scholarly journals Assembly of the Nuclear Transcription and Processing Machinery: Cajal Bodies (Coiled Bodies) and Transcriptosomes

1999 ◽  
Vol 10 (12) ◽  
pp. 4385-4402 ◽  
Author(s):  
Joseph G. Gall ◽  
Michel Bellini ◽  
Zheng’an Wu ◽  
Christine Murphy
Keyword(s):  
Germinal Vesicle ◽  
Cajal Bodies ◽  
Oocyte Nucleus ◽  
Electron Microscopic ◽  
Pol Ii ◽  
Processing Machinery ◽  
Small Nuclear Ribonucleoprotein ◽  
Coiled Bodies ◽  
Pol I ◽  
Pol Iii

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem–loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.

scholarly journals Cajal Bodies, Nucleoli, and Speckles in the Xenopus Oocyte Nucleus Have a Low-Density, Sponge-like Structure

2005 ◽  
Vol 16 (1) ◽  
pp. 202-211 ◽  
Author(s):  
Korie E. Handwerger ◽  
Jason A. Cordero ◽  
Joseph G. Gall
Keyword(s):  
Xenopus Oocyte ◽  
Aqueous Media ◽  
Germinal Vesicle ◽  
Physical Structure ◽  
Cajal Bodies ◽  
Oocyte Nucleus ◽  
Refractive Indices ◽  
Low Density ◽  
Penetration Data ◽  
Fibrillar Region

Nuclear organelles, unlike many cytoplasmic organelles, lack investing membranes and are thus in direct contact with the surrounding nucleoplasm. Because the properties of the nucleoplasm and nuclear organelles influence the exchange of molecules from one compartment to another, it is important to understand their physical structure. We studied the density of the nucleoplasm and the density and permeability of nucleoli, Cajal bodies (CBs), and speckles in the Xenopus oocyte nucleus or germinal vesicle (GV). Refractive indices were measured by interferometry within intact GVs isolated in oil. The refractive indices were used to estimate protein concentrations for nucleoplasm (0.106 g/cm3), CBs (0.136 g/cm3), speckles (0.162 g/cm3), and the dense fibrillar region of nucleoli (0.215 g/cm3). We determined similar protein concentrations for nuclear organelles isolated in aqueous media, where they are no longer surrounded by nucleoplasm. To examine the permeability of nuclear organelles, we injected fluorescent dextrans of various molecular masses (3–2000 kDa) into the cytoplasm or directly into the GV and measured the extent to which they penetrated the organelles. Together, the interferometry and dextran penetration data show that organelles in the Xenopus GV have a low-density, sponge-like structure that provides access to macromolecules from the nucleoplasm.

Heat shock induces mini-Cajal bodies in the Xenopus germinal vesicle

2002 ◽  
Vol 115 (10) ◽  
pp. 2011-2020 ◽  
Author(s):  
Korie E. Handwerger ◽  
Zheng'an Wu ◽  
Christine Murphy ◽  
Joseph G. Gall
Keyword(s):  
Heat Shock ◽  
Germinal Vesicle ◽  
Model System ◽  
Cajal Body ◽  
Self Organization ◽  
Cajal Bodies ◽  
Oocyte Nucleus ◽  
Evolutionarily Conserved ◽  
And Behavior ◽  
Heat Shock Induction

Cajal bodies are evolutionarily conserved nuclear organelles that are believed to play a central role in assembly of RNA transcription and processing complexes. Although knowledge of Cajal body composition and behavior has greatly expanded in recent years, little is known about the molecules and mechanisms that lead to the formation of these organelles in the nucleus. The Xenopus oocyte nucleus or germinal vesicle is an excellent model system for the study of Cajal bodies, because it is easy to manipulate and it contains 50-100 Cajal bodies with diameters up to 10 μm. In this study we show that numerous mini-Cajal bodies (less than 2 μm in diameter) form in the germinal vesicle after oocytes recover from heat shock. The mechanism for heat shock induction of mini-Cajal bodies is independent of U7 snRNA and does not require transcription or import of newly translated proteins from the cytoplasm. We suggest that Cajal bodies originate by self-organization of preformed components, preferentially on the surface of B-snurposomes.

