RESUMEN
Small nucleolar (sno)RNAs guide posttranscriptional modifications essential for the biogenesis and function of their target. The majority of snoRNAs in higher eukaryotes are encoded within introns. They are first released from nascent transcripts in the form of a lariat and rapidly targeted by the debranching enzyme and nuclear exonucleases for linearization and further trimming. In this study, we report that some snoRNAs are encoded within unusually stable intronic RNAs. These intronic sequences can escape the debranching enzyme and accumulate as lariats. Stable lariats bearing a snoRNA, or slb-snoRNA, are associated with snoRNA binding proteins but do not guide posttranscriptional modification. While most slb-snoRNAs accumulate in the nucleus, some can be exported to the cytoplasm. We find that this export competes with snoRNA maturation. Slb-snoRNAs provide a previously unknown layer of regulation to snoRNA and snoRNA binding proteins.
Asunto(s)
ARN Circular/metabolismo , ARN Nucleolar Pequeño/metabolismo , Células 3T3 , Animales , Femenino , Regulación de la Expresión Génica , Células HeLa , Humanos , Intrones , Ratones , ARN Guía de Kinetoplastida , Saccharomyces cerevisiae , Xenopus laevisRESUMEN
In eukaryotes, rRNAs and spliceosomal snRNAs are heavily modified post-transcriptionally. Pseudouridylation and 2'-O-methylation are the most abundant types of RNA modifications. They are mediated by modification guide RNAs, also known as small nucleolar (sno)RNAs and small Cajal body-specific (sca)RNAs. We used yeast and vertebrate cells to test guide activities predicted for a number of snoRNAs, based on their regions of complementarity with rRNAs. We showed that human SNORA24 is a genuine guide RNA for 18S-Ψ609, despite some noncanonical base-pairing with its target. At the same time, we found quite a few snoRNAs that have the ability to base-pair with rRNAs and can induce predicted modifications in artificial substrate RNAs, but do not modify the same target sequence within endogenous rRNA molecules. Furthermore, certain fragments of rRNAs can be modified by the endogenous yeast modification machinery when inserted into an artificial backbone RNA, even though the same sequences are not modified in endogenous yeast rRNAs. In Xenopus cells, a guide RNA generated from scaRNA, but not from snoRNA, could induce an additional pseudouridylation of U2 snRNA at position 60; both guide RNAs were equally active on a U2 snRNA-specific substrate in yeast cells. Thus, post-transcriptional modification of functionally important RNAs, such as rRNAs and snRNAs, is highly regulated and more complex than simply strong base-pairing between a guide RNA and substrate RNA. We discuss possible regulatory roles for these unexpected modifications.
Asunto(s)
Cuerpos Enrollados/metabolismo , Seudouridina/metabolismo , Procesamiento Postranscripcional del ARN , ARN Guía de Kinetoplastida/metabolismo , ARN Nucleolar Pequeño/metabolismo , Empalmosomas/metabolismo , Secuencia de Bases , Cuerpos Enrollados/genética , Humanos , Metilación , ARN Guía de Kinetoplastida/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , Empalmosomas/genéticaRESUMEN
Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB)-specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at â¼80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2'-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.
Asunto(s)
Células Intersticiales de Cajal/metabolismo , Metilación , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Empalme del ARN , ARN Nuclear Pequeño/metabolismo , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Colangiocarcinoma/tratamiento farmacológico , Neoplasias Hematológicas/tratamiento farmacológico , Humanos , Fosforilación , ARN Nuclear Pequeño/química , Ribonucleoproteínas/metabolismo , Empalmosomas/genética , Tratamiento Farmacológico de COVID-19RESUMEN
Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB) specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at some 80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2'-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.
