RESUMO
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.
Assuntos
Computação em Nuvem , Bases de Dados Genéticas , Vírus de RNA/genética , Vírus de RNA/isolamento & purificação , Alinhamento de Sequência/métodos , Virologia/métodos , Viroma/genética , Animais , Arquivos , Bacteriófagos/enzimologia , Bacteriófagos/genética , Biodiversidade , Coronavirus/classificação , Coronavirus/enzimologia , Coronavirus/genética , Evolução Molecular , Vírus Delta da Hepatite/enzimologia , Vírus Delta da Hepatite/genética , Humanos , Modelos Moleculares , Vírus de RNA/classificação , Vírus de RNA/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , SoftwareRESUMO
Fungi harbor a vast diversity of mobile genetic elements (MGEs). Recently, novel fungal MGEs, tentatively referred to as 'ambiviruses,' were described. 'Ambiviruses' have single-stranded RNA genomes of about 4-5 kb in length that contain at least two open reading frames (ORFs) in non-overlapping ambisense orientation. Both ORFs are conserved among all currently known 'ambiviruses,' and one of them encodes a distinct viral RNA-directed RNA polymerase (RdRP), the hallmark gene of ribovirian kingdom Orthornavirae. However, 'ambivirus' genomes are circular and predicted to replicate via a rolling-circle mechanism. Their genomes are also predicted to form rod-like structures and contain ribozymes in various combinations in both sense and antisense orientations-features reminiscent of viroids, virusoids, ribozyvirian kolmiovirids, and yet-unclassified MGEs (such as 'epsilonviruses,' 'zetaviruses,' and some 'obelisks'). As a first step toward the formal classification of 'ambiviruses,' the International Committee on Taxonomy of Viruses (ICTV) recently approved the establishment of a novel ribovirian phylum, Ambiviricota, to accommodate an initial set of 20 members with well-annotated genome sequences.
Assuntos
Genoma Viral , Fases de Leitura Aberta , Viroides , Viroides/genética , Viroides/classificação , Filogenia , RNA Viral/genética , Vírus de RNA/genética , Vírus de RNA/classificação , Fungos/genética , Fungos/virologia , RNA Polimerase Dependente de RNA/genética , Micovírus/genética , Micovírus/classificação , Micovírus/isolamento & purificaçãoRESUMO
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.
Assuntos
Vírus Auxiliares , Viroides , Animais , Humanos , Evolução Biológica , Vírus de RNA de Sentido Negativo , RNA Polimerase II , MamíferosRESUMO
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 â¼170-400 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.
Assuntos
RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Circular/metabolismo , Ambystoma mexicanum/genética , Animais , Antozoários/genética , Bivalves/genética , Genoma , RNA Catalítico/química , RNA Circular/biossíntese , Retroelementos , Sequências de Repetição em Tandem , TranscriptomaRESUMO
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.
Assuntos
Eucariotos/genética , Splicing de RNA , RNA Catalítico/metabolismo , RNA/biossíntese , Animais , Eucariotos/metabolismo , Previsões , Regulação da Expressão Gênica , Genoma , Conformação de Ácido Nucleico , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA/genética , RNA Circular , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , Retroelementos/genéticaRESUMO
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.
Assuntos
RNA Catalítico/genética , RNA de Plantas/genética , RNA Viral/genética , RNA/genética , Retroelementos , Animais , Pareamento de Bases , Sequência de Bases , Humanos , Conformação de Ácido Nucleico , Plantas/virologia , RNA/química , RNA/metabolismo , RNA Catalítico/química , RNA Catalítico/metabolismo , RNA Circular , RNA de Plantas/metabolismo , RNA Satélite/genética , RNA Satélite/metabolismo , RNA Viral/metabolismo , Sequências Repetidas Terminais , Viroides/genética , Viroides/metabolismoRESUMO
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.
Assuntos
Plantas/química , RNA Catalítico/química , RNA/química , Schistosoma mansoni/química , Animais , Pareamento de Bases , Sequência de Bases , Biocatálise , Domínio Catalítico , História do Século XX , História do Século XXI , Hidrólise , Modelos Moleculares , Conformação de Ácido Nucleico , RNA/história , RNA/fisiologia , RNA/ultraestrutura , RNA Catalítico/história , RNA Catalítico/fisiologia , RNA Catalítico/ultraestrutura , RNA CircularRESUMO
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.
