The curious case of the disappearing piRNAs.

Small non-coding RNAs are key regulators of gene expression across eukaryotes. Piwi-interacting small RNAs (piRNAs) are a specific type of small non-coding RNAs, conserved across animals, which are best known as regulators of genome stability through their ability to target transposable elements for silencing. Despite the near ubiquitous presence of piRNAs in animal lineages, there are some examples where the piRNA pathway has been lost completely, most dramatically in nematodes where loss has occurred in at least four independent lineages. In this perspective I will provide an evaluation of the presence of piRNAs across animals, explaining how it is known that piRNAs are missing from certain organisms. I will then consider possible explanations for why the piRNA pathway might have been lost and evaluate the evidence in favor of each possible mechanism. While it is still impossible to provide definitive answers, these theories will prompt further investigations into why such a highly conserved pathway can nevertheless become dispensable in certain lineages. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

An Overview of Current Detection Methods for RNA Methylation.

Epitranscriptomic mechanisms, which constitute an important layer in post-transcriptional gene regulation, are involved in numerous cellular processes under health and disease such as stem cell development or cancer. Among various such mechanisms, RNA methylation is considered to have vital roles in eukaryotes primarily due to its dynamic and reversible nature. There are numerous RNA methylations that include, but are not limited to, 2'-O-dimethyladenosine (m6Am), N7-methylguanosine (m7G), N6-methyladenosine (m6A) and N1-methyladenosine (m1A). These biochemical modifications modulate the fate of RNA by affecting the processes such as translation, target site determination, RNA processing, polyadenylation, splicing, structure, editing and stability. Thus, it is highly important to quantitatively measure the changes in RNA methylation marks to gain insight into cellular processes under health and disease. Although there are complicating challenges in identifying certain methylation marks genome wide, various methods have been developed recently to facilitate the quantitative measurement of methylated RNAs. To this end, the detection methods for RNA methylation can be classified in five categories such as antibody-based, digestion-based, ligation-based, hybridization-based or direct RNA-based methods. In this review, we have aimed to summarize our current understanding of the detection methods for RNA methylation, highlighting their advantages and disadvantages, along with the current challenges in the field.

From cyanobacteria and cyanophages to chloroplasts: the fate of the genomes of oxyphototrophs and the genes encoding photosystem II proteins.

Chloroplasts are the result of endosymbiosis of cyanobacterial organisms with proto-eukaryotes. The psbA, psbD and psbO genes are present in all oxyphototrophs and encode the D1/D2 proteins of photosystem II (PSII) and PsbO, respectively. PsbO is a peripheral protein that stabilizes the O2-evolving complex in PSII. Of these genes, psbA and psbD remained in the chloroplastic genome, while psbO was transferred to the nucleus. The genomes of selected cyanobacteria, chloroplasts and cyanophages carrying psbA and psbD, respectively, were analysed. The highest density of genes and coding sequences (CDSs) was estimated for the genomes of cyanophages, cyanobacteria and chloroplasts. The synonymous mutation rate (rS) of psbA and psbD in chloroplasts remained almost unchanged and is lower than that of psbO. The results indicate that the decreasing genome size in chloroplasts is more similar to the genome reduction observed in contemporary endosymbiotic organisms than in streamlined genomes of free-living cyanobacteria. The rS of atpA, which encodes the α-subunit of ATP synthase in chloroplasts, suggests that psbA and psbD, and to a lesser extent psbO, are ancient and conservative and arose early in the evolution of oxygenic photosynthesis. The role of cyanophages in the evolution of oxyphototrophs and chloroplastic genomes is discussed.

Discovery of a Potent and Cell-Active Inhibitor of DNA 6mA Demethylase ALKBH1.

