Perfect and imperfect views of ultraconserved sequences.

Across the human genome, there are nearly 500 'ultraconserved' elements: regions of at least 200 contiguous nucleotides that are perfectly conserved in both the mouse and rat genomes. Remarkably, the majority of these sequences are non-coding, and many can function as enhancers that activate tissue-specific gene expression during embryonic development. From their first description more than 15 years ago, their extreme conservation has both fascinated and perplexed researchers in genomics and evolutionary biology. The intrigue around ultraconserved elements only grew with the observation that they are dispensable for viability. Here, we review recent progress towards understanding the general importance and the specific functions of ultraconserved sequences in mammalian development and human disease and discuss possible explanations for their extreme conservation.

Conserved long-range base pairings are associated with pre-mRNA processing of human genes.

The ability of nucleic acids to form double-stranded structures is essential for all living systems on Earth. Current knowledge on functional RNA structures is focused on locally-occurring base pairs. However, crosslinking and proximity ligation experiments demonstrated that long-range RNA structures are highly abundant. Here, we present the most complete to-date catalog of conserved complementary regions (PCCRs) in human protein-coding genes. PCCRs tend to occur within introns, suppress intervening exons, and obstruct cryptic and inactive splice sites. Double-stranded structure of PCCRs is supported by decreased icSHAPE nucleotide accessibility, high abundance of RNA editing sites, and frequent occurrence of forked eCLIP peaks. Introns with PCCRs show a distinct splicing pattern in response to RNAPII slowdown suggesting that splicing is widely affected by co-transcriptional RNA folding. The enrichment of 3'-ends within PCCRs raises the intriguing hypothesis that coupling between RNA folding and splicing could mediate co-transcriptional suppression of premature pre-mRNA cleavage and polyadenylation.

Intrinsic toxicity of the cellular prion protein is regulated by its conserved central region.

The conserved central region (CR) of PrPC has been hypothesized to serve as a passive linker connecting the protein's toxic N-terminal and globular C-terminal domains. Yet, deletion of the CR causes neonatal fatality in mice, implying the CR possesses a protective function. The CR encompasses the regulatory α-cleavage locus, and additionally facilitates a regulatory metal ion-promoted interaction between the PrPC N- and C-terminal domains. To elucidate the role of the CR and determine why CR deletion generates toxicity, we designed PrPC constructs wherein either the cis-interaction or α-cleavage are selectively prevented. These constructs were interrogated using nuclear magnetic resonance, electrophysiology, and cell viability assays. Our results demonstrate the CR is not a passive linker and the native sequence is crucial for its protective role over the toxic N-terminus, irrespective of α-cleavage or the cis-interaction. Additionally, we find that the CR facilitates homodimerization of PrPC , attenuating the toxicity of the N-terminus.

Plant organellar DNA polymerases bypass thymine glycol using two conserved lysine residues.

Plant organelles cope with endogenous DNA damaging agents, byproducts of respiration and photosynthesis, and exogenous agents like ultraviolet light. Plant organellar DNA polymerases (DNAPs) are not phylogenetically related to yeast and metazoan DNAPs and they harbor three insertions not present in any other DNAPs. Plant organellar DNAPs from Arabidopsis thaliana (AtPolIA and AtPolIB) are translesion synthesis (TLS) DNAPs able to bypass abasic sites, a lesion that poses a strong block to replicative polymerases. Besides abasic sites, reactive oxidative species and ionizing radiation react with thymine resulting in thymine glycol (Tg), a DNA adduct that is also a strong block to replication. Here, we report that AtPolIA and AtPolIB bypass Tg by inserting an adenine opposite the lesion and efficiently extend from a Tg-A base pair. The TLS ability of AtPolIB is mapped to two conserved lysine residues: K593 and K866. Residue K593 is situated in insertion 1 and K866 is in insertion 3. With basis on the location of both insertions on a structural model of AtPolIIB, we hypothesize that the two positively charged residues interact to form a clamp around the primer-template. In contrast with nuclear and bacterial replication, where lesion bypass involves an interplay between TLS and replicative DNA polymerases, we postulate that plant organellar DNAPs evolved to exert replicative and TLS activities.

Conservation and innovation: Plastome evolution during rapid radiation of Rhodiola on the Qinghai-Tibetan Plateau.

