epigenetic mark in the human gut microbiome, with implications for inflammatory conditions of the gut.

Structural basis of tRNA recognition by the m3C RNA methyltransferase METTL6 in complex with SerRS seryl-tRNA synthetase.

Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m3C) is crucial for cellular translation fidelity. Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances. Here, we report the cryo-electron microscopy structure of the human m3C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNASer. Through the complex structure, we identify the tRNA-binding domain of METTL6. We show that SerRS acts as the tRNASer substrate selection factor for METTL6. We demonstrate that SerRS augments the methylation activity of METTL6 and that direct contacts between METTL6 and SerRS are necessary for efficient tRNASer methylation. Finally, on the basis of the structure of METTL6 in complex with SerRS and tRNASer, we postulate a universal tRNA-binding mode for m3C RNA methyltransferases, including METTL2 and METTL8, suggesting that these mammalian paralogs use similar ways to engage their respective tRNA substrates and cofactors.

First evidence of prickly shark, Echinorhinus cookei Pietschmann 1928, aggregation on seamounts in the eastern Pacific, Panama.

In May 2022, twelve prickly sharks, Echinorhinus cookei Pietschmann 1928, were sighted at 151-350 m depth in the Cordillera de Coiba seamounts, Pacific Panama. This discovery expands our knowledge of the distribution and habitat use of this rare deep-sea species. It underscores the potential significance of the Cordillera de Coiba seamounts, an offshore marine protected area, as a critical habitat for E. cookei, a species threatened by commercial fishing. Although unverified reports exist on its presence in the tropical eastern Pacific, this publication represents the first documented record of live specimens of E. cookei in Panama.

Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications.

The chemical biology of native nucleic acid modifications has seen an intense upswing, first concerning DNA modifications in the field of epigenetics and then concerning RNA modifications in a field that was correspondingly rebaptized epitranscriptomics by analogy. The German Research Foundation (DFG) has funded several consortia with a scientific focus in these fields, strengthening the traditionally well-developed nucleic acid chemistry community and inciting it to team up with colleagues from the life sciences and data science to tackle interdisciplinary challenges. This Perspective focuses on the genesis, scientific outcome, and downstream impact of the DFG priority program SPP1784 and offers insight into how it fecundated further consortia in the field. Pertinent research was funded from mid-2015 to 2022, including an extension related to the coronavirus pandemic. Despite being a detriment to research activity in general, the pandemic has resulted in tremendously boosted interest in the field of RNA and RNA modifications as a consequence of their widespread and successful use in vaccination campaigns against SARS-CoV-2. Funded principal investigators published over 250 pertinent papers with a very substantial impact on the field. The program also helped to redirect numerous laboratories toward this dynamic field. Finally, SPP1784 spawned initiatives for several funded consortia that continue to drive the fields of nucleic acid modification.

Pitfalls in RNA Modification Quantification Using Nucleoside Mass Spectrometry.

ConspectusIn recent years, there has been a high interest in researching RNA modifications, as they are involved in many cellular processes and in human diseases. A substantial set of enzymes within the cell, called RNA writers, place RNA modifications selectively and site-specifically. Another set of enzymes, called readers, recognize these modifications which guide the fate of the modified RNA. Although RNA is a transient molecule and RNA modification could be removed by RNA degradation, a subclass of enzymes, called RNA erasers, remove RNA modifications selectively and site-specifically to alter the characteristics of the RNA. The detection of RNA modifications can be done by various methods including second and next generation sequencing but also mass spectrometry. An approach capable of both qualitative and quantitative RNA modification analysis is liquid chromatography coupled to mass spectrometry of enzymatic hydrolysates of RNA into nucleosides. However, for successful detection and quantification, various factors must be considered to avoid biased identification and inaccurate quantification. In this Account, we identify three classes of errors that may distort the analysis. These classes comprise (I) errors related to chemical instabilities, (II) errors revolving around enzymatic hydrolysis to nucleosides, and (III) errors arising from issues with chromatographic separation and/or subsequent mass spectrometric analysis.A prominent example for class 1 is Dimroth rearrangement of m1A to m6A, but class 1 also comprises hydrolytic reactions and reactions with buffer components. Here, we also present the conversion of m3C to m3U under mild alkaline conditions and propose a practical solution to overcome these instabilities. Class 2 errors-such as contaminations in hydrolysis reagents or nuclease specificities-have led to erroneous discoveries of nucleosides in the past and possess the potential for misquantification of nucleosides. Impurities in the samples may also lead to class 3 errors: For instance, issues with chromatographic separation may arise from residual organic solvents, and salt adducts may hamper mass spectrometric quantification. This Account aims to highlight various errors connected to mass spectrometry analysis of nucleosides and presents solutions for how to overcome or circumnavigate those issues. Therefore, the authors anticipate that many scientists, but especially those who plan on doing nucleoside mass spectrometry, will benefit from the collection of data presented in this Account as a raised awareness, toward the variety of potential pitfalls, may further enhance the quality of data.