RESUMEN
Small nucleolar RNAs (snoRNAs) function primarily as guide RNAs for posttranscriptional modification of rRNAs and spliceosomal snRNAs, both of which are functionally important and evolutionarily conserved molecules. It is commonly believed that snoRNAs and the modifications they mediate are highly conserved across species. However, most relevant data on snoRNA annotation and RNA modification are limited to studies on human and yeast. Here, we used RNA-sequencing data from the giant oocyte nucleus of the frog Xenopus tropicalis to annotate a nearly complete set of snoRNAs. We compared the frog data with snoRNA sets from human and other vertebrate genomes, including mammals, birds, reptiles, and fish. We identified many Xenopus-specific (or nonhuman) snoRNAs and Xenopus-specific domains in snoRNAs from conserved RNA families. We predicted that some of these nonhuman snoRNAs and domains mediate modifications at unexpected positions in rRNAs and snRNAs. These modifications were mapped as predicted when RNA modification assays were applied to RNA from nine vertebrate species: frogs X. tropicalis and X. laevis, newt Notophthalmus viridescens, axolotl Ambystoma mexicanum, whiptail lizard Aspidoscelis neomexicana, zebrafish Danio rerio, chicken, mouse, and human. This analysis revealed that only a subset of RNA modifications is evolutionarily conserved and that modification patterns may vary even between closely related species. We speculate that each functional domain in snoRNAs (half of an snoRNA) may evolve independently and shuffle between different snoRNAs.
Asunto(s)
ARN Nucleolar Pequeño/genética , Xenopus/genética , Animales , Genoma , Humanos , Anotación de Secuencia Molecular , Mutación PuntualRESUMEN
Site-specific 2'-O-ribose methylation is an abundant post-transcriptional modification mediated by small non-coding nuclear RNAs known as box C/D modification guide RNAs. The minimal structural requirements for these guide RNAs to function in higher eukaryotes are still unclear. To address this question, we generated a series of mutant variants of Drosophila box C/D scaRNA:MeU2-C28 and tested their modification guide activities in the Xenopus oocyte system. Our data suggest that box C/D guide RNA function requires either a terminal or an internal consensus kink-turn structure. We identified the minimal functional box C/D guide RNA. It consists of a single-domain molecule with (i) a terminal stem with a consensus kink-turn domain, (ii) one box C and box D connected by a 14-nucleotide antisense element and (iii) a one-nucleotide spacer between the box C and the antisense element. In this single domain RNA, the sequence of the spacer is more important than its length. We suggest that the secondary structure of box C/D RNAs, essential for guide RNA function, is more complex than generally supposed. At the same time, the expression of functional extremely short single-domain box C/D RNAs is possible in higher eukaryotes.
Asunto(s)
ARN Guía de Kinetoplastida/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Femenino , Conformación de Ácido Nucleico , ARN Largo no Codificante/química , Xenopus laevisRESUMEN
Posttranscriptional modifications of rRNA occur in the nucleolus where rRNA modification guide RNAs, or snoRNAs, concentrate. On the other hand, scaRNAs, the modification guide RNAs for spliceosomal snRNAs, concentrate in the Cajal body (CB). It is generally assumed, therefore, that snRNAs must accumulate in CBs to be modified by scaRNAs. Here we demonstrate that the evidence for the latter postulate is not consistent. In the nucleus, scaRNA localization is not limited to CBs. Furthermore, canonical scaRNAs can modify rRNAs. We suggest that the conventional view that scaRNAs function only in the CB needs revision.
Asunto(s)
Cuerpos Enrollados/metabolismo , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/metabolismo , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/metabolismo , Animales , Secuencia de Bases , Células HeLa , Humanos , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , ARN Guía de Kinetoplastida/genética , ARN Ribosómico/química , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , ARN Nucleolar Pequeño/genética , Empalmosomas/genética , Empalmosomas/metabolismo , Xenopus/genética , Xenopus/metabolismoRESUMEN