N6-Methyladenine (6mA) of DNA has emerged as a novel epigenetic mark in eukaryotes, and several 6mA effector proteins have been identified. However, efforts to selectively inhibit the biological functions of these effector proteins with small molecules are unsuccessful to date. Here we report the first potent and selective small molecule inhibitor (13h) of AlkB homologue 1 (ALKBH1), the only validated 6mA demethylase. 13h showed an IC50 of 0.026 ± 0.013 µM and 1.39 ± 0.13 µM in the fluorescence polarization (FP) and enzyme activity assay, respectively, and a KD of 0.112 ± 0.017 µM in the isothermal titration calorimetry (ITC) assay. The potency of 13h was well explained by the cocrystal structure of the 13h-ALKBH1 complex. Furthermore, 13h displayed excellent selectivity for ALKBH1. In cells, compound 13h and its derivative 16 were able to engage ALKBH1 and modulate the 6mA levels. Collectively, our study identified the first potent, isoform selective, and cell-active ALKBH1 inhibitor, providing a tool compound for exploring the biological functions of ALKBH1 and DNA 6mA.

Poly(A) tale: From A to A; RNA polyadenylation in prokaryotes and eukaryotes.

Most eukaryotic mRNAs and different non-coding RNAs undergo a form of 3' end processing known as polyadenylation. Polyadenylation machinery is present in almost all organisms except few species. In bacteria, the machinery has evolved from PNPase, which adds heteropolymeric tails, to a poly(A)-specific polymerase. Differently, a complex machinery for accurate polyadenylation and several non-canonical poly(A) polymerases are developed in eukaryotes. The role of poly(A) tail has also evolved from serving as a degradative signal to a stabilizing modification that also regulates translation. In this review, we discuss poly(A) tail emergence in prokaryotes and its development into a stable, yet dynamic feature at the 3' end of mRNAs in eukaryotes. We also describe how appearance of novel poly(A) polymerases gives cells flexibility to shape poly(A) tail. We explain how poly(A) tail dynamics help regulate cognate RNA metabolism in a context-dependent manner, such as during oocyte maturation. Finally, we describe specific mRNAs in metazoans that bear stem-loops instead of poly(A) tails. We conclude with how recent discoveries about poly(A) tail can be applied to mRNA technology. This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Processing > 3' End Processing RNA Turnover and Surveillance > Regulation of RNA Stability.

The unique dual targeting of AGO1 by two types of PRMT enzymes promotes phasiRNA loading in Arabidopsis thaliana.

Arginine/R methylation (R-met) of proteins is a widespread post-translational modification (PTM), deposited by a family of protein arginine/R methyl transferase enzymes (PRMT). Regulations by R-met are involved in key biological processes deeply studied in metazoan. Among those, post-transcriptional gene silencing (PTGS) can be regulated by R-met in animals and in plants. It mainly contributes to safeguard processes as protection of genome integrity in germlines through the regulation of piRNA pathway in metazoan, or response to bacterial infection through the control of AGO2 in plants. So far, only PRMT5 has been identified as the AGO/PIWI R-met writer in higher eukaryotes. We uncovered that AGO1, the main PTGS effector regulating plant development, contains unique R-met features among the AGO/PIWI superfamily, and outstanding in eukaryotes. Indeed, AGO1 contains both symmetric (sDMA) and asymmetric (aDMA) R-dimethylations and is dually targeted by PRMT5 and by another type I PRMT in Arabidopsis thaliana. We showed also that loss of sDMA didn't compromise AtAGO1 subcellular trafficking in planta. Interestingly, we underscored that AtPRMT5 specifically promotes the loading of phasiRNA in AtAGO1. All our observations bring to consider this dual regulation of AtAGO1 in plant development and response to environment, and pinpoint the complexity of AGO1 post-translational regulation.

CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

Nucleosomes at the Dawn of Eukaryotes.

Genome regulation in eukaryotes revolves around the nucleosome, the fundamental building block of eukaryotic chromatin. Its constituent parts, the four core histones (H3, H4, H2A, H2B), are universal to eukaryotes. Yet despite its exceptional conservation and central role in orchestrating transcription, repair, and other DNA-templated processes, the origins and early evolution of the nucleosome remain opaque. Histone-fold proteins are also found in archaea, but the nucleosome we know-a hetero-octameric complex composed of histones with long, disordered tails-is a hallmark of eukaryotes. What were the properties of the earliest nucleosomes? Did ancestral histones inevitably assemble into nucleosomes? When and why did the four core histones evolve? This review will look at the evolution of the eukaryotic nucleosome from the vantage point of archaea, focusing on the key evolutionary transitions required to build a modern nucleosome. We will highlight recent work on the closest archaeal relatives of eukaryotes, the Asgardarchaea, and discuss what their histones can and cannot tell us about the early evolution of eukaryotic chromatin. We will also discuss how viruses have become an unexpected source of information about the evolutionary path toward the nucleosome. Finally, we highlight the properties of early nucleosomes as an area where new tools and data promise tangible progress in the not-too-distant future.

