Localisation of RNAs and proteins in nucleolar precursor bodies of early mouse embryos

2017 ◽  
Vol 29 (3) ◽  
pp. 509 ◽  
Author(s):  
Elena Lavrentyeva ◽  
Kseniya Shishova ◽  
German Kagarlitsky ◽  
Olga Zatsepina
Keyword(s):  
Ribosomal Proteins ◽  
Rna Binding ◽  
Mammalian Species ◽  
In Situ Hybridisation ◽  
Mouse Embryos ◽  
28S Rrna ◽  
Fluorescence In Situ Hybridisation ◽  
Rdna Transcription ◽  
Early Mouse ◽  
Nucleolar Precursor Bodies

Early embryos of all mammalian species contain morphologically distinct but transcriptionally silent nucleoli called the nucleolar precursor bodies (NPBs), which, unlike normal nucleoli, have been poorly studied at the biochemical level. To bridge this gap, here we examined the occurrence of RNA and proteins in early mouse embryos with two fluorochromes – an RNA-binding dye pyronin Y (PY) and the protein-binding dye fluorescein-5′-isothiocyanate (FITC). The staining patterns of zygotic NPBs were then compared with those of nucleolus-like bodies (NLBs) in fully grown surrounded nucleolus (SN)-type oocytes, which are morphologically similar to NPBs. We show that both entities contain proteins, but unlike NLBs, NPBs are significantly impoverished for RNA. Detectable amounts of RNA appear on the NPB surface only after resumption of rDNA transcription and includes pre-rRNAs and 28S rRNA as evidenced by fluorescence in situ hybridisation with specific oligonucleotide probes. Immunocytochemical assays demonstrate that zygotic NPBs contain rRNA processing factors fibrillarin, nucleophosmin and nucleolin, while UBF (the RNA polymerase I transcription factor) and ribosomal proteins RPL26 and RPS10 are not detectable. Based on the results obtained and data in the contemporary literature, we suggest a scheme of NPB assembly and maturation to normal nucleoli that assumes utilisation of maternally derived nucleolar proteins but of nascent rRNAs.

scholarly journals Association of Nonribosomal Nucleolar Proteins in Ribonucleoprotein Complexes during Interphase and Mitosis

1999 ◽  
Vol 10 (1) ◽  
pp. 77-90 ◽  
Author(s):  
Serafı́n Piñol-Roma
Keyword(s):  
Ribosomal Proteins ◽  
Rna Binding ◽  
Rrna Synthesis ◽  
28S Rrna ◽  
Ribosomal Subunits ◽  
Interphase Cell ◽  
Ribonucleoprotein Complexes ◽  
Nucleolar Proteins ◽  
M Phase ◽  
Nucleolar Rna

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin—a major nucleolar RNA-binding protein—contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

scholarly journals Insight into Mammalian Selenocysteine Insertion: Domain Structure and Ribosome Binding Properties of Sec Insertion Sequence Binding Protein 2

2001 ◽  
Vol 21 (5) ◽  
pp. 1491-1498 ◽  
Author(s):  
Paul R. Copeland ◽  
Vincent A. Stepanik ◽  
Donna M. Driscoll
Keyword(s):  
Ribosomal Proteins ◽  
Insertion Sequence ◽  
Rna Binding ◽  
Stop Codon ◽  
Binding Protein ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Functional Domain ◽  
28S Rrna ◽  
Rna Binding Domain

ABSTRACT The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec. The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3′ untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis. We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1. We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding. Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity. This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA. SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.

scholarly journals Rapid Multi-Hybridisation FISH Screening for Balanced Porcine Reciprocal Translocations Suggests a Much Higher Abnormality Rate Than Previously Appreciated

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 250
Author(s):  
Rebecca E O’Connor ◽  
Lucas G Kiazim ◽  
Claudia C Rathje ◽  
Rebecca L Jennings ◽  
Darren K Griffin
Keyword(s):  
Semen Analysis ◽  
In Situ Hybridisation ◽  
Economic Loss ◽  
Environmental Damage ◽  
Fluorescence In Situ Hybridisation ◽  
Reciprocal Translocations ◽  
Chromosome Translocations ◽  
Farrowing Rate ◽  
Significant Economic Loss