The branch point recognition region of spliceosomal snRNA U2 is heavily modified post-transcriptionally in most eukaryotic species. We focused on this region to learn how nearby positions may interfere with each other when targeted for modification. Using an in vivo yeast Saccharomyces cerevisiae cell system, we tested the modification activity of several guide RNAs from human, mouse, the frog Xenopus tropicalis, the fruit fly Drosophila melanogaster, and the worm Caenorhabditis elegans We experimentally verified predictions for vertebrate U2 modification guide RNAs SCARNA4 and SCARNA15, and identified a C. elegans ortholog of SCARNA15. We observed crosstalk between sites in the heavily modified regions, such that modification at one site may inhibit modification at nearby sites. This is true for the branch point recognition region of U2 snRNA, the 5' loop of U5 snRNA, and certain regions of rRNAs, when tested either in yeast or in HeLa cells. The position preceding a uridine targeted for isomerization by a box H/ACA guide RNA is the most sensitive for noncanonical base-pairing and modification (either pseudouridylation or 2'-O-methylation). Based on these findings, we propose that modification must occur stepwise starting with the most vulnerable positions and ending with the most inhibiting modifications. We discuss possible strategies that cells use to reach complete modification in heavily modified regions.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Nuclear Pequeño/genética , Animales , Secuencia de Bases , Sitios de Unión , Caenorhabditis elegans , Drosophila melanogaster , Células HeLa , Humanos , Ratones , Seudouridina/metabolismo , ARN Nuclear Pequeño/metabolismo , Saccharomyces cerevisiae , XenopusRESUMEN
The pseudouridine at position 43 in vertebrate U2 snRNA is one of the most conserved post-transcriptional modifications of spliceosomal snRNAs; the equivalent position is pseudouridylated in U2 snRNAs in different phyla including fungi, insects, and worms. Pseudouridine synthase Pus1p acts alone on U2 snRNA to form this pseudouridine in yeast Saccharomyces cerevisiae and mouse. Furthermore, in S. cerevisiae, Pus1p is the only pseudouridine synthase for this position. Using an in vivo yeast cell system, we tested enzymatic activity of Pus1p from the fission yeast Schizosaccharomyces pombe, the worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the frog Xenopus tropicalis We demonstrated that Pus1p from C. elegans has no enzymatic activity on U2 snRNA when expressed in yeast cells, whereas in similar experiments, position 44 in yeast U2 snRNA (equivalent to position 43 in vertebrates) is a genuine substrate for Pus1p from S. cerevisiae, S. pombe, Drosophila, Xenopus, and mouse. However, when we analyzed U2 snRNAs from Pus1 knockout mice and the pus1Δ S. pombe strain, we could not detect any changes in their modification patterns when compared to wild-type U2 snRNAs. In S. pombe, we found a novel box H/ACA RNA encoded downstream from the RPC10 gene and experimentally verified its guide RNA activity for positioning Ψ43 and Ψ44 in U2 snRNA. In vertebrates, we showed that SCARNA8 (also known as U92 scaRNA) is a guide for U2-Ψ43 in addition to its previously established targets U2-Ψ34/Ψ44.
Asunto(s)
Transferasas Intramoleculares/metabolismo , ARN Guía de Kinetoplastida/metabolismo , ARN Nuclear Pequeño/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnicas de Inactivación de Genes , Transferasas Intramoleculares/genética , Ratones , Seudouridina/metabolismo , ARN de Hongos/metabolismo , ARN Nuclear Pequeño/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Xenopus/genética , Xenopus/metabolismoRESUMEN
Despite long-term exploration into ribosomal RNA gene functioning during the oogenesis of various organisms, many intriguing problems remain unsolved. In this review, we describe nucleolus organizer region (NOR) activity in avian oocytes. Whereas oocytes from an adult avian ovary never reveal the formation of the nucleolus in the germinal vesicle (GV), an ovary from juvenile birds possesses both nucleolus-containing and non-nucleolus-containing oocytes. The evolutionary diversity of oocyte NOR functioning and the potential non-rRNA-related functions of the nucleolus in oocytes are also discussed.
Asunto(s)
Aves/genética , Genes de ARNr , Oogénesis/genética , Animales , Nucléolo Celular/metabolismo , Cariotipificación , Oocitos/metabolismoRESUMEN
The spliceosomal small nuclear RNAs (snRNAs) are modified post-transcriptionally by introduction of pseudouridines and 2'-O-methyl modifications, which are mediated by box H/ACA and box C/D guide RNAs, respectively. Because of their concentration in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific (sca) RNAs. In the cell, scaRNAs are associated with the WD-repeat protein WDR79. We used coimmunoprecipitation with WDR79 to recover seven new scaRNAs from Drosophila cell lysates. We demonstrated concentration of these new scaRNAs in the CB by in situ hybridization, and we verified experimentally that they can modify their putative target RNAs. Surprisingly, one of the new scaRNAs targets U6 snRNA, whose modification is generally assumed to occur in the nucleolus, not in the CB. Two other scaRNAs have dual guide functions, one for an snRNA and one for 28S rRNA. Again, the modification of 28S rRNA is assumed to take place in the nucleolus. These findings suggest that canonical scaRNAs may have functions in addition to their established role in modifying U1, U2, U4, and U5 snRNAs. We discuss the likelihood that processing by scaRNAs is not limited to the CB.