The Sphinx and the egg: Evolutionary enigmas of the (glyco)sphingolipid biosynthetic pathway.

In eukaryotes, the de novo synthesis of sphingolipids (SLs) consists of multiple sequential steps which are compartmentalized between the endoplasmic reticulum and the Golgi apparatus. Studies over many decades have identified the enzymes in the pathway, their localization, topology and an array of regulatory mechanisms. However, little is known about the evolutionary forces that underly the generation of this complex pathway or of its anteome, i.e., the metabolic pathways that converge on the SL biosynthetic pathway and are essential for its activity. After briefly describing the pathway, we discuss the mechanisms by which the enzymes of the SL biosynthetic pathway are targeted to their different subcellular locations, how the pathway per se may have evolved, including its compartmentalization, and the relationship of the pathway to eukaryogenesis. We discuss the circular interdependence of the evolution of the SL pathway, and comment on whether current Darwinian evolutionary models are able to provide genuine mechanistic insight into how the pathway came into being.

TOR in plants: Multidimensional regulators of plant growth and signaling pathways.

Target Of Rapamycin (TOR) represents a ubiquitous kinase complex that has emerged as a central regulator of cell growth and metabolism in nearly all eukaryotic organisms. TOR is an evolutionarily conserved protein kinase, functioning as a central signaling hub that integrates diverse internal and external cues to regulate a multitude of biological processes. These processes collectively exert significant influence on plant growth, development, nutrient assimilation, photosynthesis, fruit ripening, and interactions with microorganisms. Within the plant domain, the TOR complex comprises three integral components: TOR, RAPTOR, and LST8. This comprehensive review provides insights into various facets of the TOR protein, encompassing its origin, structure, function, and the regulatory and signaling pathways operative in photosynthetic organisms. Additionally, we explore future perspectives related to this pivotal protein kinase.

Prevotella copri variants among a single host diverge in sphingolipid production.

Sphingolipids serve as vital structural and signaling components of the cell membranes in both eukaryotes and prokaryotes. Within the gut microbiome, Bacteroides species have been identified as major producers of sphingolipids, and Bacteroides-produced sphingolipids have been shown to be modulators of host immune and metabolic functions. While Bacteroides species are a prominent feature of the gut microbiomes of populations living in industrialized countries, Prevotella copri, a member of the same phyla, albeit a different family, is the dominant feature across the remainder of the global population, although their sphingolipid-producing capabilities have not been as thoroughly investigated. To fill this gap, we examined the genomes of over 60 diverse isolates of P. copri and identified several key enzymes involved in sphingolipid synthesis in P. copri. Combining bioorthogonal labeling and liquid chromatography-mass spectrometry (LC-MS) based lipidomics, we functionally characterized the first step in P. copri de novo sphingolipid synthesis in addition to profiling the sphingolipidomes of P. copri strains, identifying key enzymes that may play roles in producing a diverse set of P. copri sphingolipids. Given the limited genetic engineering tools amenable for use in P. copri, our approach takes advantage of comparative genomics and phenotypic profiling to explore sphingolipid production in these understudied, yet highly prevalent, organisms.IMPORTANCESphingolipids are important signaling molecules for maintaining metabolic and immune homeostasis in the host. These lipids are also produced by gut commensals, most notably by Bacteroides species. Despite the global prevalence of Prevotella copri in gut microbiomes of individuals, little is known about the types of sphingolipids they produce and whether they are similar in composition and structure to those produced by Bacteroides. Given the varied associations of P. copri with diverse sphingolipid-related health outcomes, such as rheumatoid arthritis and glucose intolerance, it is important to first characterize the specific sphingolipids produced by individual strains of P. copri and to identify the genes involved in their pathways of production. This characterization of P. copri-derived sphingolipids provides further insight into how bacterial sphingolipid production can serve as a mechanism for microbial modulation of host phenotypes.

