ICTV Virus Taxonomy Profile: Kolmioviridae 2024.

Kolmioviridae is a family for negative-sense RNA viruses with circular, viroid-like genomes of about 1.5-1.7 kb that are maintained in mammals, amphibians, birds, fish, insects and reptiles. Deltaviruses, for instance, can cause severe hepatitis in humans. Kolmiovirids encode delta antigen (DAg) and replicate using host-cell DNA-directed RNA polymerase II and ribozymes encoded in their genome and antigenome. They require evolutionary unrelated helper viruses to provide envelopes and incorporate helper virus proteins for infectious particle formation. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Kolmioviridae, which is available at ictv.global/report/kolmioviridae.

Viroid-like colonists of human microbiomes.

Here, we describe the "Obelisks," a previously unrecognised class of viroid-like elements that we first identified in human gut metatranscriptomic data. "Obelisks" share several properties: (i) apparently circular RNA ~1kb genome assemblies, (ii) predicted rod-like secondary structures encompassing the entire genome, and (iii) open reading frames coding for a novel protein superfamily, which we call the "Oblins". We find that Obelisks form their own distinct phylogenetic group with no detectable sequence or structural similarity to known biological agents. Further, Obelisks are prevalent in tested human microbiome metatranscriptomes with representatives detected in ~7% of analysed stool metatranscriptomes (29/440) and in ~50% of analysed oral metatranscriptomes (17/32). Obelisk compositions appear to differ between the anatomic sites and are capable of persisting in individuals, with continued presence over >300 days observed in one case. Large scale searches identified 29,959 Obelisks (clustered at 90% nucleotide identity), with examples from all seven continents and in diverse ecological niches. From this search, a subset of Obelisks are identified to code for Obelisk-specific variants of the hammerhead type-III self-cleaving ribozyme. Lastly, we identified one case of a bacterial species (Streptococcus sanguinis) in which a subset of defined laboratory strains harboured a specific Obelisk RNA population. As such, Obelisks comprise a class of diverse RNAs that have colonised, and gone unnoticed in, human, and global microbiomes.

Hybrids of RNA viruses and viroid-like elements replicate in fungi.

Earth's life may have originated as self-replicating RNA, and it has been argued that RNA viruses and viroid-like elements are remnants of such pre-cellular RNA world. RNA viruses are defined by linear RNA genomes encoding an RNA-dependent RNA polymerase (RdRp), whereas viroid-like elements consist of small, single-stranded, circular RNA genomes that, in some cases, encode paired self-cleaving ribozymes. Here we show that the number of candidate viroid-like elements occurring in geographically and ecologically diverse niches is much higher than previously thought. We report that, amongst these circular genomes, fungal ambiviruses are viroid-like elements that undergo rolling circle replication and encode their own viral RdRp. Thus, ambiviruses are distinct infectious RNAs showing hybrid features of viroid-like RNAs and viruses. We also detected similar circular RNAs, containing active ribozymes and encoding RdRps, related to mitochondrial-like fungal viruses, highlighting fungi as an evolutionary hub for RNA viruses and viroid-like elements. Our findings point to a deep co-evolutionary history between RNA viruses and subviral elements and offer new perspectives in the origin and evolution of primordial infectious agents, and RNA life.

A life of research on circular RNAs and ribozymes: towards the origin of viroids, deltaviruses and life.

The first examples of circular RNAs (circRNAs) were reported in the '70s as a family of minimal infectious agents of flowering plants; the viroids and viral satellites of circRNA. In some cases, these small circular genomes encode self-cleaving RNA motifs or ribozymes, including an exceptional circRNA infecting not plants but humans: the Hepatitis Delta Virus. Autocatalytic ribozymes not only allowed to propose a common rolling-circle replication mechanism for all these subviral agents, but also a tentative link with the origin of life as molecular fossils of the so-called RNA world. Despite the weak biologic connection between angiosperm plants and the human liver, diverse scientists, and most notably Ricardo Flores, firmly supported an evolutionary relationship between plant viroids and human deltavirus agents. The tireless and inspiring work done by Ricardo's lab in the field of infectious circRNAs fuelled multiple hypotheses for the origin of these entities, allowing advances in other fields, from eukaryotic circRNAs to small ribozymes in genomes from all life kingdoms. The recent discovery of a plethora of viral-like circRNAs with ribozymes in disparate biological samples may finally allow us to connect plant and animal subviral agents, confirming again that Ricardo's eye for science was always a keen eye.