Asunto(s)
Cuerpos Enrollados/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de Unión al ARN/metabolismo , Animales , Emparejamiento Base , Secuencia de Bases , Células Cultivadas , Proteínas de Drosophila/genética , Femenino , Genes de Insecto , Secuencias Invertidas Repetidas , Masculino , Anotación de Secuencia Molecular , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae , Xenopus , ARN Pequeño no TraducidoRESUMEN
Chicken (Gallus gallus domesticus, GGA) and Japanese quail (Coturnix coturnix japonica, CCO) karyotypes are very similar. They have identical chromosome number (2n = 78) and show a high degree of synteny. Centromere positions on the majority of orthologous chromosomes are different in these two species. To explore the nature of this divergence, we used high-resolution comparative fluorescent in situ hybridization mapping on giant lampbrush chromosomes (LBCs) from growing oocytes. We applied 41 BAC clones specific for GGA1, 2, 3, 11, 12, 13, 14, and 15 to chicken and quail LBCs. This approach allowed us to rule out a pericentric inversion earlier proposed to explain the difference between GGA1 and CCO1. In addition to a well-established large-scale pericentric inversion that discriminates GGA2 and CCO2, we identified another, smaller one in the large inverted region. For the first time, we described in detail inversions that distinguish GGA3 from CCO3 and GGA11 from CCO11. Despite the newly identified and confirmed inversions, our data suggest that, in chicken and Japanese quail, the difference in centromere positions is not mainly caused by pericentric inversions but is instead due to centromere repositioning events and the formation of new centromeres. We also consider the formation of short arms of quail microchromosomes by heterochromatin accumulation as a third scenario that could explain the discrepancy in centromeric indexes.
Asunto(s)
Centrómero/ultraestructura , Pollos/genética , Cromosomas/genética , Animales , Centrómero/genética , Inversión Cromosómica , Mapeo Cromosómico , Cromosomas/ultraestructura , Coturnix/genética , Técnica del Anticuerpo Fluorescente , Heterocromatina , Hibridación Fluorescente in Situ , CariotipificaciónRESUMEN
The survival of motor neuron (SMN) protein plays an important role in the biogenesis of spliceosomal snRNPs and is one factor required for the integrity of nuclear Cajal bodies (CBs). CBs are enriched in small CB-specific (sca) RNAs, which guide the formation of pseudouridylated and 2'-O-methylated residues in the snRNAs. Because SMN-deficient cells lack typical CBs, we asked whether the modification of internal residues of major and minor snRNAs is defective in these cells. We mapped modified nucleotides in the major U2 and the minor U4atac and U12 snRNAs. Using both radioactive and fluorescent primer extension approaches, we found that modification of major and minor spliceosomal snRNAs is normal in SMN-deficient cells. Our experiments also revealed a previously undetected pseudouridine at position 60 in human U2 and 2'-O-methylation of A1, A2, and G19 in human U4atac. These results confirm, and extend to minor snRNAs, previous experiments showing that scaRNPs can function in the absence of typical CBs. Furthermore, they show that the differential splicing defects in SMN-deficient cells are not due to failure of post-transcriptional modification of either major or minor snRNAs.
Asunto(s)
Procesamiento Postranscripcional del ARN/genética , ARN Nuclear Pequeño/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Cuerpos Enrollados/química , Cuerpos Enrollados/metabolismo , Células HeLa , Humanos , Metilación , ARN Nuclear Pequeño/química , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
The Cajal body (CB) is a nuclear organelle present in all eukaryotes that have been carefully studied. It is identified by the signature protein coilin and by CB-specific RNAs (scaRNAs). CBs contain high concentrations of splicing small nuclear ribonucleoproteins (snRNPs) and other RNA processing factors, suggesting that they are sites for assembly and/or posttranscriptional modification of the splicing machinery of the nucleus. The histone locus body (HLB) contains factors required for processing histone pre-mRNAs. As its name implies, the HLB is associated with the genes that code for histones, suggesting that it may function to concentrate processing factors at their site of action. CBs and HLBs are present throughout the interphase of the cell cycle, but disappear during mitosis. The biogenesis of CBs shows the features of a self-organizing structure.