Functional analysis of the AUG initiator codon context reveals novel conserved sequences that disfavor mRNA translation in eukaryotes.

mRNA translation is a fundamental process for life. Selection of the translation initiation site (TIS) is crucial, as it establishes the correct open reading frame for mRNA decoding. Studies in vertebrate mRNAs discovered that a purine at -3 and a G at +4 (where A of the AUG initiator codon is numbered + 1), promote TIS recognition. However, the TIS context in other eukaryotes has been poorly experimentally analyzed. We analyzed in vitro the influence of the -3, -2, -1 and + 4 positions of the TIS context in rabbit, Drosophila, wheat, and yeast. We observed that -3A conferred the best translational efficiency across these species. However, we found variability at the + 4 position for optimal translation. In addition, the Kozak motif that was defined from mammalian cells was only weakly predictive for wheat and essentially non-predictive for yeast. We discovered eight conserved sequences that significantly disfavored translation. Due to the big differences in translational efficiency observed among weak TIS context sequences, we define a novel category that we termed 'barren AUG context sequences (BACS)', which represent sequences disfavoring translation. Analysis of mRNA-ribosomal complexes structures provided insights into the function of BACS. The gene ontology of the BACS-containing mRNAs is presented.

Dynamics and interactions of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are widespread in eukaryotes and participate in a variety of important cellular processes. Numerous studies using state-of-the-art experimental and theoretical methods have advanced our understanding of IDPs and revealed that disordered regions engage in a large repertoire of intra- and intermolecular interactions through their conformational dynamics, thereby regulating many intracellular functions in concert with folded domains. The mechanisms by which IDPs interact with their partners are diverse, depending on their conformational propensities, and include induced fit, conformational selection, and their mixtures. In addition, IDPs are implicated in many diseases, and progress has been made in designing inhibitors of IDP-mediated interactions. Here we review these recent advances with a focus on the dynamics and interactions of IDPs.

Bimodular architecture of bacterial effector SAP05 that drives ubiquitin-independent targeted protein degradation.

In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously identified that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05-Rpn10vWA complex at 2.17 Å resolution and of the SAP05-SPL5ZnF complex at 2.20 Å resolution. Structural analyses revealed that SAP05 displays a remarkable bimodular architecture with two distinct nonoverlapping surfaces, a "loop surface" with three protruding loops that form electrostatic interactions with ZnF, and a "sheet surface" featuring two ß-sheets, loops, and α-helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical ubiquitin-proteasome proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of TPD technologies.

Review of Eukaryote Cellular Membrane Lipid Composition, with Special Attention to the Fatty Acids.

Biological membranes, primarily composed of lipids, envelop each living cell. The intricate composition and organization of membrane lipids, including the variety of fatty acids they encompass, serve a dynamic role in sustaining cellular structural integrity and functionality. Typically, modifications in lipid composition coincide with consequential alterations in universally significant signaling pathways. Exploring the various fatty acids, which serve as the foundational building blocks of membrane lipids, provides crucial insights into the underlying mechanisms governing a myriad of cellular processes, such as membrane fluidity, protein trafficking, signal transduction, intercellular communication, and the etiology of certain metabolic disorders. Furthermore, comprehending how alterations in the lipid composition, especially concerning the fatty acid profile, either contribute to or prevent the onset of pathological conditions stands as a compelling area of research. Hence, this review aims to meticulously introduce the intricacies of membrane lipids and their constituent fatty acids in a healthy organism, thereby illuminating their remarkable diversity and profound influence on cellular function. Furthermore, this review aspires to highlight some potential therapeutic targets for various pathological conditions that may be ameliorated through dietary fatty acid supplements. The initial section of this review expounds on the eukaryotic biomembranes and their complex lipids. Subsequent sections provide insights into the synthesis, membrane incorporation, and distribution of fatty acids across various fractions of membrane lipids. The last section highlights the functional significance of membrane-associated fatty acids and their innate capacity to shape the various cellular physiological responses.

Comparison of cysteine content in whole proteomes across the three domains of life.

