RESUMO
Dental pain is a persistent, detrimental public health issue that requires a better understanding of the mechanisms of tooth pain and inflammation in order to develop more effective treatments. Calcitonin gene-related peptide (CGRP) and dental pulp cells are promising candidates for mediating tooth pain and generating reparative dental tissues, respectively, but their behavior in the context of pulpitis remains elusive. The mouse incisor requires Sonic hedgehog (Shh) secreted from sensory nerves to continuously regenerate. However, it is unknown whether sensory nerves also regulate the comparatively nonregenerative mouse molar through CGRP and Shh. This is an important knowledge gap to fill since mouse incisors differ biologically from human teeth, while mouse and human molars are similar. In this work, we identified that molar pulp cells express CGRP receptor and Gli1, a Hedgehog (Hh) signaling protein found to label a dental stem cell population in the mouse incisor. We also observed in a mouse molar injury model that Hh signaling was activated and Shh expression was upregulated in vivo. We then determined in vitro that Shh and CGRP regulate differentiation of primary mouse molar and incisor pulp cells and a human dental pulp stem cell line. Furthermore, conditioned media from stimulated sensory neurons induced Hh signaling activation and inflammatory gene expression in primary molar pulp cells, which was abolished by inhibition of either Shh or CGRP. Our results suggest that CGRP and Shh signaling may promote an inflammatory response after injury in the molar and that activated sensory nerves secrete CGRP and Shh to regulate molar pulp cell expansion and differentiation into odontoblast-like cells for dentin repair. Thus, CGRP/Shh signaling should be considered for new strategies that seek to manage pain or dentin regeneration in the molar.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Polpa Dentária , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Polpa Dentária/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Incisivo , Camundongos , Neurônios Aferentes/metabolismo , DorRESUMO
Dental pulp stem cells (DPSCs) are important in tooth physiology, contributing to development, repair, regeneration, and immunomodulatory processes. However, their role in inflammatory mechanisms underlying pulpitis is not well understood. We evaluated the influence of DPSCs stimulated with calcitonin gene-related peptide (CGRP), a proinflammatory neuropeptide, on the expression of mediators released from DPSCs and the effect of these mediators on sensory neuron activity. Human DPSCs were treated with either control media or media containing CGRP (10-8 M) for 7 d, and the conditioned media (CM) containing DPSC-released mediators was collected. The expression of cytokines and chemokines from DPSCs was evaluated by reverse transcription quantitative polymerase chain reaction. The effects of the CM from CGRP-primed DPSCs (primed DPSC-CM) were evaluated on sensory afferents by using primary cultures of mouse trigeminal neurons and an organotypic model of cultured human pulp slices. Mouse trigeminal neurons and human pulp explants were pretreated for 24 h with control or primed DPSC-CM and then stimulated with capsaicin. Afferent activity was measured by quantifying the response to capsaicin via live cell calcium imaging in mouse neurons and CGRP released from pulp explants. Gene expression analysis showed that primed DPSCs overexpressed some proinflammatory cytokines and chemokines, including chemokines CXCL1 and CXCL8, which are both agonists of the receptor CXCR2 expressed in sensory neurons. Primed DPSC-CM increased human pulp sensory afferent activity as compared with control DPSC-CM. Similarly, primed DPSC-CM increased the intensity of calcium responses in cultured mouse trigeminal neurons. Furthermore, the CXCR2 antagonist SB225002 prevented trigeminal neuron sensitization to capsaicin induced by primed DPSC-CM. In conclusion, mediators released by DPSCs, primed with the proinflammatory mediator CGRP, induce neuronal sensitization through CXCR2 receptor. These data suggest that DPSCs might contribute to pain symptoms that develop in pulpitis.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Polpa Dentária , Animais , Calcitonina , Diferenciação Celular , Células Cultivadas , Camundongos , Células Receptoras Sensoriais , Células-TroncoRESUMO
