RESUMEN

Piwi proteins and Piwi interacting RNAs, piRNAs, presented in germline cells play a role in transposon silencing during germline development. In contrast, the role of somatic Piwi proteins and piRNAs still remains obscure. Here, we characterize the expression pattern and distribution of piRNAs in human renal cells in terms of their potential role in kidney development. Further, we show that all PIWI genes are expressed at the RNA level, however, only PIWIL1 gene is detected at the protein level by western blotting in healthy and cancerous renal cells. So far, the expression of human Piwil1 protein has only been shown in testes and cancer cells, but not in healthy somatic cell lines. Since we observe only Piwil1 protein, the regulation of other PIWI genes is probably more intricated, and depends on environmental conditions. Next, we demonstrate that downregulation of Piwil1 protein results in a decrease in the rate of cell proliferation, while no change in the level of apoptotic cells is observed. Confocal microscopy analysis reveals that Piwil1 protein is located in both cellular compartments, cytoplasm and nucleus in renal cells. Interestingly, in nucleus region Piwil1 is observed close to the spindle during all phases of mitosis in all tested cell lines. It strongly indicates that Piwil1 protein plays an essential role in proliferation of somatic cells. Moreover, involvement of Piwil1 in cell division could, at least partly, explain invasion and metastasis of many types of cancer cells with upregulation of PIWIL1 gene expression. It also makes Piwil1 protein as a potential target in the anticancer therapy.

Asunto(s)

Proteínas Argonautas , Riñón , Mitosis , ARN de Interacción con Piwi , Humanos , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Proliferación Celular , Riñón/citología , Riñón/crecimiento & desarrollo , Riñón/metabolismo , Mitosis/genética , ARN de Interacción con Piwi/genética , ARN de Interacción con Piwi/metabolismo

RESUMEN

tRNA-derived fragments participate in the regulation of many processes, such as gene silencing, splicing and translation in many organisms, ranging from bacteria to humans. We were interested to know how tRF abundance changes during the different stages of renal cell development. The research model used here consisted of the following human renal cells: hESCs, HEK-293T, HK-2 and A-489 kidney tumor cells, which, together, mimic the different stages of kidney development. The characteristics of the most abundant tRFs, tRFGly(CCC), tRFVal(AAC) and tRFArg(CCU), were presented. It was found that these parental tRNAs present in cells are the source of many tRFs, thus increasing the pool of potential regulatory RNAs. Indeed, a bioinformatic analysis showed the possibility that tRFGly(CCC) and tRRFVal(AAC) could regulate the activity of a range of kidney proteins. Moreover, the distribution of tRFs and the efficiency of their expression is similar in adult and embryonic stem cells. During the formation of tRFs, HK-2 cells resemble A-498 cancer cells more than other cells. Additionally, we postulate the involvement of Dicer nuclease in the formation of tRF-5b in all the analyzed tRNAs. To confirm this, 293T NoDice cells, which in the absence of Dicer activity do not generate tRF-5b, were used.

Asunto(s)

Biología Computacional , ARN de Transferencia , Adulto , Humanos , Riñón/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo

RESUMEN

Long noncoding RNAs exceeding a length of 200 nucleotides play an important role in ensuring cell functions and proper organism development by interacting with cellular compounds such as miRNA, mRNA, DNA and proteins. However, there is an additional level of lncRNA regulation, called lncRNA epigenetics, in gene expression control. In this review, we describe the most common modified nucleosides found in lncRNA, 6-methyladenosine, 5-methylcytidine, pseudouridine and inosine. The biosynthetic pathways of these nucleosides modified by the writer, eraser and reader enzymes are important to understanding these processes. The characteristics of the individual methylases, pseudouridine synthases and adenine-inosine editing enzymes and the methods of lncRNA epigenetics for the detection of modified nucleosides, as well as the advantages and disadvantages of these methods, are discussed in detail. The final sections are devoted to the role of modifications in the most abundant lncRNAs and their functions in pathogenic processes.