scholarly journals RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure

2020 ◽  
Vol 295 (15) ◽  
pp. 4782-4795 ◽  
Author(s):  
Philipp E. Merkl ◽  
Michael Pilsl ◽  
Tobias Fremter ◽  
Katrin Schwank ◽  
Christoph Engel ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Dna Templates ◽  
Pol Ii ◽  
Naked Dna ◽  
Pol I ◽  
Intrinsic Capacity ◽  
Pol Iii ◽  
Polymerase I

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I–associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I–specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.

scholarly journals A TATA-Binding Protein Mutant Defective for TFIID Complex Formation In Vivo

1999 ◽  
Vol 19 (6) ◽  
pp. 3951-3957 ◽  
Author(s):  
Ryan T. Ranallo ◽  
Kevin Struhl ◽  
Laurie A. Stargell
Keyword(s):  
Binding Protein ◽  
Tata Binding Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Pol Ii ◽  
Pol I ◽  
Pol Iii ◽  
Polymerase I ◽  
Tfiid Complex

ABSTRACT Using an intragenic complementation screen, we have identified a temperature-sensitive TATA-binding protein (TBP) mutant (K151L,K156Y) that is defective for interaction with certain yeast TBP-associated factors (TAFs) at the restrictive temperature. The K151L,K156Y mutant appears to be functional for RNA polymerase I (Pol I) and Pol III transcription, and it is capable of supporting Gal4-activated and Gcn4-activated transcription by Pol II. However, transcription from certain TATA-containing and TATA-less Pol II promoters is reduced at the restrictive temperature. Immunoprecipitation analysis of extracts prepared after culturing cells at the restrictive temperature for 1 h indicates that the K151L,K156Y derivative is severely compromised in its ability to interact with TAF130, TAF90, TAF68/61, and TAF25 while remaining functional for interaction with TAF60 and TAF30. Thus, a TBP mutant that is compromised in its ability to form TFIID can support the response to Gcn4 but is defective for transcription from specific promoters in vivo.

The phylogenetic relations of DNA-dependent RNA polymerases of archaebacteria, eukaryotes, and eubacteria

1989 ◽  
Vol 35 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Wolfram Zillig ◽  
Hans-Peter Klenk ◽  
Peter Palm ◽  
Gabriela Pühler ◽  
Felix Gropp ◽  
...  
Keyword(s):  
Nuclear Genome ◽  
Distance Matrix ◽  
Gene Organization ◽  
Amino Acid Sequences ◽  
Rna Polymerases ◽  
Matrix Analysis ◽  
Pol Ii ◽  
Phylogenetic Relations ◽  
Pol I ◽  
Pol Iii

Unrooted phylogenetic dendrograms were calculated by two independent methods, parsimony and distance matrix analysis, from an alignment of the derived amino acid sequences of the A and C subunits of the DNA-dependent RNA polymerases of the archaebacteria Sulfolobus acidocaldarius and Halobacterium halobium with 12 corresponding sequences including a further set of archaebacterial A + C subunits, eukaryotic nuclear RNA polymerases, pol I, pol II, and pol III, eubacterial β′ and chloroplast β′ and β″ subunits. They show the archaebacteria as a coherent group in close neighborhood of and sharing a bifurcation with eukaryotic pol II and (or) pol IIIA components. The most probable trees show pol IA branching off from the tree separately at a bifurcation with the eubacterial β′ lineage. The implications of these results, especially for understanding the possibly chimeric origin of the eukaryotic nuclear genome, are discussed.Key words: transcription, evolution, taxonomy, subunits, gene organization.

scholarly journals RNA Polymerase III in Cajal Bodies and Lampbrush Chromosomes of the Xenopus Oocyte Nucleus

2002 ◽  
Vol 13 (10) ◽  
pp. 3466-3476 ◽  
Author(s):  
Christine Murphy ◽  
Zhengxin Wang ◽  
Robert G. Roeder ◽  
Joseph G. Gall
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Cajal Bodies ◽  
Oocyte Nucleus ◽  
Xenopus Laevis Oocyte ◽  
Lampbrush Chromosomes ◽  
Oocyte Cytoplasm ◽  
Pol Iii