The amount of plastome sequence data available has soared in the last decade, but the nature of plastome evolution during rapid radiations is largely unknown. Moreover, although there is increasing evidence showing that plastomes may have undergone adaptive evolution in order to allow adaptation to various environments, few studies have systematically investigated the role of the plastome in alpine adaptation. To address these questions, we sequenced and analyzed 12 representative species of Rhodiola, a genus which includes ca. 70 perennial herbs mainly growing in alpine habitats in the Qinghai-Tibet Plateau and the Hengduan Mountains. Rapid radiation in this genus was triggered by the uplift of the Qinghai-Tibet Plateau. We also included nine species of Crassulaceae as the outgroups. All plastomes were conserved with respect to size, structure, and gene content and order, with few variations: each contained 134 genes, including 85 protein-coding genes, 37 tRNAs, 8 rRNAs, and 4 potential pseudogenes. Four types of repeat sequence were detected. Slight contraction and expansion of the inverted repeats were also revealed. Both the genome-wide alignment and sequence polymorphism analyses showed that the inverted repeats and coding regions were more conserved than the single-copy regions and the non-coding regions. Positive selection analyses identified three genes containing sites of positive selection (rpl16, ndhA, ndhH), and one gene with a faster than average rate of evolution (psaA). The products of these genes may be involved in the adaptation of Rhodiola to alpine environments such as low CO2 concentration and high-intensity light.

A Hox-TALE regulatory circuit for neural crest patterning is conserved across vertebrates.

In jawed vertebrates (gnathostomes), Hox genes play an important role in patterning head and jaw formation, but mechanisms coupling Hox genes to neural crest (NC) are unknown. Here we use cross-species regulatory comparisons between gnathostomes and lamprey, a jawless extant vertebrate, to investigate conserved ancestral mechanisms regulating Hox2 genes in NC. Gnathostome Hoxa2 and Hoxb2 NC enhancers mediate equivalent NC expression in lamprey and gnathostomes, revealing ancient conservation of Hox upstream regulatory components in NC. In characterizing a lamprey hoxα2 NC/hindbrain enhancer, we identify essential Meis, Pbx, and Hox binding sites that are functionally conserved within Hoxa2/Hoxb2 NC enhancers. This suggests that the lamprey hoxα2 enhancer retains ancestral activity and that Hoxa2/Hoxb2 NC enhancers are ancient paralogues, which diverged in hindbrain and NC activities. This identifies an ancestral mechanism for Hox2 NC regulation involving a Hox-TALE regulatory circuit, potentiated by inputs from Meis and Pbx proteins and Hox auto-/cross-regulatory interactions.

Intracellular Binding Site for a Positive Allosteric Modulator of the Dopamine D1 Receptor.

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a ß2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state ß2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.

TM8 represses developmental timing in Nicotiana benthamiana and has functionally diversified in angiosperms.

BACKGROUND: MADS-box genes are key regulators of plant reproductive development and members of most lineages of this gene family have been extensively studied. However, the function and diversification of the ancient TM8 lineage remains elusive to date. The available data suggest a possible function in flower development in tomato and fast evolution through numerous gene loss events in flowering plants. RESULTS: We show the broad conservation of TM8 within angiosperms and find that in contrast to other MADS-box gene lineages, no gene duplicates have been retained after major whole genome duplication events. Through knock-down of NbTM8 by virus induced gene silencing in Nicotiana benthamiana, we show that NbTM8 represses miR172 together with another MADS-box gene, SHORT VEGETATIVE PHASE (NbSVP). In the closely related species Petunia hybrida, PhTM8 is not expressed under the conditions we investigated and consistent with this, a knock-out mutant did not show a phenotype. Finally, we generated transgenic tomato plants in which TM8 was silenced or ectopically expressed, but these plants did not display a clear phenotype. Therefore, no clear function could be confirmed for Solanum lycopersium. CONCLUSIONS: While the presence of TM8 is generally conserved, it remains difficult to propose a general function in angiosperms. Based on all the available data to date, supplemented with our own results, TM8 function seems to have diversified quickly throughout angiosperms and acts as repressor of miR172 in Nicotiana benthamiana, together with NbSVP.

The evolutionarily conserved genes: Tex37, Ccdc73, Prss55 and Nxt2 are dispensable for fertility in mice.

There are more than 2300 genes that are predominantly expressed in mouse testes. The role of hundreds of these genes has been studied in mouse spermatogenesis but still there are many genes whose function is unknown. Gene knockout (KO) strategy in mice is widely used for in vivo study of gene function. The present study was designed to explore the function of the four genes: Tex37, Ccdc73, Prss55 and Nxt2, which were evolutionarily conserved in eutherians. We found that these genes had a testis-enriched expression pattern in mice except Nxt2. We knocked out these genes by CRISPR/Cas9 individually and found that all the KO mice had normal fertility with no detectable difference in testis/body weight ratios, epididymal sperm counts, as well as testicular and epididymal histology from wild type mice. Although these genes are evolutionarily conserved in eutherians including human and mouse, they are not individually essential for spermatogenesis, testis development and male fertility in mice in laboratory conditions. Our report of these fertile KO data could avoid the repetition and duplication of efforts which will help in prioritizing efforts to focus on genes that are indispensable for male reproduction.