Asunto(s)

Enfermedad/etiología , Epigénesis Genética , MicroARNs/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , Humanos

RESUMEN

The application of a new generation of sequencing techniques has revealed that most of the genome has already been transcribed. However, only a small part of the genome codes proteins. The rest of the genome "dark matter" belongs to divergent groups of non-coding RNA (ncRNA), that is not translated into proteins. There are two groups of ncRNAs, which include small and long non-coding RNAs (sncRNA and lncRNA respectively). Over the last decade, there has been an increased interest in lncRNAs and their interaction with cellular components. In this review, we presented the newest information about the human lncRNA interactome. The term lncRNA interactome refers to cellular biomolecules, such as nucleic acids, proteins, and peptides that interact with lncRNA. The lncRNA interactome was characterized in the last decade, however, understanding what role the biomolecules associated with lncRNA play and the nature of these interactions will allow us to better understand lncRNA's biological functions in the cell. We also describe a set of methods currently used for the detection of lncRNA interactome components and the analysis of their interactions. We think that such a holistic and integrated analysis of the lncRNA interactome will help to better understand its potential role in the development of organisms and cancers.

Asunto(s)

ARN Largo no Codificante/genética , Genoma/genética , Humanos , Ácidos Nucleicos/genética , Péptidos/genética , Proteínas/genética

RESUMEN

The recently discovered group of noncoding RNAs, which are fragments of tRNA molecules (tRFs), has not been fully characterized and its potential functions still require investigation. Porcine tRFs were characterized and compared to mouse and human tRFs. Two tRFs, 5' 32-33 nt and 3' 41-42 nt that are derived from the mature tRNAVal(CAC) and tRNAGly(GCC) were detected with the use of bioinformatics and the Northern blot method. The abundance of these tRFs in the case of Sus scrofa is restricted to the ovary and the kidney. The same tRFs were found in human cancer cells and in mouse sperm, circulating blood and its serum. The binding of selected sncRNAs (piRNA, 5'tRFVal(CAC) and miRNA) to the overexpressed PAZ domain of the PIWIL4 protein was also studied. It is noteworthy that porcine 5'tRFVal(CAC) and human 5'tRFVal(CAC)as well as 5'tRFGly(GCC) are bound to the PIWIL4 protein. The potential role of the analyzed tRFs in the development of mammals is also discussed.

Asunto(s)

Mamíferos/crecimiento & desarrollo , Mamíferos/genética , ARN de Transferencia/genética , Sus scrofa/crecimiento & desarrollo , Sus scrofa/genética , Animales , Proteínas Argonautas/química , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Secuencia Conservada , Evolución Molecular , Femenino , Humanos , Masculino , Mamíferos/metabolismo , Ratones , MicroARNs/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Dominios Proteicos , Estructura Terciaria de Proteína , ARN Interferente Pequeño/genética , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN , Especificidad de la Especie , Sus scrofa/metabolismo

RESUMEN

Here, we describe the characterization of new RNA-cleaving DNAzymes that showed the highest catalytic efficiency at pH 4.0 to 4.5, and were completely inactive at pH values higher than 5.0. Importantly, these DNAzymes did not require any divalent metal ion cofactors for catalysis. This clearly suggests that protonated nucleic bases are involved in the folding of the DNAzymes into catalytically active structures and/or in the cleavage mechanism. The trans-acting DNAzyme variants were also catalytically active. Mutational analysis revealed a conservative character of the DNAzyme catalytic core that underpins the high structural requirements of the cleavage mechanism. A significant advantage of the described DNAzymes is that they are inactive at pH values close to physiological pH and under a wide range of conditions in the presence of monovalent and divalent metal ions. These pH-dependent DNAzymes could be used as molecular cassettes in biotechnology or nanotechnology, in molecular processes that consist of several steps. The results expand the repertoire of DNAzymes that are active under nonphysiological conditions and shed new light on the possible mechanisms of catalysis.

RESUMEN

Colistin and transition metal ions are commonly used as feed additives for livestock animals. This work presents the results of an analysis of combined potentiometric and spectroscopic (UV-vis, EPR, CD, NMR) data which lead to conclude that colistin is able to effectively chelate copper(II) ions. In cell-free system the oxidative activity of the complex manifests itself in the plasmid DNA destruction with simultaneous generation of reactive OH species, when accompanied by hydrogen peroxide or ascorbic acid. The degradation of RNA occurs most likely via a hydrolytic mechanism not only for complexed compound but also colistin alone. Therefore, huge amounts of the used antibiotic for nontherapeutic purposes might have a potential influence on livestock health.