Sangivamycin is preferentially incorporated into viral RNA by the SARS-CoV-2 polymerase.

Sangivamycin (S) is an adenosine (A) nucleoside analog with low nanomolar antiviral activity against SARS-CoV-2 in vitro. Previously, low nanomolar antiviral efficacy was revealed when tested against multiple viral variants in several cell types. SARS-CoV-2 RNA isolated from live virus infected cells and the virions released from these cells was analyzed by mass spectrometry (MS) for S incorporation. Dose-dependent incorporation occurred up to 1.8 S per 1,000 nucleotides (49 S per genome) throughout the viral genomes isolated from both infected cells and viral particles, but this incorporation did not change the viral mutation rate. In contrast, host mRNA, affinity purified from the same infected and treated cells, contained little or no S. Sangivamycin triphosphate (STP) was synthesized to evaluate its incorporation into RNA by recombinant SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) under defined in vitro conditions. SARS-CoV-2 RdRp showed that S was not a chain terminator and S containing oligonucleotides templated as A. Though the antiviral mechanism remains to be determined, the data suggests that SARS-CoV-2 RdRp incorporates STP into SARS-CoV-2 RNA, which does not significantly impair viral RNA synthesis or the mutation rate.

Different modification pathways for m1A58 incorporation in yeast elongator and initiator tRNAs.

As essential components of the protein synthesis machinery, tRNAs undergo a tightly controlled biogenesis process, which include the incorporation of numerous posttranscriptional modifications. Defects in these tRNA maturation steps may lead to the degradation of hypomodified tRNAs by the rapid tRNA decay (RTD) and nuclear surveillance pathways. We previously identified m1A58 as a late modification introduced after modifications Ψ55 and T54 in yeast elongator tRNAPhe. However, previous reports suggested that m1A58 is introduced early during the tRNA modification process, in particular on primary transcripts of initiator tRNAiMet, which prevents its degradation by RNA decay pathways. Here, aiming to reconcile this apparent inconsistency on the temporality of m1A58 incorporation, we examined its introduction into yeast elongator and initiator tRNAs. We used specifically modified tRNAs to report on the molecular aspects controlling the Ψ55 → T54 → m1A58 modification circuit in elongator tRNAs. We also show that m1A58 is efficiently introduced on unmodified tRNAiMet, and does not depend on prior modifications. Finally, we show that m1A58 has major effects on the structural properties of initiator tRNAiMet, so that the tRNA elbow structure is only properly assembled when this modification is present. This observation provides a structural explanation for the degradation of hypomodified tRNAiMet lacking m1A58 by the nuclear surveillance and RTD pathways.

Combining morphological and mitochondrial DNA data to describe a new species of Austroniscus Vanhöffen, 1914 (Isopoda, Janiroidea, Nannoniscidae) linking abyssal and hadal depths of the Puerto Rico Trench.