Assuntos
Sequência Conservada , RNA Catalítico/genética , Retroelementos , Anfíbios/genética , Animais , Sequência de Bases , Evolução Biológica , Biologia Computacional , Dimerização , Peixes/genética , Humanos , Insetos/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plantas/genética , RNA Catalítico/química , Schistosoma mansoni/genéticaRESUMO
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.
Assuntos
RNA Catalítico/química , RNA Catalítico/genética , Variação Genética , Genoma , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , RNA Catalítico/metabolismo , Homologia de Sequência , Sequências de Repetição em TandemRESUMO
Members of the genus Armillaria are widespread forest pathogens against which effective protection has not yet been developed. Due to their longevity and the creation of large-scale cloning of Armillaria individuals, the use of mycoviruses as biocontrol agents (BCAs) against these pathogens could be an effective alternative. This work describes the detection and characterization of viruses in Armillaria spp. collected in the Czech Republic through the application of stranded total RNA sequencing. A total of five single-stranded RNA viruses were detected in Armillaria ostoyae and A. cepistipes, including viruses of the family Tymoviridae and four viruses belonging to the recently described "ambivirus" group with a circular ambisense genome arrangement. Both hammerhead (HHRz) and hairpin (HpRz) ribozymes were detected in all the ambiviricot sequences. Armillaria viruses were compared through phylogenetic analysis and confirmed their specific host by direct RT-PCR. One virus appears to infect both Armillaria species, suggesting the occurrence of interspecies transmission in nature.
Assuntos
Armillaria , Micovírus , Genoma Viral , Filogenia , RNA Viral , República Tcheca , Armillaria/genética , Armillaria/virologia , Micovírus/classificação , Micovírus/genética , Micovírus/isolamento & purificação , RNA Viral/genética , Vírus de RNA/genética , Vírus de RNA/classificação , Vírus de RNA/isolamento & purificação , Doenças das Plantas/virologia , Doenças das Plantas/microbiologia , Análise de Sequência de RNARESUMO
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.
RESUMO
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.
Assuntos
Vírus de RNA , RNA Catalítico , Viroides , Viroides/genética , RNA Catalítico/genética , RNA Viral/genética , Replicação Viral/genética , RNA/genética , Vírus de RNA/genética , RNA Polimerase Dependente de RNA/genética , Fungos/genéticaRESUMO
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.
Assuntos
Íntrons/genética , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Mensageiro/biossíntese , Animais , Sequência de Bases , Sequência Conservada , Humanos , Dados de Sequência Molecular , Splicing de RNA , RNA Catalítico/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Examples of small self-cleaving RNAs embedded in noncoding regions already have been found to be involved in the control of gene expression, although their origin remains uncertain. In this work, we show the widespread occurrence of the hammerhead ribozyme (HHR) motif among genomes from the Bacteria, Chromalveolata, Plantae, and Metazoa kingdoms. Intergenic HHRs were detected in three different bacterial genomes, whereas metagenomic data from Galapagos Islands showed the occurrence of similar ribozymes that could be regarded as direct relics from the RNA world. Among eukaryotes, HHRs were detected in the genomes of three water molds as well as 20 plant species, ranging from unicellular algae to vascular plants. These HHRs were very similar to those previously described in small RNA plant pathogens and, in some cases, appeared as close tandem repetitions. A parallel situation of tandemly repeated HHR motifs was also detected in the genomes of lower metazoans from cnidarians to invertebrates, with special emphasis among hematophagous and parasitic organisms. Altogether, these findings unveil the HHR as a widespread motif in DNA genomes, which would be involved in new forms of retrotransposable elements.