Asunto(s)

Colistina/química , Cobre/química , Cobre/farmacología , Ácidos Nucleicos/química , Sistema Libre de Células/efectos de los fármacos , Dicroismo Circular , Complejos de Coordinación , Iones/farmacología , Estructura Molecular , Oxidación-Reducción/efectos de los fármacos

RESUMEN

In vitro selection was performed to search for RNA-cleaving DNAzymes catalytically active with Cd(2+) ions from the oligonucleotide combinatorial library with a 23-nucleotide random region. All the selected, catalytically active variants turned out to belong to the 8-17 type DNAzyme. Three DNAzymes were prepared in shortened, cis-acting versions which were subjected to a detailed study of the kinetic properties and metal ion preferences. Although the selection protocol was designed for Cd(2+)-dependent DNAzymes, the variants showed broader metal ion specificity. They preferred Cd(2+) but were also active with Mn(2+) and Zn(2+), suggesting that binding of the catalytic ion does not require an extremely specific coordination pattern. The unexpected decrease of the catalytic activity of the variants along with the temperature increase suggested that some changes occurred in their structures or the rate-limiting step of the reaction was changed. Two elements of the catalytic core of DNAzyme 1/VIIWS, the nucleotide at position 12 and the three-base-pair hairpin motif, were mutated. The presence of a purine residue at position 12 was crucial for the catalytic activity but the changes at that position had a relatively small influence on the metal ion preferences of this variant. The middle base pair of the three-base-pair hairpin was changed from A-T to C-G interaction. The catalytic activity of the mutated variant was increased with Zn(2+), decreased with Mn(2+), and was not changed in the presence of Cd(2+) ions. Clearly, this base pair was important for defining the metal ion preferences of the DNAzyme 1/VIIWS.

Asunto(s)

Cadmio/química , ADN Catalítico/química , Secuencia de Bases , Catalasa/química , Catálisis , Dominio Catalítico , Clonación Molecular , ADN/química , Biblioteca de Genes , Iones , Manganeso/química , Metales/química , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligonucleótidos/química , ARN/química , Temperatura , Zinc/química

RESUMEN

Small non-coding RNAs (sncRNAs) are indispensable for proper germ cell development, emphasizing the need for greater elucidation of the mechanisms of germline development and regulation of this process by sncRNAs. We used deep sequencing to characterize three families of small non-coding RNAs (piRNAs, miRNAs, and tRFs) present in Sus scrofa gonads and focused on the small RNA fraction present in both male and female gonads. Although similar numbers of reads were obtained from both types of gonads, the number of unique RNA sequences in the ovaries was several times lower. Of the sequences detected in the testes, 2.6% of piRNAs, 9% of miRNAs, and 10% of tRFs were also present in the ovaries. Notably, the majority of the shared piRNAs mapped to ribosomal RNAs and were derived from clustered loci. In addition, the most abundant miRNAs present in the ovaries and testes are conserved and are involved in many biological processes such as the regulation of homeobox genes, the control of cell proliferation, and carcinogenesis. Unexpectedly, we detected a novel sncRNA type, the tRFs, which are 30-36-nt RNA fragments derived from tRNA molecules, in gonads. Analysis of S. scrofa piRNAs show that testes specific piRNAs are biased for 5' uracil but both testes and ovaries specific piRNAs are not biased for adenine at the 10th nucleotide position. These observations indicate that adult porcine piRNAs are predominantly produced by a primary processing pathway or other mechanisms and secondary piRNAs generated by ping-pong mechanism are absent.