Hadal trenches are perceived as a unique deep-sea ecosystem with fundamentally different communities compared to the nearby abyss. So far, however, scarce information exists about how populations are genetically linked within a trench and about mechanisms for species divergence. The present study presents the morphological and molecular-genetic characterization and description of a new nannoniscid species within the genus Austroniscus Vanhöffen, 1914 obtained from abyssal and hadal depths of the Puerto Rico Trench, NW Atlantic. Samples were collected as part of the Vema-TRANSIT expedition onboard RV Sonne in January 2015. Because of the large depth differences between sampling locations (4,552-8,338 m), we expected to find different species within the genus inhabiting abyssal and hadal sites. Initial morphological examination using traditional light microscopy and Confocal Laser Scanning Microscopy was paired with subsequent molecular analysis based on mtDNA (COI and 16S). Contrary to our assumptions, combined morphological and molecular species delimitation analyses (sGMYC, mPTP, ABGD) revealed the presence of only one species spanning the abyssal and hadal seafloor of the Puerto Rico Trench. In addition, comparison with type material could show that this species belongs to a new species, Austroniscus brandtae n. sp., which is described herein. Incongruence between some species delimitation methods suggesting the presence of multiple species is interpreted as strong genetic population structuring within the trench, which is also supported by the analysis of the haplotype networks. The geographic and bathymetric distribution of Austroniscus species is discussed. The species described herein represents the first in the genus Austroniscus from the Atlantic Ocean and the deepest record of the genus to date, and hence significantly expanding previously known limits of its geographic and bathymetric range.

The plant cytosolic m6A RNA methylome stabilizes photosynthesis in the cold.

The sessile lifestyle of plants requires an immediate response to environmental stressors that affect photosynthesis, growth, and crop yield. Here, we showed that three abiotic perturbations-heat, cold, and high light-triggered considerable changes in the expression signatures of 42 epitranscriptomic factors (writers, erasers, and readers) with putative chloroplast-associated functions that formed clusters of commonly expressed genes in Arabidopsis. The expression changes under all conditions were reversible upon deacclimation, identifying epitranscriptomic players as modulators in acclimation processes. Chloroplast dysfunctions, particularly those induced by the oxidative stress-inducing norflurazon in a largely GENOME UNCOUPLED-independent manner, triggered retrograde signals to remodel chloroplast-associated epitranscriptomic expression patterns. N6-methyladenosine (m6A) is known as the most prevalent RNA modification and impacts numerous developmental and physiological functions in living organisms. During cold treatment, expression of components of the primary nuclear m6A methyltransferase complex was upregulated, accompanied by a significant increase in cellular m6A mRNA marks. In the cold, the presence of FIP37, a core component of the writer complex, played an important role in positive regulation of thylakoid structure, photosynthetic functions, and accumulation of photosystem I, the Cytb6f complex, cyclic electron transport proteins, and Curvature Thylakoid1 but not that of photosystem II components and the chloroplast ATP synthase. Downregulation of FIP37 affected abundance, polysomal loading, and translation of cytosolic transcripts related to photosynthesis in the cold, suggesting m6A-dependent translational regulation of chloroplast functions. In summary, we identified multifaceted roles of the cellular m6A RNA methylome in coping with cold; these were predominantly associated with chloroplasts and served to stabilize photosynthesis.

Temporal resolution of NAIL-MS of tRNA, rRNA and Poly-A RNA is overcome by actinomycin D.

RNA is dynamically modified and has the potential to respond to environmental changes and tune translation. The objective of this work is to uncover the temporal limitation of our recently developed cell culture NAIL-MS (nucleic acid isotope labelling coupled mass spectrometry) technology and overcome it. Actinomycin D (AcmD), an inhibitor of transcription, was used in the NAIL-MS context to reveal the origin of hybrid nucleoside signals composed of unlabelled nucleosides and labelled methylation marks. We find that the formation of these hybrid species depends exclusively on transcription for Poly-A RNA and rRNA but is partly transcription-independent for tRNA. This finding suggests that tRNA modifications adapt and are dynamically regulated by cells to overcome e.g. stress. Future studies on the tRNA modification mediated stress response are now accessible and the temporal resolution of NAIL-MS is improved by the use of AcmD.

Northernmost (Subarctic) and deepest record of Paleodictyon: paleoecological and biological implications.