BACKGROUND: Previous data showed that, in rats, anti-migraine drugs (triptans, olcegepant) significantly reduced mechanical allodynia induced by infraorbital nerve (ION) ligation but not that evoked by sciatic nerve (SN) ligation. Whether this also occurs with MK-8825, a novel anti-migraine drug also acting through CGRP receptor blockade (but chemically unrelated to olcegepant) was tested in the present study, which also investigated possible anti-neuroinflammatory effects of this drug. METHODS: Adult male Sprague-Dawley rats underwent unilateral chronic constriction injury (CCI) to either the ION or the SN, and mechanical allodynia was assessed 2 weeks later within the ipsilateral vibrissae territory or hindpaw, respectively. Transcripts of neuroinflammatory markers were quantified by real-time quantitative RT-PCR in ipsilateral trigeminal ganglion and spinal trigeminal nucleus in CCI-ION rats. RESULTS: Acute as well as repeated (for 4 days) administration of MK-8825 (30-100 mg/kg, i.p.) significantly reduced CCI-ION-induced mechanical allodynia but was ineffective in CCI-SN rats. CCI-ION was associated with marked up-regulation of neuronal and glial inflammatory markers (ATF3, IL6, iNOS, COX2) in ipsilateral trigeminal ganglion but not spinal trigeminal nucleus. MK-8825-induced inhibition of iNOS mRNA up-regulation probably underlay its anti-allodynic effect because pharmacological blockade of iNOS by AMT (6 mg/kg, s.c.) mimicked this effect. CONCLUSIONS: These data further support the idea that CGRP receptor blockade might be a valuable approach to alleviate trigeminal, but not spinal, neuropathic pain through, at least partly, an inhibitory effect on neuropathic pain-associated increase in NO production in trigeminal ganglion.
Assuntos
Hiperalgesia/etiologia , Piridinas/farmacologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Nervo Isquiático/lesões , Neuropatia Ciática/tratamento farmacológico , Compostos de Espiro/farmacologia , Animais , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Ligadura/métodos , Masculino , Neuralgia/induzido quimicamente , Ratos Sprague-Dawley , Gânglio Trigeminal/efeitos dos fármacos , Regulação para CimaRESUMO
BACKGROUND: Convergent data showed that neuropathic pain has specific characteristics at cephalic versus extra-cephalic level, where single-targeted drugs differentially alleviate pain. Because the novel analgesic drug, tapentadol, is acting at two targets, µ-opioid receptors (as agonist) and noradrenaline reuptake (as inhibitor), we tested its effects on neuropathic pain at both cephalic and extra-cephalic levels. METHODS: Sprague-Dawley rats underwent unilateral constriction injury (CCI) to the infraorbital nerve (ION; cephalic territory) or the sciatic nerve (SN; extra-cephalic territory), and alleviation of nerve lesion-induced mechanical allodynia/hyperalgesia was assessed after acute or repeated (for 4 days) treatment with tapentadol compared with morphine and/or reboxetine (noradrenaline reuptake inhibitor) 2 weeks after surgery. Possible changes in the expression of the neuroinflammatory markers activating transcription factor 3 (ATF3), interleukin-6 (IL-6) and brain-derived neurotrophic factor (BDNF) by repeated tapentadol treatment were quantified by real-time reverse transcription polymerase chain reaction in ganglia and central tissues. RESULTS: Acute administration of tapentadol (1-10 mg/kg, i.p.) significantly reduced allodynia in both CCI-SN and CCI-ION rats. Although morphine (3 mg/kg, s.c.) or reboxetine (10 mg/kg, i.p.) alone was only marginally active, the combination of both drugs produced supra-additive effects like those observed with tapentadol. In contrast to repeated morphine whose effects vanished, the anti-allodynic effects of tapentadol remained unchanged after a 4-day treatment. However, the latter treatment with tapentadol did not affect nerve lesion-evoked overexpression of ATF3, IL-6 and BDNF transcripts. CONCLUSIONS: The dual synergistic pharmacological properties of tapentadol, which result in clear-cut anti-neuropathic pain effects at both cephalic and extra-cephalic levels, probably involve mechanisms downstream of nerve injury-induced neuroinflammatory reaction.