Asunto(s)

Ovario/metabolismo , ARN Pequeño no Traducido/genética , Sus scrofa/genética , Testículo/metabolismo , Animales , Femenino , Gametogénesis , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Ovario/citología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Pequeño no Traducido/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Testículo/citología , Transcripción Genética

RESUMEN

BACKGROUND: Bacitracin is a polypeptide antibiotic active against Gram-positive bacterial strains. Its mechanism of action postulates disturbing the cell wall synthesis by inhibiting dephosphorylation of the lipid carrier. We have discovered that bacitracin induces degradation of nucleic acids, being particularly active against RNA. METHODS: In the examination of the nucleolytic activity of bacitracin several model RNA and DNA oligomers were used. The oligomers were labeled at their 5' ends with (32)P radioisotope and following treatment with bacitracin the cleavage sites and efficiency were determined. RESULTS AND CONCLUSIONS: Bacitracin induces degradation of RNA at guanosine residues, preferentially in single-stranded RNA regions. Bacitracin is also able to degrade DNA to some extent but comparable effects to those observed with RNA require its 10-fold higher concentration. The sites of degradation in DNA are very infrequent and preferentially occur near cytidine residues. Free radicals are not involved in the reaction, and which probably proceeds via a hydrolytic mechanism. The phosphate groups at the cleavage sites are present at the 3' ends of RNA products and at the 5' ends of DNA fragments. Importantly, the presence of EDTA does not influence RNA degradation but completely inhibits the degradation of DNA. For DNA degradation divalent metal ions like Mg(2+), Mn(2+) or Zn(2+) are absolutely necessary. GENERAL SIGNIFICANCE: The ability of bacitracin to degrade nucleic acids via a hydrolytic mechanism was a surprising observation, and it is of interest whether these properties can contribute to its mechanisms of action during antibiotic treatment.

Asunto(s)

Antibacterianos/farmacología , Bacitracina/farmacología , ADN/química , ARN/química , Hidrólisis

RESUMEN

Three representatives of the distinct antibiotics groups: amoxicillin, apramycin and ristomycin A were studied regarding their impact on hepatitis D virus (HDV) ribozyme both in the metal-free form and complexed with copper(II) ions. Hence the Cu(II)-ristomycin A complex has been characterized by means of NMR, EPR, CD and UV-visible spectroscopic techniques and its binding pattern has been compared with the coordination modes estimated previously for Cu(II)-amoxicillin and Cu(II)-apramycin complexes. It has thus been found that all three antibiotics bind the Cu(II) ion in a very similar manner, engaging two nitrogen and two oxygen donors into coordination with the square planar symmetry in physiological conditions. All three tested antibiotics were able to inhibit the HDV ribozyme catalysis. However, in the presence of the complexes, the catalytic reactions were almost completely inhibited. It was important therefore to check whether the complexes used in lower concentrations could inhibit the HDV ribozyme catalytic activity, thus creating opportunities for their practical application. It turned out that the complexes used in the concentrations of 50µM influenced the catalysis much less effectively comparing to the 200 micromolar concentration. The kobs values were lower than those observed in the control reaction, in the absence of potential inhibitors: 2-fold for amoxicillin, ristomycin A and 3.3-fold for apramycin, respectively.

Asunto(s)

Amoxicilina/química , Cobre/química , Virus de la Hepatitis Delta/enzimología , Nebramicina/análogos & derivados , ARN Catalítico/química , ARN Viral/química , Ristocetina/química , Catálisis , Espectroscopía de Resonancia por Spin del Electrón , Espectroscopía de Resonancia Magnética , Nebramicina/química

RESUMEN

The interactions of selected antibiotics with the trans-acting antigenomic delta ribozyme were mapped. Ribozyme with two oligonucleotide substrates was used, one uncleavable with deoxycytidine at the cleavage site, mimicking the initial state of ribozyme, and the other with an all-RNA substrate mimicking, after cleavage, the product state. Mapping was performed with a set of RNA structural probing methods: Pb(2+) -induced cleavage, nuclease digestion, and the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) approach. The experimental results combined with molecular modeling revealed different binding sites for neomycin B, amikacin and actinomycin D inside the ribozyme structure. Neomycin B, an aminoglycoside antibiotic, which strongly inhibited the catalytic properties of delta ribozyme, was bound to the pocket formed by the P1 stem, the P1.1 pseudoknot, and the J4/2 junction. Amikacin showed less effective binding to the ribozyme catalytic core, resulting in weak inhibition. Complexes of these aminoglycosides with Cu(2+) ions were bound to the same ribozyme regions, but more effectively, showing lower Kd values. On the other hand, the Cu(2+) complex of the cyclopeptide antibiotic actinonomycin D was preferentially intercalated into the P2 and the P4 double-stranded region, and was three times more potent in ribozyme inhibition than the free antibiotic. In addition, some differences in SHAPE reactivities between the ribozyme forms containing all-RNA and deoxycytidine-modified substrates in the J4/2 region were detected, pointing to different ribozyme conformations before and after the cleavage event.