Genome-wide identification of conserved and novel microRNAs in one bud and two tender leaves of tea plant (Camellia sinensis) by small RNA sequencing, microarray-based hybridization and genome survey scaffold sequences.

BACKGROUND: MicroRNAs (miRNAs) are important for plant growth and responses to environmental stresses via post-transcriptional regulation of gene expression. Tea, which is primarily produced from one bud and two tender leaves of the tea plant (Camellia sinensis), is one of the most popular non-alcoholic beverages worldwide owing to its abundance of secondary metabolites. A large number of miRNAs have been identified in various plants, including non-model species. However, due to the lack of reference genome sequences and/or information of tea plant genome survey scaffold sequences, discovery of miRNAs has been limited in C. sinensis. RESULTS: Using small RNA sequencing, combined with our recently obtained genome survey data, we have identified and analyzed 175 conserved and 83 novel miRNAs mainly in one bud and two tender leaves of the tea plant. Among these, 93 conserved and 18 novel miRNAs were validated using miRNA microarray hybridization. In addition, the expression pattern of 11 conserved and 8 novel miRNAs were validated by stem-loop-qRT-PCR. A total of 716 potential target genes of identified miRNAs were predicted. Further, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the target genes were primarily involved in stress response and enzymes related to phenylpropanoid biosynthesis. The predicted targets of 4 conserved miRNAs were further validated by 5'RLM-RACE. A negative correlation between expression profiles of 3 out of 4 conserved miRNAs (csn-miR160a-5p, csn-miR164a, csn-miR828 and csn-miR858a) and their targets (ARF17, NAC100, WER and MYB12 transcription factor) were observed. CONCLUSION: In summary, the present study is one of few such studies on miRNA detection and identification in the tea plant. The predicted target genes of majority of miRNAs encoded enzymes, transcription factors, and functional proteins. The miRNA-target transcription factor gene interactions may provide important clues about the regulatory mechanism of these miRNAs in the tea plant. The data reported in this study will make a huge contribution to knowledge on the potential miRNA regulators of the secondary metabolism pathway and other important biological processes in C. sinensis.

Potent influenza A virus entry inhibitors targeting a conserved region of hemagglutinin.

Influenza A viruses (IAVs) induce acute respiratory disease and cause significant morbidity and mortality throughout the world. With the emergence of drug-resistant viral strains, new and effective anti-IAV drugs with different modes of action are urgently needed. In this study, by conjugating cholesterol to the N-terminus of the short peptide KKWK, a lipopeptide named S-KKWK was created. The anti-IAV test indicated that S-KKWK and its derivatives displayed potent antiviral activities against a broad variety of influenza A viral strains including oseltamivir-resistant strains and clinically relevant isolates with IC50 values ranging from 0.7 to 3.0µM. An extensive mechanistic study showed that these peptides functioned as viral "entry blockers" by inhibiting the conformational rearrangements of HA2 subunit, thereby interrupting the fusion of virus-host cell membranes. Significantly, a computer-aided docking simulation and protein sequence alignment identified conserved residues in the stem region of HA2 as the possible binding site of S-KKWK, which may be employed as a potential drug target for designing anti-IAVs with a broad-spectrum of activity. By targeting this region, a potent anti-IAV agent was subsequently created. In addition, the anti-IAV activity of S-KKWK was assessed by experiments with influenza A virus-infected mice, in which S-KKWK reduced the mortality of infected animals and extended survival time significantly. Overall, in addition to providing a strategy for designing broad-spectrum anti-IAV agents, these results indicate that S-KKWK and its derivatives are prospective candidates for potent antivirals.

PvGAMA reticulocyte binding activity: predicting conserved functional regions by natural selection analysis.

BACKGROUND: Adhesin proteins are used by Plasmodium parasites to bind and invade target cells. Hence, characterising molecules that participate in reticulocyte interaction is key to understanding the molecular basis of Plasmodium vivax invasion. This study focused on predicting functionally restricted regions of the P. vivax GPI-anchored micronemal antigen (PvGAMA) and characterising their reticulocyte binding activity. RESULTS: The pvgama gene was initially found in P. vivax VCG-I strain schizonts. According to the genetic diversity analysis, PvGAMA displayed a size polymorphism very common for antigenic P. vivax proteins. Two regions along the antigen sequence were highly conserved among species, having a negative natural selection signal. Interestingly, these regions revealed a functional role regarding preferential target cell adhesion. CONCLUSIONS: To our knowledge, this study describes PvGAMA reticulocyte binding properties for the first time. Conserved functional regions were predicted according to natural selection analysis and their binding ability was confirmed. These findings support the notion that PvGAMA may have an important role in P. vivax merozoite adhesion to its target cells.