Assuntos
Hipersensibilidade/tratamento farmacológico , Nervo Maxilar/efeitos dos fármacos , Neuralgia/tratamento farmacológico , Fenóis/uso terapêutico , Nervo Isquiático/efeitos dos fármacos , Animais , Humanos , Hiperalgesia/tratamento farmacológico , Ligadura , Masculino , Nervo Maxilar/lesões , Morfina/uso terapêutico , Medição da Dor/métodos , Ratos , Ratos Sprague-Dawley , Receptores Opioides mu/metabolismo , Nervo Isquiático/lesões , Tapentadol , Resultado do TratamentoAssuntos
Antígenos de Neoplasias/urina , Neoplasias da Bexiga Urinária/diagnóstico , Bexiga Urinária/imunologia , Urina/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/urinaRESUMO
Molecular mechanisms of ITS2 processing, a eukaryotic insertion between the 5.8S and LSU rRNA, remain largely elusive even in yeast. To delineate ITS2 structural and functional features which could be common to eukaryotes, we first produced phylo-genetically supported folding models in the vertebrate lineage, then tested them in deeper branchings and, more particularly, among yeasts. ITS2 comparisons between four Teleostei, a Chondrichthyes specimen and two jawless organisms have revealed a common folding architecture in four to five domains of secondary structure emerging from a preserved structural core. This folding, largely reminiscent of ITS2 architecture in mammals, is also preserved in amphibia and in chicken, despite dramatic sequence variations. Preferential conservation is located around a central loop and at the apex of a long stem in the ITS2 3'-half. Interestingly, these two independent structural features contain, respectively, the 3'-ends of the two transient rRNA precursors 8S and 12S RNA identified in mammals, suggesting a preservation of these intermediates of processing over the entire vertebrate group. Surprising similarities between the vertebrate ITS2 folding shape and that of invertebrates as well as protista have made intriguing the significant differences from the yeast model. A detailed comparative analysis including four relatively close species and Schizosaccharomyces pombe, a deep yeast branching, has revealed an alternative phylogenetically supported four-domain folding presenting strong similarities to the vertebrate model. Remarkably, the two best conserved regions of vertebrates have unambiguously preserved counterparts which are also sites for internal processing in yeast. Therefore, molecular mechanisms involved in ITS2 excision in vertebrates and yeast might be more closely related than currently believed and might require a very similar trans -acting machinery.
Assuntos
Conformação de Ácido Nucleico , RNA Ribossômico/química , Vertebrados/genética , Leveduras/genética , Animais , Sequência de Bases , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
Mechanisms of ITS2 excision from pre-rRNA remain largely elusive. In mammals, at least two endonucleolytic cleavages are involved, which result in the transient accumulation of precursors to 5.8S rRNA termed 8S and 12S RNAs. We have sequenced ITS2 in four new species of the Mus genus and investigated its secondary structure using thermodynamic prediction and comparative approach. Phylogenetic evidence supports an ITS2 folding organized in four domains of secondary structure extending from a preserved structural core. This folding is also largely conserved for the previously available mammalian ITS2 sequences, rat and human, despite their extensive sequence divergence relative to the Mus species. Conserved structural features include the structural core, containing the 3' end of 8S pre-rRNA within a single-stranded sequence, and a stem containing the 3' end of the 12S pre-rRNA species. A putative, phylogenetically preserved pseudoknot has been detected 1 nt downstream from the 12S 3' end. Two long complementarities have also been identified, in sequences conserved among vertebrates, between the pre-rRNA 32S and the snoRNA (small nucleolar RNA) U8 which is required for the excision of Xenopus ITS2. The first complementarity involves the 5.8S-ITS2 junction and 13 nt at the 5' end of U8, whereas the other one occurs between a mature 28S rRNA segment known to be required for ITS2 excision and positions 15-25 of snoRNA U8. These two potential interactions, in combination with ITS2 folding, could organize a functional pocket containing three cleavage sites and key elements for pre-rRNA processing, suggesting a chaperone role for the snoRNA U8.