Petabase-scale sequence alignment catalyses viral discovery.

Public databases contain a planetary collection of nucleic acid sequences, but their systematic exploration has been inhibited by a lack of efficient methods for searching this corpus, which (at the time of writing) exceeds 20 petabases and is growing exponentially1. Here we developed a cloud computing infrastructure, Serratus, to enable ultra-high-throughput sequence alignment at the petabase scale. We searched 5.7 million biologically diverse samples (10.2 petabases) for the hallmark gene RNA-dependent RNA polymerase and identified well over 105 novel RNA viruses, thereby expanding the number of known species by roughly an order of magnitude. We characterized novel viruses related to coronaviruses, hepatitis delta virus and huge phages, respectively, and analysed their environmental reservoirs. To catalyse the ongoing revolution of viral discovery, we established a free and comprehensive database of these data and tools. Expanding the known sequence diversity of viruses can reveal the evolutionary origins of emerging pathogens and improve pathogen surveillance for the anticipation and mitigation of future pandemics.

Hepatitis delta virus-like circular RNAs from diverse metazoans encode conserved hammerhead ribozymes.

Human hepatitis delta virus (HDV) is a unique infectious agent whose genome is composed of a small circular RNA. Recent data, however, have reported the existence of highly divergent HDV-like circRNAs in the transcriptomes of diverse vertebrate and invertebrate species. The HDV-like genomes described in amniotes such as birds and reptiles encode self-cleaving RNA motifs or ribozymes similar to the ones present in the human HDV, whereas no catalytic RNA domains have been reported for the HDV-like genomes detected in metagenomic data from some amphibians, fish, and invertebrates. Herein, we describe the self-cleaving motifs of the HDV-like genomes reported in newts and fish, which belong to the characteristic class of HDV ribozymes. Surprisingly, HDV-like genomes from a toad and a termite show conserved type III hammerhead ribozymes, which belong to an unrelated class of catalytic RNAs characteristic of plant genomes and plant subviral circRNAs, such as some viral satellites and viroids. Sequence analyses revealed the presence of similar HDV-like hammerhead ribozymes encoded in two termite genomes, but also in the genomes of several dipteran species. In vitro transcriptions confirmed the cleaving activity for these motifs, with moderate rates of self-cleavage. These data indicate that all described HDV-like agents contain self-cleaving motifs from either the HDV or the hammerhead class. Autocatalytic ribozymes in HDV-like genomes could be regarded as interchangeable domains and may have arisen from cellular transcriptomes, although we still cannot rule out some other evolutionary explanations.

Cloning and Detection of Genomic Retrozymes and Their circRNA Intermediates.

Retrozymes are a novel family of non-autonomous retrotransposable elements that contain hammerhead ribozyme motifs. These retroelements are found widespread in eukaryotic genomes, with active copies present in many species, which rely on other autonomous transposons for mobilization. Contrary to other retrotransposons, transcription of retrozymes in vivo leads to the formation and accumulation of circular RNAs, which can be readily detected by RNA blotting. In this chapter, we describe the procedures needed to carry out the cloning of genomic retrozymes, and to detect by northern blot their circular RNA retrotransposition intermediates.

A Singular and Widespread Group of Mobile Genetic Elements: RNA Circles with Autocatalytic Ribozymes.