Assuntos
Evolução Molecular , RNA Catalítico/genética , Animais , Artrópodes/enzimologia , Artrópodes/genética , Bactérias/enzimologia , Bactérias/genética , Sequência de Bases , Cnidários/enzimologia , Cnidários/genética , Biologia Computacional , Metagenômica , Conformação de Ácido Nucleico , Oomicetos/enzimologia , Oomicetos/genética , Plantas/enzimologia , Plantas/genética , RNA Catalítico/química , RNA Catalítico/metabolismoRESUMO
Small ribozymes have been regarded as living fossils of a prebiotic RNA world that would have remained in the genomes of modern organisms. In this study, we report the ultraconserved occurrence of hammerhead ribozymes in Amniota genomes (reptiles, birds and mammals, including humans), similar to those described previously in amphibians and platyhelminth parasites. The ribozymes mapped to intronic regions of different genes, such as the tumour suppressor RECK in birds and mammals, a mammalian tumour antigen and the dystrobrevin beta in lizards and birds. In vitro characterization confirmed a high self-cleavage activity, whereas analysis of RECK-expressed sequence tags revealed fusion events between the in vivo self-cleaved intron and U5 or U6 small nuclear RNA fragments. Together, these results suggest a conserved role for these ribozymes in messenger RNA biogenesis.
Assuntos
Genoma Humano , RNA Catalítico/genética , Animais , Antígenos de Neoplasias/genética , Sequência de Bases , Biologia Computacional , Evolução Molecular , Genes Supressores de Tumor , Humanos , Íntrons , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Catalítico/química , Alinhamento de SequênciaRESUMO
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.
Assuntos
RNA Catalítico , Viroides , Animais , Vírus Delta da Hepatite/genética , Plantas , RNA Catalítico/genética , RNA Circular , RNA Viral/genética , Viroides/genética , Replicação ViralRESUMO
The vaccinia virus mRNA capping enzyme is a multifunctional heterodimeric protein associated with the viral polymerase that both catalyses the three steps of mRNA capping and regulates gene transcription. The structure of a subcomplex comprising the C-terminal N7-methyl-transferase (MT) domain of the large D1 subunit, the stimulatory D12 subunit and bound S-adenosyl-homocysteine (AdoHcy) has been determined at 2.7 A resolution and reveals several novel features of the poxvirus capping enzyme. The structure shows for the first time the critical role played by the proteolytically sensitive N-terminus of the MT domain in binding the methyl donor and in catalysis. In addition, the poxvirus enzyme has a completely unique mode of binding of the adenosine moiety of AdoHcy, a feature that could be exploited for design of specific anti-poxviral compounds. The structure of the poxvirus-specific D12 subunit suggests that it was originally an RNA cap 2'O-MT that has evolved to a catalytically inactive form that has been retained for D1 stabilisation and MT activity enhancement through an allosteric mechanism.
Assuntos
Metiltransferases/química , Metiltransferases/fisiologia , S-Adenosil-Homocisteína/química , Vaccinia virus/enzimologia , Vaccinia virus/genética , Sítio Alostérico , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Capuzes de RNARESUMO
Three-way junction RNAs adopt a recurrent Y shape when two of the helices form a coaxial stack and the third helix establishes one or more tertiary contacts several base pairs away from the junction. In this review, the structure, distribution, and functional relevance of these motifs are examined. Structurally, the folds exhibit conserved junction topologies, and the distal tertiary interactions play a crucial role in determining the final shape of the structures. The junctions and remote tertiary contacts behave as flexible hinge motifs that respond to changes in the other region, providing these folds with switching mechanisms that have been shown to be functionally useful in a variety of contexts. In addition, the juxtaposition of RNA domains at the junction and at the distal tertiary complexes enables the RNA helices to adopt unusual conformations that are frequently used by proteins, RNA molecules, and antibiotics as platforms for specific binding. As a consequence of these properties, Y-shaped junctions are widely distributed in all kingdoms of life, having been observed in small naked RNAs such as riboswitches and ribozymes or embedded in complex ribonucleoprotein systems like ribosomal RNAs, RNase P, or the signal recognition particle. In all cases, the folds were found to play an essential role for the functioning or assembly of the RNA or ribonucleoprotein systems that contain them.
Assuntos
Conformação de Ácido Nucleico , RNA/química , Animais , Humanos , Modelos Moleculares , Proteínas/metabolismo , RNA/genética , RNA/metabolismo , Ribossomos/química , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Loop-loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (-) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5' side and an extrahelical U at its 3'-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3'-side. These structural features promote a specific loop-loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5' U of loop 1 and the 3' A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.
Assuntos
Vírus de Plantas/enzimologia , RNA Catalítico/química , Viroides/enzimologia , Sequência de Bases , Sequência Conservada , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , RNA Catalítico/metabolismo , Relação Estrutura-Atividade , Uracila/química , Viroides/fisiologiaRESUMO
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.