Assuntos
DNA Ribossômico/química , Evolução Molecular , Precursores de RNA/química , RNA Nuclear Pequeno , Animais , Sequência de Bases , Humanos , Mamíferos , Camundongos , Dados de Sequência Molecular , Muridae , Conformação de Ácido Nucleico , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
Microsporidia are eukaryotic parasites lacking mitochondria, the ribosomes of which present prokaryote-like features. In order to better understand the structural evolution of rRNA molecules in microsporidia, the 5S and rDNA genes were investigated in Encephalitozoon cuniculi . The genes are not in close proximity. Non-tandemly arranged rDNA units are on every one of the 11 chromosomes. Such a dispersion is also shown in two other Encephalitozoon species. Sequencing of the 5S rRNA coding region reveals a 120 nt long RNA which folds according to the eukaryotic consensus structural shape. In contrast, the LSU rRNA molecule is greatly reduced in length (2487 nt). This dramatic shortening is essentially due to truncation of divergent domains, most of them being removed. Most variable stems of the conserved core are also deleted, reducing the LSU rRNA to only those structural features preserved in all living cells. This suggests that the E.cuniculi LSU rRNA performs only the basic mechanisms of translation. LSU rRNA phylogenetic analysis with the BASEML program favours a relatively recent origin of the fast evolving microsporidian lineage. Therefore, the prokaryote-like ribosomal features, such as the absence of ITS2, may be derived rather than primitive characters.
Assuntos
Encephalitozoon cuniculi/genética , Conformação de Ácido Nucleico , RNA de Protozoário , RNA Ribossômico 5S , RNA Ribossômico , Animais , Sequência de Bases , Mapeamento Cromossômico , DNA Ribossômico , Células Eucarióticas , Dados de Sequência Molecular , Filogenia , RNA de Protozoário/química , RNA Ribossômico/química , RNA Ribossômico 5S/química , RibossomosRESUMO
With ESSA, we propose an approach of RNA secondary structure analysis based on extensive viewing within a friendly graphical interface. This computer program is organized around the display of folding models produced by two complementary methods suitable to draw long RNA molecules. Any feature of interest can be managed directly on the display and highlighted by a rich combination of colours and symbols with emphasis given to structural probe accessibilities. ESSA also includes a word searching procedure allowing easy visual identification of structural features even complex and degenerated. Analysis functions make it possible to calculate the thermodynamic stability of any part of a folding using several models and compare homologous aligned RNA both in primary and secondary structure. The predictive capacities of ESSA which brings together the experimental, thermodynamic and comparative methods, are increased by coupling it with a program dedicated to RNA folding prediction based on constraints management and propagation. The potentialities of ESSA are illustrated by the identification of a possible tertiary motif in the LSU rRNA and the visualization of a pseudoknot in S15 mRNA.
Assuntos
Conformação de Ácido Nucleico , RNA/química , Software , Composição de Bases , Sequência de Bases , Sequência Consenso , Bases de Dados Factuais , Estabilidade de Medicamentos , Dados de Sequência Molecular , RNA Ribossômico/química , TermodinâmicaRESUMO
A growing number of small nucleolar RNAs (snoRNAs) are intron-encoded, contain the characteristic box C (UGAUGA) and box D (CUGA) motifs and exhibit long complementarities to conserved sequences in mature rRNAs. We have identified nine additional members of this family, U32 to U40. All but one are encoded in introns of ribosomal protein genes in vertebrates: U32 to U35 in rpL13a, U36 in rpL7a and U38 to U40 in rpS8. By contrast, U37 is encoded in elongation factor 2 gene. Interestingly, U32 and U36 each contain two complementarities (one to 18 S and the other to 28 S rRNA). U32 to U40 are fibrillarin-associated, devoid of a 5'-trimethyl-cap and display an exclusively nucleolar localization. They are all metabolically stable and roughly as abundant as previously reported members of this family. Characterization of their homologs in distant species shows that their 10 to 14 nt long rRNA complementarities are conserved. A clue on the function of this snoRNA family is provided by the comparative analysis of the largely expanded collection of their conserved duplexes with rRNA. Not only does each duplex span at least one site of 2'-O-ribose methylation in the rRNA but the modification site is always at the same position in the duplex, paired to the fifth nucleotide upstream from a box D motif in the snoRNA. Consistent with the notion that each snoRNA of this family guides one particular methylation along the rRNA sequence, we have detected several pairs of snoRNAs with overlapping complementarities to rRNA tracts with vicinal sites of ribose methylations. In each case, the two overlapping complementarities are shifted from each other by a distance equal to the spacing between the methylated sites which are thus found at the same position within each of the mutually exclusive duplexes. Finally, we have also identified, within three previously known snoRNAs, novel antisense elements able to form a canonical duplex around ribose-methylated sites in rRNA, which further supports the conclusion that the duplex structure provides the 2'-O-methyltransferase with the appropriate site-specificity on the substrate.