Asunto(s)

Antibacterianos/química , Antibacterianos/metabolismo , Virus de la Hepatitis Delta/enzimología , ARN Catalítico/química , ARN Catalítico/metabolismo , Amicacina/química , Amicacina/metabolismo , Secuencia de Bases , Simulación por Computador , Cobre/metabolismo , Dactinomicina/química , Dactinomicina/metabolismo , Framicetina/química , Framicetina/metabolismo , Virus de la Hepatitis Delta/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Catalítico/genética

RESUMEN

During in vitro run-off transcription with T7 RNA polymerase, transcripts with heterogenous 3' ends are commonly synthesized. Here, we describe an efficient procedure for correct processing of transcript 3' ends with the use of antigenomic HDV ribozyme. The procedure involves the extension of nascent transcripts with seven nucleotides complementary to the ribozyme's recognition site and, subsequently, the removal of those nucleotides with the HDV ribozyme acting in trans. Sufficient reaction rates and final cleavage extents of approx. 90% can be obtained with just twofold excess of the ribozyme. The highest concentration of RNA substrate suggested for practical applications turns out to be 3 µM. The procedure is an alternative to the use of ribozymes as cis-cleaving autocatalytic cassettes attached to transcript 3' ends.

Asunto(s)

Técnicas Genéticas , Virus de la Hepatitis Delta/enzimología , ARN Catalítico/metabolismo , Secuencia de Bases , ADN Complementario/biosíntesis , ADN Complementario/genética , Técnicas de Amplificación de Ácido Nucleico , División del ARN , ARN Mensajero/biosíntesis , ARN Mensajero/química , ARN Mensajero/genética , Transcripción Reversa

RESUMEN

Three Sus scrofa Piwi genes (Piwil1, Piwil2 and Piwil4) encoding proteins of 861, 985 and 853 aminoacids, respectively, were cloned and sequenced. Alignment of the Piwi proteins showed the high identity between Sus scrofa and Homo sapiens. Relative transcript abundance of porcine Piwil1, Piwil2 and Piwil4 genes in testes, ovaries and oocytes derived from sexually immature and mature animals was examined using Real-Time PCR. Expression of the three Piwi mRNAs was proved to be tissue specific and restricted exclusively to the gonads. In testes of adult pigs the highest relative transcript abundance was observed for the Sus scrofa Piwil1 gene. On the other hand, in testes of neonatal pigs the Piwil1 transcript level was over 2-fold reduced while the level of Piwil2 transcript was higher. As regards the expression of the Piwil4 transcript, its level was 34-fold elevated in testes of neonatal piglet when compared to adult male. In ovaries of prepubertal and pubertal female pigs transcript abundance of the three Piwi genes was significantly reduced in comparison with testes. However, similarly to testes, in ovaries of neonatal pigs the Piwil2 gene was characterized by the highest relative transcript abundance among the three Piwi genes analysed. In prepubertal and pubertal oocytes Piwil1 transcript was the most abundant whereas the expression of Piwil4 was undetectable. We also demonstrated that expression of piRNA occurs preferentially in the gonads of adult male and female pigs. Moreover, a piRNA subset isolated from ovaries was 2-3 nucleotides longer than the piRNA from testes.

Asunto(s)

Proteínas Argonautas/genética , Regulación del Desarrollo de la Expresión Génica , ARN Interferente Pequeño/genética , Porcinos , Secuencia de Aminoácidos , Animales , Proteínas Argonautas/química , Secuencia Conservada , Evolución Molecular , Femenino , Genómica , Masculino , Datos de Secuencia Molecular , Oocitos/metabolismo , Especificidad de Órganos , Ovario/crecimiento & desarrollo , Ovario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Maduración Sexual/genética , Testículo/crecimiento & desarrollo , Testículo/metabolismo