An empirical observation suggests that Giardia lamblia proteins have larger cysteine content than their counterparts in other organisms. As this parasite lacks conventional antioxidant stress systems, it is generally accepted that high cysteine content helps G. lamblia cope with oxygen toxicity, a strategy apparently shared by other organisms. Here, we question whether the high cysteine content in some organisms is genuine or just a simple assumption based on singular observations. To this end, we analyzed the cysteine content in 78 proteomes of organisms spanning the three domains of life. The results indicate that the cysteine content in eukaryota is approximately double that in archaea and bacteria, with G. lamblia among the highest. Atypical cysteine contents were found in a few organisms correlating with specific environmental conditions, supporting the evolutionary amino acid-level selection of amino acid composition.

Phylogenetic and Protein Structure Analyses Provide Insight into the Evolution and Diversification of the CD36 Domain "Apex" among Scavenger Receptor Class B Proteins across Eukarya.

The cluster of differentiation 36 (CD36) domain defines the characteristic ectodomain associated with class B scavenger receptor (SR-B) proteins. In bilaterians, SR-Bs play critical roles in diverse biological processes including innate immunity functions such as pathogen recognition and apoptotic cell clearance, as well as metabolic sensing associated with fatty acid uptake and cholesterol transport. Although previous studies suggest this protein family is ancient, SR-B diversity across Eukarya has not been robustly characterized. We analyzed SR-B homologs identified from the genomes and transcriptomes of 165 diverse eukaryotic species. The presence of highly conserved amino acid motifs across major eukaryotic supergroups supports the presence of a SR-B homolog in the last eukaryotic common ancestor. Our comparative analyses of SR-B protein structure identify the retention of a canonical asymmetric beta barrel tertiary structure within the CD36 ectodomain across Eukarya. We also identify multiple instances of independent lineage-specific sequence expansions in the apex region of the CD36 ectodomain-a region functionally associated with ligand-sensing. We hypothesize that a combination of both sequence expansion and structural variation in the CD36 apex region may reflect the evolution of SR-B ligand-sensing specificity between diverse eukaryotic clades.

Autophagy and Inflammation: Regulatory Roles in Viral Infections.

Autophagy is a highly conserved intracellular degradation pathway in eukaryotic organisms, playing an adaptive role in various pathophysiological processes throughout evolution. Inflammation is the immune system's response to external stimuli and tissue damage. However, persistent inflammatory reactions can lead to a range of inflammatory diseases and cancers. The interaction between autophagy and inflammation is particularly evident during viral infections. As a crucial regulator of inflammation, autophagy can either promote or inhibit the occurrence of inflammatory responses. In turn, inflammation can establish negative feedback loops by modulating autophagy to suppress excessive inflammatory reactions. This interaction is pivotal in the pathogenesis of viral diseases. Therefore, elucidating the regulatory roles of autophagy and inflammation in viral infections will significantly enhance our understanding of the mechanisms underlying related diseases. Furthermore, it will provide new insights and theoretical foundations for disease prevention, treatment, and drug development.

Systematic identification of conditionally folded intrinsically disordered regions by AlphaFold2.

The AlphaFold Protein Structure Database contains predicted structures for millions of proteins. For the majority of human proteins that contain intrinsically disordered regions (IDRs), which do not adopt a stable structure, it is generally assumed that these regions have low AlphaFold2 confidence scores that reflect low-confidence structural predictions. Here, we show that AlphaFold2 assigns confident structures to nearly 15% of human IDRs. By comparison to experimental NMR data for a subset of IDRs that are known to conditionally fold (i.e., upon binding or under other specific conditions), we find that AlphaFold2 often predicts the structure of the conditionally folded state. Based on databases of IDRs that are known to conditionally fold, we estimate that AlphaFold2 can identify conditionally folding IDRs at a precision as high as 88% at a 10% false positive rate, which is remarkable considering that conditionally folded IDR structures were minimally represented in its training data. We find that human disease mutations are nearly fivefold enriched in conditionally folded IDRs over IDRs in general and that up to 80% of IDRs in prokaryotes are predicted to conditionally fold, compared to less than 20% of eukaryotic IDRs. These results indicate that a large majority of IDRs in the proteomes of human and other eukaryotes function in the absence of conditional folding, but the regions that do acquire folds are more sensitive to mutations. We emphasize that the AlphaFold2 predictions do not reveal functionally relevant structural plasticity within IDRs and cannot offer realistic ensemble representations of conditionally folded IDRs.