Assuntos
Nucléolo Celular/química , Íntrons/genética , Processamento Pós-Transcricional do RNA , RNA Antissenso/metabolismo , RNA Ribossômico/metabolismo , RNA Nuclear Pequeno/metabolismo , Animais , Sequência de Bases , Northern Blotting , Proteínas Cromossômicas não Histona/metabolismo , Sequência Conservada , Bases de Dados Factuais , Evolução Molecular , Humanos , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Precursores de RNA/metabolismo , RNA Antissenso/química , RNA Antissenso/genética , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Ribose/metabolismoRESUMO
One of the four ribosomal RNA operons (rrnA) from the Agrobacterium vitis vitopine strain S4 was sequenced, rrnA is most closely related to the rrn operons of Bradyrhizobium japonicum and Rhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case of R. sphaeroides. The 16S rRNA sequence of S4 differs from the A. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3' ends of the three other rrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3' ends of the four repeats and defines two groups: rrnA/rrnB and rrnC/rrnD.
Assuntos
Óperon , RNA Ribossômico/genética , Rhizobium/genética , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/genética , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/genética , Sequências Repetitivas de Ácido Nucleico , Rhizobiaceae/genética , Rhodobacter sphaeroides/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Recently, several new snoRNAs encoded in introns of genes coding for ribosomal, ribosome-associated, or nucleolar proteins have been discovered. We are presently studying four of these intronic snoRNAs. Three of them, U20, U21, and U24, are closely related to each other on a structural basis. They are included in genes encoding nucleolin and ribosomal proteins L5 and L7a, respectively, in warm-blooded vertebrates. These three metabolically stable snoRNAs interact with nucleolar protein fibrillarin. In addition, they display common features that make them strikingly related to snoRNA U14. U14 contains two tracts of complementarity to 18S rRNA, which are required for the production of 18S rRNA. U20 displays a 21 nucleotide (nt) long complementarity to 18S rRNA. U21 contains a 13 nt complementarity to an invariant sequence in eukaryotic 28S rRNA. U24 has two separate 12 nt long complementarities to a highly conserved tract of 28S rRNA. Phylogenetic evidences support the fundamental importance of the pairings of these three snoRNAs to pre-rRNA, which could be involved in a control of pre-rRNA folding during preribosome assembly. By transfection of mouse cells, we have also analyzed the processing of U20 and found that the -cis acting signals for its processing from intronic RNA are restricted to the mature snoRNA sequence. Finally, we have documented changes of host genes for these three intronic snoRNAs during the evolution of eukaryotes.
Assuntos
Nucléolo Celular/genética , Íntrons , RNA Ribossômico/genética , RNA Nuclear Pequeno/genética , Animais , Sequência de Bases , Código Genético , Camundongos , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
A growing subset of small nucleolar RNAs (snoRNAs) contains long stretches of sequence complementarity to conserved sequences in mature ribosomal RNA (rRNA). This article reviews current knowledge about these complementarities and proposes that these antisense snoRNAs might function in pre-rRNA folding, base modification and ribosomal ribonucleoprotein assembly, in some cases acting as RNA chaperones.