We used immunofluorescence to study the distribution and targeting of RNA polymerase (pol) III subunits and pol III transcription factors in the Xenopus laevis oocyte nucleus. Antibodies against several of these proteins stained Cajal bodies and ∼90 specific sites on the lampbrush chromosomes. Some of the chromosomal sites had been identified previously by in situ hybridization as the genes for 5S rRNA. The remaining sites presumably encode tRNAs and other pol III transcripts. Pol III sites were often resolvable as loops similar to the much more abundant pol II loops, but without a matrix detectable by phase contrast or differential interference contrast. This morphology is consistent with the transcription of short repeated sequences. Hemagglutinin-tagged transcripts encoding core subunits and transcription factors were injected into the oocyte cytoplasm, and the distribution of newly translated proteins inside the nucleus was monitored by immunostaining. Cajal bodies were preferentially targeted by these proteins, and in some cases the chromosomal sites were also weakly stained. The existence of pol III subunits and pol III transcription factors in Cajal bodies and their targeting to these organelles are consistent with a model of Cajal bodies as sites for preassembly of the nuclear transcription machinery.

TAFII70 protein in Cajal bodies of the amphibian germinal vesicle

Genome ◽  
2001 ◽  
Vol 44 (6) ◽  
pp. 1100-1103 ◽  
Author(s):  
Stefania Bucci ◽  
Letizia Giani ◽  
Giorgio Mancino ◽  
Mario Pellegrino ◽  
Matilde Ragghianti
Keyword(s):  
Monoclonal Antibody ◽  
Germinal Vesicle ◽  
Tata Binding Protein ◽  
Cajal Body ◽  
Cajal Bodies ◽  
Oocyte Nucleus ◽  
Lampbrush Chromosomes ◽  
Rna Transcription ◽  
Associated Factor ◽  
Oocyte Cytoplasm

The localization of the TATA-binding protein (TBP) associated factor II70 (TAFII70) in the germinal vesicle (GV) of newt oocytes was investigated. In spreads of GV content, anti-hTAFII70 monoclonal antibody (mAb) stained Cajal bodies (CBs) that were either attached to specific sites on the lampbrush chromosomes or free in the nucleoplasm. To confirm this localization the PwTAFII70 cDNA was cloned and myc-tagged transcripts injected into the oocyte cytoplasm. Newly translated PwTAFII70 protein was detected a few hours later in the Cajal bodies. These data support the hypothesis that Cajal bodies are the assembly sites of the transcription machinery of the oocyte nucleus. TAFII70 protein can play a role in lampbrush transcription; alternatively TAFII70 can be considered a component in the subset of TFIID complexes that do not function during oogenesis, but are accumulated in the oocyte for later use during early development.Key words: TAFII70, Cajal body, lampbrush chromosomes, RNA transcription and processing, newts, Pleurodeles.

scholarly journals The Stem-Loop Binding Protein (SLBP1) Is Present in Coiled Bodies of the Xenopus Germinal Vesicle

1999 ◽  
Vol 10 (2) ◽  
pp. 487-499 ◽  
Author(s):  
Jennifer Abbott ◽  
William F. Marzluff ◽  
Joseph G. Gall
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Germinal Vesicle ◽  
Xenopus Laevis Oocytes ◽  
Coiled Body ◽  
Stem Loop ◽  
Subnuclear Localization ◽  
Small Nuclear Ribonucleoprotein ◽  
Coiled Bodies ◽  
U7 Snrnp

The stem-loop binding protein (SLBP1) binds the 3′ stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the germinal vesicle of Xenopus laevis oocytes. In spread preparations of germinal vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection ofmyc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 3′ end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

scholarly journals The promoter for the procyclic acidic repetitive protein (PARP) genes of Trypanosoma brucei shares features with RNA polymerase I promoters.

