RESUMEN
Noncoding RNAs play essential roles in genetic regulation in all organisms. In eukaryotic cells, many small noncoding RNAs act in complex with Argonaute proteins and regulate gene expression by recognizing complementary RNA targets. The complexes of Argonaute proteins with small RNAs also play a key role in silencing of mobile genetic elements and, in some cases, viruses. These processes are collectively called RNA interference. RNA interference is a powerful tool for specific gene silencing in both basic research and therapeutic applications. Argonaute proteins are also found in prokaryotic organisms. Recent studies have shown that prokaryotic Argonautes can also cleave their target nucleic acids, in particular DNA. This activity of prokaryotic Argonautes might potentially be used to edit eukaryotic genomes. However, the molecular mechanisms of small nucleic acid biogenesis and the functions of Argonaute proteins, in particular in bacteria and archaea, remain largely unknown. Here we briefly review available data on the RNA interference processes and Argonaute proteins in eukaryotes and prokaryotes.
Asunto(s)
Proteínas Argonautas/metabolismo , Eucariontes/metabolismo , Células Procariotas/metabolismo , Interferencia de ARN , Animales , HumanosRESUMEN
Tandem Stellate genes organized into two clusters in heterochromatin and euchromatin of the X-chromosome are part of the Ste-Su(Ste) genetic system required for maintenance of male fertility and reproduction of Drosophila melanogaster. Stellate genes encode a regulatory subunit of protein kinase CK2 and are the main targets of germline-specific piRNA-silencing; their derepression leads to appearance of protein crystals in spermatocytes, meiotic disturbances, and male sterility. A short promoter region of 134 bp appears to be sufficient for testis-specific transcription of Stellate, and it contains three closely located cis-regulatory elements called E-boxes. By using reporter analysis, we confirmed a strong functionality of the E-boxes in the Stellate promoter for in vivo transcription. Using selective mutagenesis, we have shown that the presence of the central E-box 2 is preferable to maintain a high-level testis-specific transcription of the reporter gene under the Stellate promoter. The Stellate promoter provides transcription even in heterochromatin, and corresponding mRNAs are translated with the generation of full-size protein products in case of disturbances in the piRNA-silencing process. We have also shown for the first time that the activity of the Stellate promoter is determined by chromatin context of the X-chromosome in male germinal cells, and it increases at about twofold when relocating in autosomes.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Elementos E-Box/genética , Secuencias Repetidas en Tándem/genética , Testículo/metabolismo , Animales , Secuencia de Bases , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Femenino , Heterocromatina/metabolismo , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Cromosoma X/metabolismoAsunto(s)
Drosophila melanogaster/genética , Drosophila/genética , Mutación , Interferencia de ARN , Retroelementos , Animales , Genoma , Modelos Genéticos , ARN/genética , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Transcripción Genética , TransgenesRESUMEN
The advent of large-scale sequencing has opened up new areas of research, such as the study of Piwi-interacting small RNAs (piRNAs). piRNAs are longer than miRNAs, close to 30 nucleotides in length, involved in various functions, such as the suppression of transposons in germline. Since a large number of them (many tens of thousands) are generated from a wide range of positions in the genome, large-scale sequencing is the only way to study them. The key to understanding their genesis and biological roles is efficient analysis, which is complicated by the large volumes of sequence data. Taking account of the underlying biology is also important. We describe here novel analyses techniques and tools applied to small RNAs from germ cells in D. melanogaster, that allowed us to infer mechanism and biological function.
Asunto(s)
ARN/genética , Análisis de Secuencia de ARN/estadística & datos numéricos , Animales , Proteínas Argonautas , Biología Computacional , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genoma de los Insectos , Células Germinativas , Familia de Multigenes , Factores de Iniciación de Péptidos/genética , ARN Interferente Pequeño/genéticaRESUMEN
During the past several years, it has become clear that small RNAs guard germ cell genomes from the activity of mobile genetic elements. Indeed, in mammals, a class of small RNAs, known as Piwi-interacting RNAs (piRNAs), forms an innate immune system that discriminates transposons from endogenous genes and selectively silences the former. piRNAs enforce silencing by directing transposon DNA methylation during male germ cell development. As such, piRNAs represent perhaps the only currently known sequence-specific factor for deposition of methylcytosine in mammals. The three mammalian Piwi proteins Miwi2, Mili, and Miwi are required at different stages of germ cell development. Moreover, distinct classes of piRNAs are expressed in developmental waves, with particular generative loci and different sequence content distinguishing piRNAs populations in embryonic germ cells from those that appear during meiosis. Although our understanding of Piwi proteins and piRNA biology have deepened substantially during the last several years, major gaps still exist in our understanding of these enigmatic RNA species.