With demand rising, pigs are the world’s leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.

scholarly journals Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Caitlin M. Singleton ◽  
Francesca Petriglieri ◽  
Jannie M. Kristensen ◽  
Rasmus H. Kirkegaard ◽  
Thomas Y. Michaelsen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Wastewater Treatment Plants ◽  
In Situ Hybridisation ◽  
Amplicon Sequencing ◽  
Rrna Genes ◽  
Fluorescence In Situ Hybridisation ◽  
Sequencing Data ◽  
High Quality ◽  
16S Rrna Amplicon Sequencing ◽  
Long Read

AbstractMicroorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

Effects of the diploidisation process upon the 5S and 35S rDNA sequences in the allopolyploid species of the Dilatata group of Paspalum (Poaceae, Paniceae)

2019 ◽  
Vol 67 (7) ◽  
pp. 521
Author(s):  
Magdalena Vaio ◽  
Cristina Mazzella ◽  
Marcelo Guerra ◽  
Pablo Speranza
Keyword(s):  
Evolutionary Dynamics ◽  
In Situ Hybridisation ◽  
5S Rdna ◽  
Its Region ◽  
Variable Number ◽  
Rrna Genes ◽  
Fluorescence In Situ Hybridisation ◽  
Rdna Sites ◽  
35S Rdna ◽  
Genome Restructuring

The Dilatata group of Paspalum includes species and biotypes native to temperate South America. Among them, five sexual allotetraploids (x = 10) share the same IIJJ genome formula: P. urvillei Steud, P. dasypleurum Kunze ex Desv., P. dilatatum subsp. flavescens Roseng., B.R. Arrill. & Izag., and two biotypes P. dilatatum Vacaria and P. dilatatum Virasoro. Previous studies suggested P. intermedium Munro ex Morong & Britton and P. juergensii Hack. or related species as their putative progenitors and donors of the I and J genome, respectively, and pointed to a narrow genetic base for their maternal origin. It has not yet been established whether the various members of the Dilatata group are the result of a single or of multiple allopolyploid formations. Here, we aimed to study the evolutionary dynamics of rRNA genes after allopolyploidisation in the Dilatata group of Paspalum and shed some light into the genome restructuring of the tetraploid taxa with the same genome formula. We used double target fluorescence in situ hybridisation of 35S and 5S rDNA probes and sequenced the nrDNA internal transcribed spacer (ITS) region. A variable number of loci at the chromosome ends were observed for the 35S rDNA, from 2 to 6, suggesting gain and loss of sites. For the 5S rDNA, only one centromeric pair of signals was observed, indicating a remarkable loss after polyploidisation. All ITS sequences generated were near identical to the one found for P. intermedium. Although sequences showed a directional homogeneisation towards the putative paternal progenitor in all tetraploid species, the observed differences in the number and loss of rDNA sites suggest independent ongoing diploidisation processes in all taxa and genome restructuring following polyploidy.

Expression of fibroblast growth factor receptors by embryonal carcinoma cells and early mouse embryos

1992 ◽  
Vol 28 (1) ◽  
pp. 61-66 ◽  
Author(s):  
Wendy J. Campbell ◽  
Keith A. Miller ◽  
Todd M. Anderson ◽  
James D. Shull ◽  
Angie Rizzino
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Embryonal Carcinoma ◽  
Growth Factor Receptors ◽  
Mouse Embryos ◽  
Carcinoma Cells ◽  
Fibroblast Growth ◽  
Embryonal Carcinoma Cells ◽  
Fibroblast Growth Factor Receptors ◽  
Early Mouse

scholarly journals RNA binding strategies of ribosomal proteins

1999 ◽  
Vol 27 (2) ◽  
pp. 381-388 ◽  
Author(s):  
D. E. Draper ◽  
L. P. Reynaldo
Keyword(s):  
Ribosomal Proteins ◽  
Rna Binding
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