1992 ◽  
Vol 12 (6) ◽  
pp. 2644-2652 ◽  
Author(s):  
S D Brown ◽  
J Huang ◽  
L H Van der Ploeg
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Protein Coding ◽  
Pol Ii ◽  
Protein Coding Genes ◽  
Pol I ◽  
Pol Iii ◽  
Repetitive Protein

All eukaryotic protein-coding genes are believed to be transcribed by RNA polymerase (Pol) II. An exception may exist in the protozoan parasite Trypanosoma brucei, in which the genes encoding the variant surface glycoprotein (VSG) and procyclic acidic repetitive protein (PARP) are transcribed by an RNA polymerase that is resistant to the Pol II inhibitor alpha-amanitin. The PARP and VSG genes were proposed to be transcribed by Pol I (C. Shea, M. G.-S. Lee, and L. H. T. Van der Ploeg, Cell 50:603-612, 1987; G. Rudenko, M. G.-S. Lee, and L. H. T. Van der Ploeg, Nucleic Acids Res. 20:303-306, 1992), a suggestion that has been substantiated by the finding that trypanosomes can transcribe protein-coding genes by Pol I (G. Rudenko, H.-M. Chung, V. P. Pham, and L. H. T. Van der Ploeg, EMBO J. 10:3387-3397, 1991). We analyzed the sequence elements of the PARP promoter by linker scanning mutagenesis and compared the PARP promoter with Pol I, Pol II, and Pol III promoters. The PARP promoter appeared to be of limited complexity and contained at least two critical regions. The first was located adjacent to the transcription initiation site (nucleotides [nt] -69 to +12) and contained three discrete domains in which linker scanning mutants affected the transcriptional efficiency: at nt -69 to -56, -37 to -11, and -11 to +12. The second region was located between nt -140 and -131, and a third region may be located between nt -228 and -205. The nucleotide sequences of these elements, and their relative positioning with respect to the transcription initiation site did not resemble those of either Pol II or Pol III promoter elements, but rather reflected the organization of Pol I promoters in (i) similarity in the positioning of essential domains in the PARP promoter and Pol I promoter, (ii) strong sequence homology between the PARP core promoter element (nt -37 to -11) and identically positioned nucleotide sequences in the trypanosome rRNA and VSG gene promoters, and (iii) moderate effects on promoter activity of mutations around the transcription initiation site.

scholarly journals Analysis of the Caenorhabditis elegans rpc-1 gene

2005 ◽  
Author(s):  
◽  
Qun Zheng
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Rna Polymerase Iii ◽  
Transgenic Animals ◽  
Rna Polymerases ◽  
Pol Ii ◽  
C Elegans ◽  
Pol I ◽  
Genes Encoding ◽  
Pol Iii

In eukaryotes, two large subunits form the core catalytic structure of RNA polymerase III (Pol III), which is conserved in other RNA polymerases, Pol I and Pol II. It has been found that Pol III activity is tightly associated to cell growth. TFIII B has been shown to be one of main mediators in this process. No regulation of the Pol III largest subunit gene has been found. In C. elegans, the rpc-1 gene encodes the largest subunit of Pol III. Here, I identified two critical structural components of RPC-1, Gly644 and Gly1055, whose mutations result in larval lethal arrestment. These two amino acid residues are universally conserved in RNA polymerases, indicating their overall involvement in gene transcription mechanism. Also, I found that maternally inherited, not embryonically expressed, rpc-1 gene products survive early development. Starvation was found to suppress rpc-1 gene expression and re-feeding treatment enhances rpc-1 gene expression rapidly. No similar regulation was detected in genes encoding largest subunits of Pol I and Pol II. This is the first time that rpc-1 gene regulation has been reported. Insulin signaling may not be involved in this regulation. Also, I found that rpc-1 promoter is not ubiquitously active in C. elegans. Using the rpc-1p::gfp transgene, the rpc-1 promoter activity is only detected in a subset of neurons in the head and the tail and the intestine. While starvation silences the rpc-1 promoter activity in most tissues and cells, ASK neurons still show GFP staining in the rpc-1p::gfp transgenic animals, indicating that rpc-1 transcription in ASK neurons is continuously active under starvation conditions. Further studies suggest that TGF-[beta] signaling is involved in mediating the rpc-1 promoter activity in ASK neurons.

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