Asunto(s)
Células Germinativas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Células Madre/metabolismo , Animales , Femenino , Masculino , Mamíferos , MicroARNs/genéticaRESUMEN
A full-length copy of the retrotransposon GATE was identified as an insertion in the tandemly repeated, heterochromatic, Stellate genes, which are expressed in the testis of Drosophila melanogaster. Sequencing of this heterochromatic GATE copy revealed that it is closely related to the BEL retrotransposon, a representative of the recently defined BEL-like group of LTR retrotransposons. This copy contains identical LTRs, indicating that the insertion is a recent event. By contrast, the euchromatic part of the D. melanogaster genome contains only profoundly damaged GATE copies or fragments of the transposon. The preferential localization of GATE sequences in heterochromatin was confirmed for the other species in the melanogaster subgroup. The level of GATE expression is dramatically increased in ovaries, but not in testes, of spn-E(1) homozygous flies. We speculate that spn-E is involved in the silencing of GATE via an RNA interference mechanism.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Heterocromatina/metabolismo , Secuencia de Aminoácidos , Animales , ADN/metabolismo , Femenino , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Mutación , Ovario/metabolismo , Filogenia , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Testículo/metabolismoRESUMEN
RNA interference consists in specific mRNA degradation in response to introduction of a double-stranded RNA, homologous in nucleotide sequence. RNA interference was found in eukaryotes and is used in genomics as a powerful method to determine the functions of genes with known nucleotide sequences. RNA interference is considered as a tool of protection against viruses and harmful consequences of mobile elements' transposals. The involvement of the components of RNA interference is considered in spermatogenesis of Drosophila melanogaster and regulation of the expression of genes in Caenorhabditis elegans responsible for temporal patterns of development. The role of RNQA interference in stem cell formation and functioning is also considered.
Asunto(s)
Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Interferencia de ARN , Animales , Drosophila melanogaster , Nematodos , Espermatogénesis/genética , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Data on RNA interference, that is, posttranscriptional gene silencing by homologous double-stranded (ds) RNA, are reviewed. Gene silencing caused by exogenous dsRNA in artificial systems and observed in transgenic organisms carrying additional gene copies is considered. Data are summarized on the mechanism that arose during evolution of the Drosophila melanogaster genome to suppress repetitive genes with the use of dsRNA and thereby to prevent male sterility. The role of dsRNA in inhibiting expression and transposition of mobile elements is discussed on the basis of authors own and published findings.
Asunto(s)
Células Eucariotas/fisiología , Silenciador del Gen , ARN Bicatenario/fisiología , Animales , Drosophila/genética , Hongos/genética , Regulación de la Expresión Génica de las Plantas , Plantas Modificadas Genéticamente , Procesamiento Postranscripcional del ARN , Supresión GenéticaRESUMEN
Specific inhibition of gene expression by exogenous homologous double-stranded RNA (dsRNA) in invertebrates and in the early development of vertebrates is termed RNA interference. Cultured cells were cotransfected with reporter plasmids and dsRNA. The inhibitor effect on reporter gene expression depended on the extent of homology between dsRNA and the target gene. RNA interference was also studied in cells cotransfected with plasmids directing synthesis of sense and antisense RNAs. Production of antisense RNA only slightly inhibited expression of the reporter gene. Simultaneous expression of both sense and antisense RNAs from a special plasmid did not inhibit expression of the reporter construct.