Assuntos
Nucléolo Celular/química , RNA Antissenso/química , RNA Ribossômico/química , RNA Nuclear Pequeno/química , Animais , Sequência de Bases , Sequência Conservada/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Antissenso/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Leveduras/genéticaRESUMO
Following computer searches of sequence banks, we have positively identified a novel intronic snoRNA, U24, encoded in the ribosomal protein L7a gene in humans and chicken. Like previously reported intronic snoRNAs, U24 is devoid of a 5'-trimethyl-cap. U24 is immunoprecipitated by an antifibrillarin antibody and displays an exclusively nucleolar localization by fluorescence microscopy after in situ hybridization with antisense oligonucleotides. In vertebrates, U24 is a 76 nt long conserved RNA which is metabolically stable, present at approximately 14,000 molecules per human HeLa cell. U24 exhibits a 5'-3' terminal stem-box C-box D structure, typical for several snoRNAs, and contains two 12 nt long conserved sequences complementary to 28S rRNA. It is, therefore, strikingly related to U14, U20 and U21 snoRNAs which also possess long sequences complementary to conserved sequences of mature 18S or 28S rRNAs. In 28S rRNA the two tracts complementary to U24 are adjacent to each other, they involve several methylated nucleotides and are surprisingly close, within the rRNA secondary structure, to complementarities to snoRNAs U18 and U21. Identification of the yeast Saccharomyces cerevisiae U24 gene directly confirms the outstanding conservation of the complementarity to 28S rRNA during evolution, suggesting a key role of U24 pairing to pre-rRNA during ribosome biogenesis, possible in the control of pre-rRNA folding. Yeast S.cerevisiae U24 is also intron-encoded but not in the same host-gene as in humans or chicken.
Assuntos
RNA Ribossômico 28S/genética , RNA Nuclear Pequeno/genética , Animais , Sequência de Bases , Nucléolo Celular/metabolismo , Galinhas , Sequência Conservada , DNA Complementar/genética , Genes Fúngicos , Células HeLa , Humanos , Íntrons , Dados de Sequência Molecular , Filogenia , RNA Fúngico/genética , Saccharomyces cerevisiae/genéticaRESUMO
RNA-d2 is a user-friendly program developed for interactively generating aesthetic and non-overlapping drawings of RNA secondary structures. It designed so that the drawings can be edited in a very natural and intuitive way, in order to emphasize structural homologies between several molecules, as well as the foldings themselves to update the base-pair sets according to new data. The program automatically produces a polygonal display in which the unpaired nucleotides are regularly positioned on circles and the stems harmoniously distributed on their periphery. RNA secondary structures can be encoded via the keyboard, but the program also automatically draws output files from thermodynamic prediction programs. The user interacts directly on different screen displays according to the editing functions. Rotation/translation of any subdomain and deletion of stems are performed on a coloured backbone view to make easier the identification of structural features, whereas addition of new base-pairings and numbering manipulation are realized on a complete polygonal view. Each modification is displayed in real time on the screen. When the display is obscured by numerous overlaps despite the colour code of the backbone view, an automatic function progressively straightens the subdomains which are highly compacted by very dissymmetric internal loops. RNA-d2 allows easy untangling and editing of RNA molecules > 1000 nucleotides long.