Asunto(s)
Cuerpos Enrollados/metabolismo , Histonas/metabolismo , Anfibios , Animales , Núcleo Celular/metabolismo , Humanos , Oocitos/metabolismoRESUMEN
During their biogenesis small nuclear RNAs (snRNAs) undergo multiple covalent modifications that require guide RNAs to direct methylase and pseudouridylase enzymes to the appropriate nucleotides. Because of their localization in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific RNAs (scaRNAs). Using a fluorescent primer extension technique, we mapped the modified nucleotides in Drosophila U1, U2, U4, and U5 snRNAs. By fluorescent in situ hybridization (FISH) we showed that seven Drosophila scaRNAs are concentrated in easily detectable CBs. We used two assays based on Xenopus oocyte nuclei to demonstrate that three of these Drosophila scaRNAs do, in fact, function as guide RNAs. In flies null for the CB marker protein coilin, CBs are absent and there are no localized FISH signals for the scaRNAs. Nevertheless, biochemical experiments show that scaRNAs are present at normal levels and snRNAs are properly modified. Our experiments demonstrate that several scaRNAs are concentrated as expected in the CBs of wild-type Drosophila, but they function equally well in the nucleoplasm of mutant flies that lack CBs. We propose that the snRNA modification machinery is not limited to CBs, but is dispersed throughout the nucleoplasm of cells in general.
Asunto(s)
Cuerpos Enrollados/genética , Drosophila melanogaster/metabolismo , ARN/metabolismo , Animales , Northern Blotting , Cuerpos Enrollados/metabolismo , Drosophila melanogaster/citología , Hibridación Fluorescente in Situ , Nucleótidos/metabolismo , Oocitos/metabolismo , Seudouridina/metabolismo , Procesamiento Postranscripcional del ARN , ARN Nuclear Pequeño/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Xenopus , ARN Pequeño no TraducidoRESUMEN
Cajal bodies (CBs) are nuclear organelles that occur in a variety of organisms, including vertebrates, insects, and plants. They are most often identified with antibodies against the marker protein coilin. Because the amino acid sequence of coilin is not strongly conserved evolutionarily, coilin orthologues have been difficult to recognize by homology search. Here, we report the identification of Drosophila melanogaster coilin and describe its distribution in tissues of the fly. Surprisingly, we found coilin not only in CBs but also in histone locus bodies (HLBs), calling into question the use of coilin as an exclusive marker for CBs. We analyzed two null mutants in the coilin gene and a piggyBac insertion mutant, which leads to specific loss of coilin from the germline. All three mutants are homozygous viable and fertile. Cells that lack coilin also lack distinct foci of other CB markers, including fibrillarin, the survival motor neuron (SMN) protein, U2 small nuclear RNA (snRNA), U5 snRNA, and the small CB-specific (sca) RNA U85. However, HLBs are not obviously affected in coilin-null flies. Thus, coilin is required for normal CB organization in Drosophila but is not essential for viability or production of functional gametes.
Asunto(s)
Cuerpos Enrollados/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Envejecimiento/fisiología , Secuencia de Aminoácidos , Animales , Ciclo Celular , Secuencia Conservada , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Histonas/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Mutación/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ovario/metabolismo , Alineación de Secuencia , Testículo/metabolismoRESUMEN
BACKGROUND: Comparative genomics is a powerful means of establishing inter-specific relationships between gene function/location and allows insight into genomic rearrangements, conservation and evolutionary phylogeny. The availability of the complete sequence of the chicken genome has initiated the development of detailed genomic information in other birds including turkey, an agriculturally important species where mapping has hitherto focused on linkage with limited physical information. No molecular study has yet examined conservation of avian microchromosomes, nor differences in copy number variants (CNVs) between birds. RESULTS: We present a detailed comparative cytogenetic map between chicken and turkey based on reciprocal chromosome painting and mapping of 338 chicken BACs to turkey metaphases. Two inter-chromosomal changes (both involving centromeres) and three pericentric inversions have been identified between chicken and turkey; and array CGH identified 16 inter-specific CNVs. CONCLUSION: This is the first study to combine the modalities of zoo-FISH and array CGH between different avian species. The first insight into the conservation of microchromosomes, the first comparative cytogenetic map of any bird and the first appraisal of CNVs between birds is provided. Results suggest that avian genomes have remained relatively stable during evolution compared to mammalian equivalents.