Asunto(s)
Drosophila melanogaster/genética , Regulación de la Expresión Génica , ARN Bicatenario/genética , Animales , Genes de Insecto , Genes Reporteros , Operón Lac , Plásmidos , ARN sin Sentido , TransfecciónRESUMEN
BACKGROUND: The injection of double-stranded RNA (dsRNA) has been shown to induce a potent sequence-specific inhibition of gene function in diverse invertebrate and vertebrate species. The homology-dependent posttranscriptional gene silencing (PTGS) caused by the introduction of transgenes in plants may be mediated by dsRNA. The analysis of Caenorhabditis elegans mutants impaired with dsRNA-mediated silencing and studies in plants implicate a biological role of dsRNA-mediated silencing as a transposon-repression and antiviral mechanism. RESULTS: We investigated the silencing of testis-expressed Stellate genes by paralogous Su(Ste) tandem repeats, which are known to be involved in the maintenance of male fertility in Drosophila melanogaster. We found that both strands of repressor Su(Ste) repeats are transcribed, producing sense and antisense RNA. The Stellate silencing is associated with the presence of short Su(Ste) RNAs. Cotransfection experiments revealed that Su(Ste) dsRNA can target and eliminate Stellate transcripts in Drosophila cell culture. The short fragment of Stellate gene that is homologous to Su(Ste) was shown to be sufficient to confer Su(Ste)-dependent silencing of a reporter construct in testes. We demonstrated that Su(Ste) dsRNA-mediated silencing affects not only Stellate expression but also the level of sense Su(Ste) RNA providing a negative autogenous regulation of Su(Ste) expression. Mutation in the spindle-E gene relieving Stellate silencing also leads to a derepression of the other genomic tandem repeats and retrotransposons in the germline. CONCLUSIONS: Homology-dependent gene silencing was shown to be used to inhibit Stellate gene expression in the D. melanogaster germline, ensuring male fertility. dsRNA-mediated silencing may provide a basis for negative autogenous control of gene expression. The related surveillance system is implicated to control expression of retrotransposons in the germline.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Silenciador del Gen , Genes de Insecto , Proteínas de Insectos/genética , Proteínas Quinasas , ARN Bicatenario/fisiología , Proteínas Represoras/genética , Adenosina Trifosfatasas/genética , Animales , Animales Modificados Genéticamente , Células Cultivadas , Femenino , Genes Reporteros , Proteínas de Insectos/biosíntesis , Masculino , Mutación , ARN sin Sentido/biosíntesis , ARN Mensajero/biosíntesis , Proteínas Represoras/biosíntesis , Retroelementos , Homología de Secuencia de Ácido Nucleico , Secuencias Repetidas en Tándem , Testículo/metabolismoRESUMEN
Testis-specific expression of tandemly repeated Stellate genes, located in eu- and heterochromatin regions of the X chromosome of Drosophila melanogaster, is suppressed by homologous Suppressor of Stellate repeats located on the Y chromosome. Using transgenic lines, we have demonstrated that three Su(Ste) copies failed to change the expression of the reporter construction carrying the bacterial beta-galactosidase gene under control of the Stellate gene regulatory sequence. Possible mechanisms of the Su(Ste) repeat suppressor activity are discussed.
Asunto(s)
Drosophila melanogaster/genética , Genes de Insecto , Genoma , Secuencias Repetidas en Tándem , AnimalesRESUMEN
Here we report the peculiarities of molecular evolution and divergence of paralogous heterochromatic clusters of the testis- expressed X-linked Stellate and Y-linked Su(Ste) tandem repeats. It was suggested that Stellate and Su(Ste) clusters affecting male fertility are the amplified derivatives of the unique euchromatic gene betaCK2tes encoding the putative testis-specific beta-subunit of protein kinase CK2. The putative Su(Ste)-like evolutionary intermediate was detected on the Y chromosome as an orphon outside of the Su(Ste) cluster. The orphon shows extensive homology to the Su(Ste) repeat, but contains several Stellate-like diagnostic nucleotide substitutions, as well as a 10-bp insertion and a 3' splice site of the first intron typical of the Stellate unit. The orphon looks like a pseudogene carrying a drastically damaged Su(Ste) open reading frame (ORF). The putative Su(Ste) ORF, as compared with the Stellate one, carries numerous synonymous substitutions leading to the major codon preference. We conclude that Su(Ste) ORFs evolved on the Y chromosome under the pressure of translational selection. Direct sequencing shows that the efficiency of concerted evolution between adjacent repeats is 5-10 times as high in the Stellate heterochromatic cluster on the X chromosome as that in the Y-linked Su(Ste) cluster, judging by the frequencies of nucleotide substitutions and single-nucleotide deletions.