Circular DNAs, such as most prokaryotic and phage genomes, are a frequent form of nucleic acids, whereas circular RNAs had been regarded as unusual macromolecules until very recently. The first reported RNA circles were the family of small infectious genomes of viroids and circular RNA (circRNA) satellites of plant viruses, some of which contain small self-cleaving RNA motifs, such as the hammerhead (HHR) and hairpin ribozymes. A similar infectious circRNA, the unique human hepatitis delta virus (HDV), is another viral satellite that also encodes self-cleaving motifs called HDV ribozymes. Very recently, different animals have been reported to contain HDV-like circRNAs with typical HDV ribozymes, but also conserved HHR motifs, as we describe here. On the other hand, eukaryotic and prokaryotic genomes encode sequences able to self-excise as circRNAs, like the autocatalytic Group I and II introns, which are widespread genomic mobile elements. In the 1990s, the first circRNAs encoded in a mammalian genome were anecdotally reported, but their abundance and importance have not been unveiled until recently. These gene-encoded circRNAs are produced by events of alternative splicing in a process generally known as backsplicing. However, we have found a second natural pathway of circRNA expression conserved in numerous plant and animal genomes, which efficiently promotes the accumulation of small non-coding RNA circles through the participation of HHRs. Most of these genome-encoded circRNAs with HHRs are the transposition intermediates of a novel family of non-autonomous retrotransposons called retrozymes, with intriguing potential as new forms of gene regulation.

Small circRNAs with self-cleaving ribozymes are highly expressed in diverse metazoan transcriptomes.

Ribozymes are catalytic RNAs present in modern genomes but regarded as remnants of a prebiotic RNA world. The paradigmatic hammerhead ribozyme (HHR) is a small self-cleaving motif widespread from bacterial to human genomes. Here, we report that most of the classical type I HHRs frequently found in the genomes of animals are contained within a novel family of non-autonomous non-LTR retrotransposons of the retrozyme class. These retroelements are expressed as abundant linear and circular RNAs of ∼170-400 nt in different animal tissues. Bioinformatic and in vitro analyses indicate an efficient self-cleavage of the HHRs harboured in most invertebrate retrozymes, whereas HHRs in retrozymes of vertebrates, such as the axolotl and other amphibians, require to act as dimeric motifs to reach higher self-cleavage rates. Ligation assays of retrozyme RNAs with a protein ligase versus HHR self-ligation indicate that, most likely, tRNA ligases and not the ribozymes are involved in the step of RNA circularization. Altogether, these results confirm the existence of a new and conserved pathway in animals and, likely, eukaryotes in general, for the efficient biosynthesis of RNA circles through small ribozymes, which opens the door for the development of new tools in the emerging field of study of circRNAs.

Simultaneous Boron Ion-Channel/Growth Factor Receptor Activation for Enhanced Vascularization.

Boron ion is essential in metabolism and its concentration is regulated by ion-channel NaBC1. NaBC1 mutations cause corneal dystrophies such as Harboyan syndrome. Here a 3D molecular model for NaBC1 is proposed and it is shown that simultaneous stimulation of NaBC1 and vascular endothelial growth factor receptors (VEGFR) promotes angiogenesis in vitro and in vivo with ultralow concentrations of VEGF. Human umbilical vein endothelial cells' (HUVEC) organization into tubular structures is shown to be indicative of vascularization potential. Enhanced cell sprouting is found only in the presence of VEGF and boron, the effect abrogated after blocking NaBC1. It is demonstrated that stimulated NaBC1 promotes angiogenesis via PI3k-independent pathways and that α5 ß1 /αv ß3 integrin binding is not essential to enhanced HUVEC organization. A novel vascularization mechanism that involves crosstalk and colocalization between NaBC1 and VEGFR receptors is described. This has important translational consequences; just by administering boron, taking advantage of endogenous VEGF, in vivo vascularization is shown in a chorioallantoic membrane assay.

Circular RNAs Biogenesis in Eukaryotes Through Self-Cleaving Hammerhead Ribozymes.