Assuntos
Conformação de Ácido Nucleico , RNA/química , Software , Algoritmos , Sequência de Bases , Gráficos por Computador , Estudos de Avaliação como Assunto , Humanos , Dados de Sequência Molecular , RNA/genética , RNA Ribossômico 28S/química , RNA Ribossômico 28S/genética , Interface Usuário-ComputadorRESUMO
Following a search of sequence data bases for intronic sequences exhibiting structural features typical of snoRNAs, we have positively identified by Northern assays and sequence analysis another intron-encoded snoRNA, termed U21. U21 RNA is a 93 nt. long, metabolically stable RNA, present at about 10(4) molecules per HeLa cell. It is encoded in intron 5 of the ribosomal protein L5 gene, both in chicken and in the two mammals studied so far, human and mouse. U21 RNA is devoid of a 5'-trimethyl-cap and is likely to result from processing of intronic RNA. The nucleolar localization of U21 has been established by fluorescence microscopy after in situ hybridization with digoxigenin-labeled oligonucleotide probes. Like most other snoRNAs U21 contains the box C and box D motifs and is precipitated by anti-fibrillarin antibodies. By the presence of a typical 5'-3' terminal stem, U21 appears more particularly related to U14, U15, U16 and U20 intron-encoded snoRNAs. Remarkably, U21 contains a long stretch (13 nt.) of complementarity to a highly conserved sequence in 28S rRNA. Sequence comparisons between chicken and mammals, together with Northern hybridizations with antisense oligonucleotides on cellular RNAs from more distant vertebrates, point to the preferential preservation of this segment of U21 sequence during evolution. Accordingly, this complementarity, which overlaps the complementarity of 28S rRNA to another snoRNA, U18, could reflect an important role of U21 snoRNA in the biogenesis of large ribosomal subunit.
Assuntos
Íntrons , RNA Ribossômico 28S/química , RNA Nuclear Pequeno/genética , Proteínas Ribossômicas/genética , Animais , Sequência de Bases , Northern Blotting , Nucléolo Celular/química , Galinhas/genética , Éxons , Células HeLa , Humanos , Hibridização In Situ , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Filogenia , RNA Nuclear Pequeno/química , Análise de SequênciaRESUMO
We have found that intron 11 of the nucleolin gene in humans and rodents encodes a previously unidentified small nucleolar RNA, termed U20. The single-copy U20 sequence is located on the same DNA strand as the nucleolin mRNA. U20 RNA, which does not possess a trimethyl cap, appears to result from intronic RNA processing and not from transcription of an independent gene. In mammals, U20 RNA is an 80-nucleotide-long, metabolically stable species, present at about 7 x 10(3) molecules per exponentially growing HeLa cell. It has a nucleolar localization, as indicated by fluorescence microscopy following in situ hybridization with digoxigenin-labeled oligonucleotides. U20 RNA contains the box C and box D sequence motifs, hallmarks of most small nucleolar RNAs reported to date, and is immunoprecipitated by antifibrillarin antibodies. It also exhibits a 5'-3' terminal stem bracketing the box C-box D motifs like U14, U15, U16, or Y RNA. A U20 homolog of similar size has been detected in all vertebrate classes by Northern (RNA) hybridization with mammalian oligonucleotide probes. U20 RNA contains an extended region (21 nucleotides) of perfect complementarity with a phylogenetically conserved sequence in 18S rRNA. This complementarity is strongly preserved among distant vertebrates, suggesting that U20 RNA may be involved in the formation of the small ribosomal subunit like nucleolin, the product of its host gene.
Assuntos
Nucléolo Celular/química , Homologia de Genes , Íntrons , Proteínas Nucleares/genética , Fosfoproteínas/genética , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA , Sequência de Bases , Proteínas Cromossômicas não Histona/metabolismo , Genes , Células HeLa , Humanos , Ligação de Hidrogênio , Técnicas In Vitro , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S/genética , Ribonucleoproteínas Nucleares Pequenas/química , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , NucleolinaRESUMO
We have developed an algorithm and a computer program for aligning new RNA sequences with a bank of aligned homologous RNA sequences. Given a common folding structure for the bank, the program performs an alignment between the bank and a new sequence, optimal both in terms of primary and secondary structure. This method is useful to align sequences that present a common folding structure despite extensive divergence of their primary structures. It allows these preserved regions to be precisely distinguished from domains with more variable secondary structure. An optimal alignment of a sequence of length N with a bank of homologous sequences of length M is produced in O (M2N3) time and O(M2N2) space. For sequences that are too long for an algorithm of this complexity, a proposed strategy is to use a classical alignment (using only primary structure data) then improve it with the new algorithm in the regions where the bank stems are not aligned with possible stems in the new sequence. The algorithm has been implemented in Turbo Pascal on a PC, and has been used to align RNA sequences of eubacterial large ribosomal subunit.