Extracting Coevolutionary Features from Protein Sequences for Predicting Protein-Protein Interactions.

Knowing the ways proteins interact with each other are crucial to our understanding of the functional mechanisms of proteins. It is for this reason that different approaches have been developed in attempts to predict protein-protein interactions (PPIs) computationally. Among them, the sequence-based approaches are preferred to the others as they do not require any information about protein properties to perform their tasks. Instead, most sequence-based approaches make use of feature extraction methods to extract features directly from protein sequences so that for each protein sequence, we can construct a feature vector. The feature vectors of every pair of proteins are then concatenated to form two classes of interacting and non-interacting proteins. The prediction of whether or not two proteins interact with each other is then formulated as a classification problem. How accurate PPI predictions can be made therefore depends on how good the features are that can be extracted from the protein sequences to allow interacting or non-interacting to be best distinguished. To do so, instead of extracting such features from individual protein sequences independently of the other protein in the same pair, we propose to jointly consider features from both sequences in a protein pair during the feature extraction process through using a novel coevolutionary feature extraction approach called CoFex. Coevolutionary features extracted by CoFex refer to the covariations found at coevolving positions. Based on the presence and absence of these coevolutionary features in the sequences of two proteins, feature vectors can be composed for pairs of proteins rather than individual proteins. The experiment results show that CoFex is a promising feature extraction approach and can improve the performance of PPI prediction.

Three conserved C-terminal residues of influenza fusion peptide alter its behavior at the membrane interface.

The N-terminal fragment of the viral hemagglutinin HA2 subunit is termed a fusion peptide (HAfp). The 23-amino acid peptide (HAfp1-23) contains three C-terminal W21-Y22-G23 residues which are highly conserved among serotypes of influenza A and has been shown to form a tight helical hairpin very distinct from the boomerang structure of HAfp1-20. We studied the effect of peptide length on fusion properties, structural dynamics, and binding to the membrane interface. We developed a novel fusion visualization assay based on FLIM microscopy on giant unilamellar vesicles (GUV). By means of molecular dynamics simulations and spectroscopic measurements, we show that the presence of the three C-terminal W21-Y22-G23 residues promotes the hairpin formation, which orients perpendicularly to the membrane plane and induces more disorder in the surrounding lipids than the less structured HAfp1-20. Moreover, we report cholesterol-enriched domain formation induced exclusively by the longer fusion peptide.

Identification of Conserved Moieties in Metabolic Networks by Graph Theoretical Analysis of Atom Transition Networks.

Conserved moieties are groups of atoms that remain intact in all reactions of a metabolic network. Identification of conserved moieties gives insight into the structure and function of metabolic networks and facilitates metabolic modelling. All moiety conservation relations can be represented as nonnegative integer vectors in the left null space of the stoichiometric matrix corresponding to a biochemical network. Algorithms exist to compute such vectors based only on reaction stoichiometry but their computational complexity has limited their application to relatively small metabolic networks. Moreover, the vectors returned by existing algorithms do not, in general, represent conservation of a specific moiety with a defined atomic structure. Here, we show that identification of conserved moieties requires data on reaction atom mappings in addition to stoichiometry. We present a novel method to identify conserved moieties in metabolic networks by graph theoretical analysis of their underlying atom transition networks. Our method returns the exact group of atoms belonging to each conserved moiety as well as the corresponding vector in the left null space of the stoichiometric matrix. It can be implemented as a pipeline of polynomial time algorithms. Our implementation completes in under five minutes on a metabolic network with more than 4,000 mass balanced reactions. The scalability of the method enables extension of existing applications for moiety conservation relations to genome-scale metabolic networks. We also give examples of new applications made possible by elucidating the atomic structure of conserved moieties.

Essential role of conserved DUF177A protein in plastid 23S rRNA accumulation and plant embryogenesis.