Circular DNAs are frequent genomic molecules, especially among the simplest life beings, whereas circular RNAs have been regarded as weird nucleic acids in biology. Now we know that eukaryotes are able to express circRNAs, mostly derived from backsplicing mechanisms, and playing different biological roles such as regulation of RNA splicing and transcription, among others. However, a second natural and highly efficient pathway for the expression in vivo of circRNAs has been recently reported, which allows the accumulation of abundant small (100-1000 nt) non-coding RNA circles through the participation of small self-cleaving RNAs or ribozymes called hammerhead ribozymes. These genome-encoded circRNAs with ribozymes seem to be a new family of small and nonautonomous retrotransposable elements of plants and animals (so-called retrozymes), which will offer functional clues to the biology and evolution of circular RNA molecules as well as new biotechnological tools in this emerging field.

Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis.

Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange ß-farnesene synthase, (Z)-ß-cubebene/α-copaene synthase, two ß-caryophyllene synthases, and three multiproduct enzymes yielding ß-cadinene/α-copaene, ß-elemene, and ß-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays.

Circular RNAs with hammerhead ribozymes encoded in eukaryotic genomes: The enemy at home.

A new family of non-autonomous retrotransposons with self-cleaving hammerhead ribozymes, the so called retrozymes, has recently been found encoded in diverse plant genomes. These retroelements can be actively transcribed, and their RNAs accumulate in the cells as abundant non-coding circular RNAs (circRNAs) of small size (600-1000 nt). Related circRNAs with self-cleaving ribozymes had already been described in plants, and belong to a group of infectious RNA agents with an uncertain origin: the viroids and viroid-like satellites of plant RNA viruses. These pathogenic circRNAs show many structural similarities with retrozyme circRNAs, and both have been found to occur in flowering plants as heterogeneous RNA molecules of positive and negative polarities. Taking all these data together, we hypothesize that circRNAs encoded by genomic retrozymes could have given origin to infectious circRNAs with self-cleaving ribozymes. Moreover, we propose that retrozymes in time could have evolved from the ancient family of Penelope-like retroelements, which also harbour hammerhead ribozymes. Putative retrozyme sequences with hammerhead ribozymes have been detected as well in metazoan genomes, opening the door to a common occurrence of circRNAs with self-cleaving motifs among eukaryotes.

The Hammerhead Ribozyme: A Long History for a Short RNA.

Small nucleolytic ribozymes are a family of naturally occurring RNA motifs that catalyse a self-transesterification reaction in a highly sequence-specific manner. The hammerhead ribozyme was the first reported and the most extensively studied member of this family. However, and despite intense biochemical and structural research for three decades since its discovery, the history of this model ribozyme seems to be far from finished. The hammerhead ribozyme has been regarded as a biological oddity typical of small circular RNA pathogens of plants. More recently, numerous and new variations of this ribozyme have been found to inhabit the genomes of organisms from all life kingdoms, although their precise biological functions are not yet well understood.

Retrozymes are a unique family of non-autonomous retrotransposons with hammerhead ribozymes that propagate in plants through circular RNAs.

BACKGROUND: Catalytic RNAs, or ribozymes, are regarded as fossils of a prebiotic RNA world that have remained in the genomes of modern organisms. The simplest ribozymes are the small self-cleaving RNAs, like the hammerhead ribozyme, which have been historically considered biological oddities restricted to some RNA pathogens. Recent data, however, indicate that small self-cleaving ribozymes are widespread in genomes, although their functions are still unknown. RESULTS: We reveal that hammerhead ribozyme sequences in plant genomes form part of a new family of small non-autonomous retrotransposons with hammerhead ribozymes, referred to as retrozymes. These elements contain two long terminal repeats of approximately 350 bp, each harbouring a hammerhead ribozyme that delimitates a variable region of 600-1000 bp with no coding capacity. Retrozymes are actively transcribed, which gives rise to heterogeneous linear and circular RNAs that accumulate differentially depending on the tissue or developmental stage of the plant. Genomic and transcriptomic retrozyme sequences are highly heterogeneous and share almost no sequence homology among species except the hammerhead ribozyme motif and two small conserved domains typical of Ty3-gypsy long terminal repeat retrotransposons. Moreover, we detected the presence of RNAs of both retrozyme polarities, which suggests events of independent RNA-RNA rolling-circle replication and evolution, similarly to that of infectious circular RNAs like viroids and viral satellite RNAs. CONCLUSIONS: Our work reveals that circular RNAs with hammerhead ribozymes are frequently occurring molecules in plant and, most likely, metazoan transcriptomes, which explains the ubiquity of these genomic ribozymes and suggests a feasible source for the emergence of circular RNA plant pathogens.