RESUMEN

Catalytic RNA molecules (ribozymes) have often been used for the testing of interactions of antibiotics with ribonucleic acids. We showed that the impact of capreomycin and hygromycin B on delta ribozyme catalysis might change dramatically, from stimulation to inhibition, depending on conditions. In order to evaluate possible mechanisms of modulation of the ribozyme catalytic activity we used our earlier data on species distribution for protonated forms of capreomycin and hygromycin B and their complexes with Cu(2+) ions at different pH values. We proposed that, upon inhibition, the protonated amino group of capreomycin was located in the ribozyme catalytic cleft interfering with binding catalytic Mg(2+). Such a mechanism was also supported by the results of ribozyme inhibition with capreomycin complexed with Cu(2+). The effects of stimulation of the delta ribozyme activity by capreomycin and hygromycin B were less pronounced than inhibition. Possibly, the amino functions of these antibiotics might be involved in a general acid-base catalysis performed by the ribozyme, acting as proton acceptors/donors.

Asunto(s)

Antibacterianos/farmacología , Capreomicina/farmacología , Cobre/metabolismo , Higromicina B/farmacología , ARN Catalítico/metabolismo , Antibacterianos/química , Secuencia de Bases , Capreomicina/química , Dominio Catalítico , Higromicina B/química , Datos de Secuencia Molecular , Protones , ARN Catalítico/química

RESUMEN

The ability of four stable hemiaminals differently substituted in the phenyl ring and their complexes with Cu(2+) ions to inhibit catalytic cleavage of the antigenomic delta ribozyme was compared. The hemiaminals were novel chiral derivatives of 1,2,4-triazole [i.e. (2,4-dinitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (2,4-dnbald), (2-nitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (2-nbald), (3-nitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (3-nbald) and (4-nitrophenyl)(4H-1,2,4-triazol-4-ylamino) methanol (4-nbald)]. The complexes of nbalds with Cu(2+) were characterized using UV and EPR methods and additionally, the formation of 2,4-dnbald-Cu(2+) complex with CuL(2) stoichiometry was confirmed by mass spectrometry. The data suggest that there are two ways in which nbalds and their Cu(2+) complexes can influence catalytic cleavage of antigenomic delta ribozyme. The coordinated Cu(2+) ions may play the role of new cationic ligands increasing the affinity of the complexes to the ribozyme. Such situation occurs in the case of 2- and 2,4-nbald. Their Cu(2+) complexes decrease ribozyme cleavage rates twice more efficiently than uncomplexed compounds. Moreover, the Cu(2+) complexes displace the catalytic divalent metal ions from their strong binding sites located in the ribozyme J4/2 region as shown by the Pb(2+)-induced cleavage approach. On the other hand, 3- and 4-nbald inhibit catalysis more strongly as compared to 2-nbald and 2,4-dnbald but the ribozyme cleavage rates are changed only slightly upon Cu(2+) complexation. The mechanism of ribozyme inhibition by interfering with the formation of a correct ribozyme tertiary structure seems to operate in this case.

Asunto(s)

Cobre/química , Metales/química , ARN Catalítico/química , Triazoles/química , Catálisis , Conformación de Ácido Nucleico

RESUMEN

Human ribonuclease Dicer is an enzyme that excises small regulatory RNAs from perfectly or partially double-stranded RNA precursors. Although Dicer substrates and products have already been quite well characterized, our knowledge about cellular factors regulating the activity of this enzyme is still limited. To learn more about this problem, we attempted to determine whether RNA could function not only as a Dicer substrate but also as its regulator. To this end, we applied an in vitro selection method. We identified 120 RNA oligomers binding human Dicer. Sixteen of them were subjected to more detailed in vitro studies. We found that 6 out of 16 oligomers affected Dicer ability to digest pre-microRNAs (miRNAs), although most of them were cleaved by this enzyme. For the 6 most active oligomers the putative mechanism of Dicer inhibition was determined. Three oligomers were classified as typical competitive inhibitors and one as an allosteric inhibitor. The remaining 2 oligomers acted as selective inhibitors. They affected the production of 1 miRNA, whereas the formation of other miRNAs was hardly influenced. In general, the data obtained suggest that one can modulate the generation of specific miRNAs by using RNA oligomers. Moreover, we found that sequences similar to those of the selected oligomers can be found within the molecules composing human transcriptome.