Asunto(s)
Drosophila melanogaster/genética , Evolución Molecular , Heterocromatina/genética , Proteínas de Insectos/genética , Familia de Multigenes , Cromosoma X , Cromosoma Y , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN/genética , Proteínas de Drosophila , Fertilidad/genética , Genes de Insecto , Intrones , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Quinasas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
The X-linked Stellate repeats, encoding a putative regulatory subunit of protein kinase CK2, are expressed in XO male testes. The Y-linked, testes-expressed paralogous Su(Ste) repeats are thought to be suppressors of Stellate transcription. The unique, testis-expressed euchromatic gene was suggested to be an ancestor of the both types of amplified paralogous repeats. A Su(Ste)-like orphon was localized on a Y chromosome, outside of the Su(Ste) cluster. Several diagnostic molecular markers peculiar for the both types of diverged Stellate and Su(Ste) units were detected in the orphon sequence. The orphon was suggested to be a close relative of the immediate ancestor of both types of paralogous repeats which initiated evolution on the Y chromosome. Selection pressure on the level of translation was shown as a driving force in the evolution of Su(Ste) repeats, which are considered as more ancient derivatives of the ancestor euchromatic gene than Stellate repeats. In a vicinity of 12E Stellate cluster the undamaged, recently originated euchromatic Stellate orphon was found at 12D, providing the poly(A) signal for the bendless gene. P-element mediated transformations reveal that the fragments of cloned Stellate and Su(Ste) clusters are able to induce variegation of a reporter mini-white gene. The observed variegation phenomenon has peculiar features: a significant increase of trans-activation of a reporter mini-white gene in homozygous state; absence of effects of several conventional modifiers of position effect variegation (PEV) and independence of a severity of variegation on a distance between insertion and centromere region.
Asunto(s)
Proteínas de Drosophila , Evolución Molecular , Silenciador del Gen , Genes Reporteros , Proteínas de Insectos/genética , Proteínas Quinasas , Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Drosophila melanogaster , Ligamiento Genético , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Cromosoma XRESUMEN
RNA editing in trypanosomatid mitochondria is a process involving the insertion and deletion of uridine residues within the coding region of maxicircle messenger RNA transcripts. Twelve of the 17 known genes need editing to produce functional molecules. We have analyzed the predicted editing sites for the Crithidia oncopelti mitochondrial NADH-ubiquinone oxidoreductase subunit 8 (ND8) gene based on known mRNAs from other trypanosomatid species. All studied ND8 mRNAs undergo editing throughout the coding (and 3' noncoding) sequences (pan-editing). The 5' part of the C. oncopelti ND8 gene undergoes editing (like in Leishmania tarentolae and Trypanosoma brucei) while the 3' part of the pre-edited gene corresponds to the 3' part of edited ND8 mRNAs from other organisms. The organization of the ND8 gene in C. oncopelti mitochondrial DNA differs from all organisms investigated so far -- this gene is not pan-edited. We have also localized the guide RNA for cytochrome b between 9S rRNA and the ND8 gene. This RNA shows high homology to the gCYb-II gene of L. tarentolae and the gCyb gene of Crithidia fasciculata. A hypothetical editing pattern for the cytochrome b gene in C. oncopelti maxicircles is proposed.
Asunto(s)
Crithidia/genética , Mitocondrias/metabolismo , NADH NADPH Oxidorreductasas/genética , Edición de ARN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Complejo I de Transporte de Electrón , Datos de Secuencia Molecular , ARN Guía de Kinetoplastida/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Euchromatic genes are often silenced by rearrangements that place them within or near heterochromatin, a phenomenon known as position effect variegation (PEV). However, little is known about molecular structure of cis-acting heterochromatic fragments responsible for PEV. Here we report that heterochromatic cluster containing Stellate repeats, that encode putative regulatory subunit of protein kinase CK2 cause PEV of a reporter white 'mini-gene'. It is the first example of an euchromatic gene being silenced because of the proximity to the natural, well-defined heterochromatic repeat cluster.