Eukaryotic penelope-like retroelements encode hammerhead ribozyme motifs.

Small self-cleaving RNAs, such as the paradigmatic Hammerhead ribozyme (HHR), have been recently found widespread in DNA genomes across all kingdoms of life. In this work, we found that new HHR variants are preserved in the ancient family of Penelope-like elements (PLEs), a group of eukaryotic retrotransposons regarded as exceptional for encoding telomerase-like retrotranscriptases and spliceosomal introns. Our bioinformatic analysis revealed not only the presence of minimalist HHRs in the two flanking repeats of PLEs but also their massive and widespread occurrence in metazoan genomes. The architecture of these ribozymes indicates that they may work as dimers, although their low self-cleavage activity in vitro suggests the requirement of other factors in vivo. In plants, however, PLEs show canonical HHRs, whereas fungi and protist PLEs encode ribozyme variants with a stable active conformation as monomers. Overall, our data confirm the connection of self-cleaving RNAs with eukaryotic retroelements and unveil these motifs as a significant fraction of the encoded information in eukaryotic genomes.

Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus.

Vaccinia virus capping enzyme is a heterodimer of D1 (844 aa) and D12 (287 aa) polypeptides that executes all three steps in m(7)GpppRNA synthesis. The D1 subunit comprises an N-terminal RNA triphosphatase (TPase)-guanylyltransferase (GTase) module and a C-terminal guanine-N7-methyltransferase (MTase) module. The D12 subunit binds and allosterically stimulates the MTase module. Crystal structures of the complete D1⋅D12 heterodimer disclose the TPase and GTase as members of the triphosphate tunnel metalloenzyme and covalent nucleotidyltransferase superfamilies, respectively, albeit with distinctive active site features. An extensive TPase-GTase interface clamps the GTase nucleotidyltransferase and OB-fold domains in a closed conformation around GTP. Mutagenesis confirms the importance of the TPase-GTase interface for GTase activity. The D1⋅D12 structure complements and rationalizes four decades of biochemical studies of this enzyme, which was the first capping enzyme to be purified and characterized, and provides new insights into the origins of the capping systems of other large DNA viruses.

Intronic hammerhead ribozymes in mRNA biogenesis.

Small self-cleaving ribozymes are a group of natural RNAs that are capable of catalyzing their own and sequence-specific endonucleolytic cleavage. One of the most studied members is the hammerhead ribozyme (HHR), a catalytic RNA originally discovered in subviral plant pathogens but recently shown to reside in a myriad of genomes along the tree of life. In eukaryotes, most of the genomic HHRs seem to be related to short interspersed retroelements, with the main exception of a group of strikingly conserved ribozymes found in the genomes of all amniotes (reptiles, birds and mammals). These amniota HHRs occur in the introns of a few specific genes, and clearly point to a preserved biological role during pre-mRNA biosynthesis. More specifically, bioinformatic analysis suggests that these intronic ribozymes could offer a new form of splicing regulation of the mRNA of higher vertebrates. We review here the latest advances in the discovery and biological characterization of intronic HHRs of vertebrates, including new conserved examples in the genomes of the primitive turtle and coelacanth fish.

The ubiquitous hammerhead ribozyme.

The hammerhead ribozyme is a small catalytic RNA motif capable of endonucleolytic (self-) cleavage. It is composed of a catalytic core of conserved nucleotides flanked by three helices, two of which form essential tertiary interactions for fast self-scission under physiological conditions. Originally discovered in subviral plant pathogens, its presence in several eukaryotic genomes has been reported since. More recently, this catalytic RNA motif has been shown to reside in a large number of genomes. We review the different approaches in discovering these new hammerhead ribozyme sequences and discuss possible biological functions of the genomic motifs.