Asunto(s)

Aptámeros de Nucleótidos/química , ARN Helicasas DEAD-box/antagonistas & inhibidores , Ribonucleasa III/antagonistas & inhibidores , Regulación Alostérica , Aptámeros de Nucleótidos/farmacología , Secuencia de Bases , Unión Competitiva , Simulación por Computador , Pruebas de Enzimas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , MicroARNs/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Técnica SELEX de Producción de Aptámeros , Transcriptoma

RESUMEN

A new group of small noncoding RNAs of 24-30 nucleotides in length, piRNAs, are mainly expressed in germline cells. They form complexes with Piwi proteins, members of the Argonaute family and unlike other small RNAs they are created without RNase Dicer participation. They are present in male and female germinal cells of numerous animals, from flies to humans. The piRNA biogenesis mechanism is unknown, however, it is postulated that they are formed from long single-stranded RNA precursors coded by repetitive sequences occurring in the genome. A large part of piRNA corresponds to retrotranspozon sequences, which may indicate their participation in silencing the mobile elements and maintaining genome integrity of germinal cells. However, disruption of the piRNA biosynthesis pathway and mutations genes encoding Piwi proteins cause the activation of transpozons and a number of defects in the course of gametogenesis, resulting in reproduction disturbance. In this review, the current state of knowledge on the structure, biogenesis and function of piRNA and their interactions with Piwi proteins is presented.

Asunto(s)

Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Células Germinativas/crecimiento & desarrollo , ARN Interferente Pequeño/genética , Animales , Femenino , Humanos , Masculino

RESUMEN

Two early nodulin 40 (enod40) genes, ENOD40-1, the shortest legume ENOD40 gene, and ENOD40-2, were isolated from Lupinus luteus, a legume with indeterminate nodules. Both genes were expressed at similar levels during symbiosis with nitrogen-fixing bacteria. ENOD40 phylogeny clustered the L. luteus genes with legumes forming determinate nodules and revealed peptide similarities. The ENOD40-1 small ORF A fused to a reporter gene was efficiently expressed in plant cells, indicating that the start codon is recognized for translation. The ENOD40-1 RNA structure predicted based on Pb(II)-induced cleavage and modeling revealed four structurally conserved domains, an absence of domain 4 characteristic for legumes of indeterminate nodules, and interactions between the conserved region I and a region located upstream of domain 6. Domain 2 contains Mg(II) ion binding sites essential for organizing RNA secondary structure. The differences between L. luteus and Glycine max ENOD40 RNA models suggest the possibility of a switch between two structural states of ENOD40 transcript.

Asunto(s)

Lupinus/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Biosíntesis de Proteínas , ARN de Planta/química , Southern Blotting , Genes Reporteros , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Co(2+) binding RNA aptamers were chosen as research models to reveal the structural basis underlying the recognition of Co(2+) by RNA, with the application of two distinct methods. Using the nucleotide analog interference mapping assay, we found strong interference effects after incorporation of the 7-deaza guanosine phosphorotioate analog into the RNA chain at equivalent positions G27 and G28 in aptamer no. 18 and G25 and G26 in aptamer no. 20. The results obtained by nucleotide analog interference mapping suggest that these guanine bases are crucial for the creation of Co(2+) binding sites and that they appear to be involved in the coordination of the ion to the exposed N7 atom of the tandem guanines. Additionally, most 7-deaza guanosine phosphorotioate and 7-deaza adenosine phosphorotioate interferences were located in the common motifs: loop E-like in aptamer no. 18 and kissing dimer in aptamer no. 20. We also found that purine-rich stretches containing guanines with the highest interference values were the targets for hybridization of 6-mers, which are members of the semi-random oligodeoxyribonucleotide library in both aptamers. It transpired that DNA oligomer directed RNase H digestions are sensitive to Co(2+) and, at an elevated metal ion concentration, the hybridization of oligomers to aptamer targets is inhibited, probably due to higher stability and complexity of the RNA structure.

Asunto(s)

Aptámeros de Nucleótidos/química , Cobalto/química , Conformación de Ácido Nucleico , Aptámeros de Nucleótidos/síntesis química , Secuencia de Bases , Sitios de Unión , Cationes Bivalentes/química , Datos de Secuencia Molecular , Fosfatos/química , Purinas/química , Soluciones , Tionucleósidos/química