Paleodictyon is one of the most iconic and widespread of trace fossils in the geological record. However, modern examples are less well known and restricted to deep-sea settings at relatively low latitudes. Here, we report the distribution of Paleodictyon at six abyssal sites near the Aleutian Trench. This study reveals for the first time the presence of Paleodictyon at Subarctic latitudes (51°-53°N) and at depths over 4500 m, although the traces were not observed at stations deeper than 5000 m suggesting that there is some bathymetric constraint for the trace maker. Two small Paleodictyon morphotypes were recognized (average mesh size of 1.81 cm), one having a central hexagonal pattern, the other being characterized by a non-hexagonal pattern. Within the study area, Paleodictyon shows no apparent correlation with local environmental parameters. Finally, based on a worldwide morphological comparison, we conclude that the new Paleodictyon specimens represent distinct ichnospecies that are associated with the relatively eutrophic conditions in this region. Their smaller size may reflect this more eutrophic setting in which sufficient food can be obtained from a smaller area in order to satisfy the energetic requirements of the tracemakers. If so, then Paleodictyon size may provide some assistance when interpreting paleoenvironmental conditions.

The SPOC domain is a phosphoserine binding module that bridges transcription machinery with co- and post-transcriptional regulators.

The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3'end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of m6A, the most abundant RNA modification. RBM15 positively regulates m6A levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes.

Comprehensive comparison between azacytidine and decitabine treatment in an acute myeloid leukemia cell line.

Azacytidine (AzaC) and decitabine (AzadC) are cytosine analogs that covalently trap DNA methyltransferases, which place the important epigenetic mark 5-methyl-2'-deoxycytidine by methylating 2'-deoxycytidine (dC) at the C5 position. AzaC and AzadC are used in the clinic as antimetabolites to treat myelodysplastic syndrome and acute myeloid leukemia and are explored against other types of cancer. Although their principal mechanism of action is known, the downstream effects of AzaC and AzadC treatment are not well understood and the cellular prerequisites that determine sensitivity toward AzaC and AzadC remain elusive. Here, we investigated the effects and phenotype of AzaC and AzadC exposure on the acute myeloid leukemia cell line MOLM-13. We found that while AzaC and AzadC share many effects on the cellular level, including decreased global DNA methylation, increased formation of DNA double-strand breaks, transcriptional downregulation of important oncogenes and similar changes on the proteome level, AzaC failed in contrast to AzadC to induce apoptosis efficiently in MOLM-13. The only cellular marker that correlated with this clear phenotypical outcome was the level of hydroxy-methyl-dC, an additional epigenetic mark that is placed by TET enzymes and repressed in cancer cells. Whereas AzadC increased hmdC substantially in MOLM-13, AzaC treatment did not result in any increase at all. This suggests that hmdC levels in cancer cells should be monitored as a response toward AzaC and AzadC and considered as a biomarker to judge whether AzaC or AzadC treatment leads to cell death in leukemic cells.

The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells.

T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.

Opportunities and Challenges to Profile mRNA Modifications in Escherichia†coli.

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. Working with bacterial mRNA is challenging because it lacks a poly(A)-tail. However, methods for detecting RNA modifications, both sequencing and mass spectrometry, rely on efficient preparation of mRNA. Here, we compared size-dependent separation by electrophoresis and rRNA depletion for enrichment of Escherichia coli mRNA. The purification success was monitored by qRT-PCR and RNA sequencing. Neither method allowed complete removal of rRNA. Nevertheless, we were able to quantitatively analyze several modified nucleosides in the different RNA types. We found evidence for stress dependent RNA modification reprofiling in rRNA, but also several modified nucleosides in the mRNA enriched fractions showed significant changes.

Habitat variability and faunal zonation at the Ægir Ridge, a canyon-like structure in the deep Norwegian Sea.