Asunto(s)
Pollos/genética , Evolución Molecular , Genoma/genética , Genómica , Pavos/genética , Animales , Mapeo Cromosómico , Cromosomas Artificiales Bacterianos/genética , Color , Citogenética , Humanos , Hibridación Fluorescente in Situ , Metafase/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Secuencia de ADNRESUMEN
The chromosomal distribution of 41-bp repeats, known as CNM and PO41 repeats in the chicken genome and BglII repeats in the Japanese quail, was analyzed precisely using giant lampbrush chromosomes (LBC) from chicken, Japanese quail, and turkey growing oocytes. The PO41 repeat is conserved in all galliform species, whereas the other repeats are species specific. In chicken and quail, the centromere and subtelomere regions share homologous satellite sequences. RNA polymerase II transcribes the 41-bp repeats in both centromere and subtelomere regions. Ongoing transcription of these repeats was demonstrated by incorporation of BrUTP injected into oocytes at the lampbrush stage. RNA complementary to both strands of CNM and PO41 repeats is present on chicken LBC loops, whereas strand-specific G-rich transcripts are characteristic of BglII repeats in the Japanese quail. The RNA from 41-bp repeats does not undergo cotranscriptional U snRNP-dependent splicing. At the same time, the ribonucleoprotein matrix of transcription units with C-rich RNA of CNM and PO41 repeats was enriched with hnRNP protein K. Potential promoters for satellite transcription are discussed.
Asunto(s)
Pollos/genética , Mapeo Cromosómico , Coturnix/genética , Genoma/genética , Hibridación Fluorescente in Situ , Secuencias Repetidas en Tándem/genética , Transcripción Genética/genética , Animales , Femenino , Oocitos , Pavos/genéticaRESUMEN
Using immunostaining with antibodies against cohesin subunits, we show here that cohesin-enriched structures analogous to the so-called centromere protein bodies (PB) are the characteristic of galliform lampbrush chromosomes. Their centromeric location was verified by FISH with certain DNA probes. PB-like structures were used as markers for centromere localization in chicken lampbrush chromosomes. The gap predicted to be centromeric in current chicken chromosome 3 sequence assembly was found to correspond to the non-centromeric cluster of CNM repeat on the q-arm of chromosome 3; the centromere is proposed to be placed at another position. The majority of chicken microchromosomes were found to be acrocentric, in contrast to Japanese quail microchromosomes which are biarmed. Centromere cohesin-enriched structures on chicken and quail lampbrush microchromosomes co-localize with pericentromeric CNM and BglII- repeats respectively. FISH to the nascent transcripts on chicken lampbrush chromosomes revealed numerous non-centromeric CNM clusters in addition to pericentromeric arrays. Complementary CNM transcripts from both C- and G-rich DNA strands were revealed during the lampbrush stage.
Asunto(s)
Proteínas de Ciclo Celular/análisis , Centrómero/ultraestructura , Pollos/genética , Proteínas Cromosómicas no Histona/análisis , Cromosomas/ultraestructura , Proteínas Nucleares/análisis , Animales , Coturnix/genética , Femenino , Hibridación Fluorescente in Situ , Profase Meiótica I , CohesinasRESUMEN
Giant lampbrush chromosomes, which are characteristic of the diplotene stage of prophase I during avian oogenesis, represent a very promising system for precise physical gene mapping. We applied 35 chicken BAC and 4 PAC clones to both mitotic metaphase chromosomes and meiotic lampbrush chromosomes of chicken (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japonica). Fluorescence in situ hybridization (FISH) mapping on lampbrush chromosomes allowed us to distinguish closely located probes and revealed gene order more precisely. Our data extended the data earlier obtained using FISH to chicken and quail metaphase chromosomes 1-6 and Z. Extremely low levels of inter- and intra-chromosomal rearrangements in the chicken and Japanese quail were demonstrated again. Moreover, we did not confirm the presence of a pericentric inversion in Japanese quail chromosome 4 as compared to chicken chromosome 4. Twelve BAC clones specific for chicken chromosome 4p and 4q showed the same order in quail as in chicken when FISH was performed on lampbrush chromosomes. The centromeres of chicken and quail chromosomes 4 seem to have formed independently after centric fusion of ancestral chromosome 4 and a microchromosome.