DUF177 proteins are nearly universally conserved in bacteria and plants except the Chlorophyceae algae. Thus far, duf177 mutants in bacteria have not established a function. In contrast, duf177a mutants have embryo lethal phenotypes in maize and Arabidopsis. In maize inbred W22, duf177a mutant embryos arrest at an early transition stage, whereas the block is suppressed in the B73 inbred background, conditioning an albino seedling phenotype. Background-dependent embryo lethal phenotypes are characteristic of maize plastid gene expression mutants. Consistent with the plastid gene expression hypothesis, quantitative real-time PCR revealed a significant reduction of 23S rRNA in an Escherichia coli duf177 knockout. Plastid 23S rRNA contents of duf177a mutant tissues were also markedly reduced compared with the wild-type, whereas plastid 16S, 5S, and 4.5S rRNA contents were less affected, indicating that DUF177 is specifically required for accumulation of prokaryote-type 23S rRNA. An AtDUF177A-green fluorescent protein (GFP) transgene controlled by the native AtDUF177A promoter fully complemented the Arabidopsis atduf177a mutant. Transient expression of AtDUF177A-GFP in Nicotiana benthamiana leaves showed that the protein was localized in chloroplasts. The essential role of DUF177A in chloroplast-ribosome formation is reminiscent of IOJAP, another highly conserved ribosome-associated protein, suggesting that key mechanisms controlling ribosome formation in plastids evolved from non-essential pathways for regulation of the prokaryotic ribosome.

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions.

UNLABELLED: FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCE: FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

Phylogenetic tree-informed microRNAome analysis uncovers conserved and lineage-specific miRNAs in Camellia during floral organ development.

In plants, miRNAs are endogenous small RNAs derived from single-stranded precursors with hairpin structures. The evolution of miRNAs and their targets represents one of the most dynamic circuits directing gene expression, which may play fundamental roles in shaping the development of distinct plant organs. Here we performed high-throughput small RNA sequencing in five organ types of Camellia azalea to capture the spatial profile of small non-coding RNA. In total we obtained >227 million high-quality reads and identified 175 miRNAs with mature and precursor sequences. We aligned the miRNAs to known miRNA databases and revealed some conserved as well as 'newly evolved' miRNA genes. Twelve miRNAs were identified to be specific in the genus Camellia, supporting the lineage-specific manner of expansion of 'young' miRNAs. Through differential expression analysis, we showed that many miRNAs were preferentially abundant in certain organ types. Moreover, hierarchical clustering analysis revealed distinctive expression patterns of tissue-specific miRNAs. Gene Ontology enrichment analysis of targets of stamen- and carpel-specific miRNA subclusters showed that miRNA-target regulatory circuits were involved in many important biological processes, enabling their proper specification and organogenesis, such as 'DNA integration' and 'fruit development'. Further, quantitative PCR of key miRNAs and their target genes revealed anti-correlated patterns, and uncovered the functions of key miRNA-target pairs in different floral organs. Taken together, this work yielded valuable information on miRNA-target regulation in the control of floral organ development and sheds light on the evolution of lineage-specific miRNAs in Camellia.

Evolution of Pentameric Ligand-Gated Ion Channels: Pro-Loop Receptors.

Pentameric ligand-gated ion channels (pLGICs) are ubiquitous neurotransmitter receptors in Bilateria, with a small number of known prokaryotic homologues. Here we describe a new inventory and phylogenetic analysis of pLGIC genes across all kingdoms of life. Our main finding is a set of pLGIC genes in unicellular eukaryotes, some of which are metazoan-like Cys-loop receptors, and others devoid of Cys-loop cysteines, like their prokaryotic relatives. A number of such "Cys-less" receptors also appears in invertebrate metazoans. Together, those findings draw a new distribution of pLGICs in eukaryotes. A broader distribution of prokaryotic channels also emerges, including a major new archaeal taxon, Thaumarchaeota. More generally, pLGICs now appear nearly ubiquitous in major taxonomic groups except multicellular plants and fungi. However, pLGICs are sparsely present in unicellular taxa, suggesting a high rate of gene loss and a non-essential character, contrasting with their essential role as synaptic receptors of the bilaterian nervous system. Multiple alignments of these highly divergent sequences reveal a small number of conserved residues clustered at the interface between the extracellular and transmembrane domains. Only the "Cys-loop" proline is absolutely conserved, suggesting the more fitting name "Pro loop" for that motif, and "Pro-loop receptors" for the superfamily. The infered molecular phylogeny shows a Cys-loop and a Cys-less clade in eukaryotes, both containing metazoans and unicellular members. This suggests new hypotheses on the evolutionary history of the superfamily, such as a possible origin of the Cys-loop cysteines in an ancient unicellular eukaryote. Deeper phylogenetic relationships remain uncertain, particularly around the split between bacteria, archaea, and eukaryotes.