The Ægir Ridge System (ARS) is an ancient extinct spreading axis in the Nordic seas extending from the upper slope east of Iceland (∼550 m depth), as part of its Exclusive Economic Zone (EEZ), to a depth of ∼3,800 m in the Norwegian basin. Geomorphologically a rift valley, the ARS has a canyon-like structure that may promote increased diversity and faunal density. The main objective of this study was to characterize benthic habitats and related macro- and megabenthic communities along the ARS, and the influence of water mass variables and depth on them. During the IceAGE3 expedition (Icelandic marine Animals: Genetics and Ecology) on RV Sonne in June 2020, benthic communities of the ARS were surveyed by means of a remotely-operated vehicle (ROV) and epibenthic sledge (EBS). For this purpose, two working areas were selected, including abyssal stations in the northeast and bathyal stations in the southwest of the ARS. Video and still images of the seabed were usedtoqualitatively describebenthic habitats based on the presence of habitat-forming taxa and the physical environment. Patterns of diversity and community composition of the soft-sediment macrofauna, retrieved from the EBS, were analyzed in a semiquantitative manner. These biological data were complemented by producing high-resolution bathymetric maps using the vessel's multi-beam echosounder system. As suspected, we were able to identify differences in species composition and number of macro- and megafaunal communities associated with a depth gradient. A biological canyon effect became evident in dense aggregates of megafaunal filter feeders and elevated macrofaunal densities. Analysis of videos and still images from the ROV transects also led to the discovery of a number ofVulnerable Marine Ecosystems (VMEs) dominated by sponges and soft corals characteristic of the Arctic region. Directions for future research encompass a more detailed, quantitative study of the megafauna and more coherent sampling over the entire depth range in order to fully capture the diversity of the habitats and biota of the region. The presence of sensitive biogenic habitats, alongside seemingly high biodiversity and naturalness are supportive of ongoing considerations of designating part of the ARS as an "Ecologically and Biologically Significant Area" (EBSA).

Niche breadth and biodiversity change derived from marine Amphipoda species off Iceland.

Understanding the ecological requirements and thresholds of individual species is crucial to better predict potential outcomes of climate change on species distribution. In particular, species optima and lower and upper limits along resource gradients require attention. Based on Huisman-Olff-Fresco (HOF) models, we determined species-specific responses along gradients of nine environmental parameters including depth in order to estimate niche attributes of 30 deep-sea benthic amphipods occurring around Iceland. We, furthermore, examined the relationships between niche breadth, occupancy, and geographic range assuming that species with a wider niche are spatially more widely dispersed and vice versa. Overall, our results reveal that species react very differently to environmental gradients, which is independent of the family affiliation of the respective species. We could infer a strong relationship between occupancy and geographic range and also relate this to differences in niche breadth; that is specialist species with a narrow niche had a more limited distribution and may thus be more threatened by changing environmental conditions than generalist species, which are more widespread. Given the preponderance of rare species in the deep sea, this implies that many species could be at risk. However, this must be carefully weighed against geographical data gaps in this area, given that many deep-sea areas are severely undersampled and the true distribution of most species is unknown. After all, our results underline that an accurate taxonomic classification is of crucial importance, without which ecological niche properties cannot be determined and which is hence fundamental for the assessment and understanding of changes in biodiversity in the face of increasing human perturbations.

Deep seabed mining and communities: A transdisciplinary approach to ecological risk assessment in the South Pacific.

Deep-sea mineral extraction is a fledgling industry whose guiding principles, legislation, protocols, and regulations are still evolving. Responsible management of the industry is difficult when it is not clearly understood what biological and environmental diversity or ecosystem services may be at risk. But the industry's infancy provides an opportunity to address this challenge by stakeholder-led development and implementation of a multidisciplinary risk assessment framework. This article aims to present the findings of a workshop held in New Zealand that hosted stakeholders from a broad range of interests and regions in the South Pacific associated with the deep-sea mineral activity. The outputs provide stakeholder-informed ecological risk assessment approaches for deep-sea mining activities, identifying tools and techniques to improve the relevance of risk assessment of deep seabed mining projects to communities in the South Pacific. Discussions highlighted the importance of trust or respect among stakeholders, valuing the "life force" of the ocean, the importance of scientific data, and the complications associated with defining acceptable change. This research highlighted the need for a holistic transdisciplinary approach that connects science, management, industry, and community, an approach most likely to provide a "social license" to operate. There is also a need to revise traditional risk assessment methods to make them more relevant to stakeholders. The development of ecotoxicological tools and approaches is an example of how existing practices could be improved to better support deep-sea mineral management. A case study is provided that highlights the current challenges within the legislative framework of New Zealand. Integr Environ Assess Manag 2022;18:664-673. © 2021 SETAC.