Perichromatin fibrils are in situ forms of nascent transcripts

1994 ◽  
Vol 4 (3) ◽  
pp. 86-90 ◽  
Author(s):  
S FAKAN
Keyword(s):  
Nascent Transcripts ◽  
Perichromatin Fibrils

Ultrastructural in situ hybridization to nascent transcripts of highly transcribed rRNA genes in chromatin spreads

Chromosoma ◽  
1994 ◽  
Vol 103 (2) ◽  
pp. 122-128
Author(s):  
Marina?M. O'Reilly ◽  
Sarah?L. French ◽  
Martha?L. Sikes ◽  
Oscar?L. Miller?Jr.
Keyword(s):  
In Situ Hybridization ◽  
Rrna Genes ◽  
Nascent Transcripts

scholarly journals Localization of HIV-1 RNA in mammalian nuclei.

1996 ◽  
Vol 135 (1) ◽  
pp. 9-18 ◽  
Author(s):  
G Zhang ◽  
M L Zapp ◽  
G Yan ◽  
M R Green
Keyword(s):  
Nuclear Export ◽  
Splice Junction ◽  
Stable Population ◽  
Beta Globin ◽  
Nuclear Retention ◽  
Viral Rnas ◽  
Immunodeficiency Virus ◽  
Nascent Transcripts ◽  
Hiv 1

The Rev protein of human immunodeficiency virus type 1 (HIV-1) facilitates the nuclear export of unspliced and partially spliced viral RNAs. In the absence of Rev, these intron-containing HIV-1 RNAs are retained in the nucleus. The basis for nuclear retention is unclear and is an important aspect of Rev regulation. Here we use in situ hybridization and digital imaging microscopy to examine the intranuclear distributions of intron-containing HIV RNAs and to determine their spatial relationships to intranuclear structures. HeLa cells were transfected with an HIV-1 expression vector, and viral transcripts were localized using oligonucleotide probes specific for the unspliced or spliced forms of a particular viral RNA. In the absence of Rev, the unspliced viral RNAs were predominantly nuclear and had two distinct distributions. First, a population of viral transcripts was distributed as approximately 10-20 intranuclear punctate signals. Actinomycin D chase experiments indicate that these signals represent nascent transcripts. A second, stable population of viral transcripts was dispersed throughout the nucleoplasm excluding nucleoli. Rev promoted the export of this stable population of viral RNAs to the cytoplasm in a time-dependent fashion. Significantly, the distributions of neither the nascent nor the stable populations of viral RNAs coincided with intranuclear speckles in which splicing factors are enriched. Using splice-junction-specific probes, splicing of human beta-globin pre-mRNA occurred cotranscriptionally, whereas splicing of HIV-1 pre-mRNA did not. Taken together, our results indicate that the nucleolus and intranuclear speckles are not involved in Rev regulation, and provide further evidence that efficient splicing signals are critical for cotranscriptional splicing.

scholarly journals Patterns of E74A RNA and protein expression at the onset of metamorphosis in Drosophila

Development ◽  
1991 ◽  
Vol 112 (4) ◽  
pp. 981-995 ◽  
Author(s):  
L. Boyd ◽  
E. O'Toole ◽  
C.S. Thummel
Keyword(s):  
Protein Expression ◽  
Polytene Chromosomes ◽  
Protein Accumulation ◽  
Spatial And Temporal Patterns ◽  
Larval Salivary Gland ◽  
Initial Appearance ◽  
Nascent Transcripts ◽  
Post Transcriptional Regulation ◽  
The Brain

Metamorphosis in Drosophila is triggered by a pulse of the steroid hormone ecdysone at the end of larval development. Ecdysone initiates a genetic hierarchy that can be visualized as a series of puffs in the larval salivary gland polytene chromosomes. The E74 gene is responsible for the early ecdysone-inducible puff at position 74EF and encodes two related DNA-binding proteins which appear to play a regulatory role in the hierarchy. Here we describe the spatial and temporal patterns of E74A RNA and protein expression at the onset of metamorphosis. We use in situ hybridization, antibody stains, and western and northern blot analyses to follow E74A expression from its initial appearance as nascent transcripts on the polytene chromosomes, to spliced mRNA, to post-translationally modified nuclear E74A protein. E74A is expressed in a wide variety of late-third instar tissues, suggesting that it plays a broad pleiotropic role in response to the hormone. In early prepupae, when the overall levels of E74A mRNA are decreasing, relatively high levels of E74A RNA persist in the gut, peripodial membranes of the imaginal discs, and proliferation centers of the brain. The spatial distribution of nuclear E74A protein correlates with the RNA distribution with the single exception that no E74A protein can be detected in the proliferation centers of the brain. There is also a temporal discrepancy between E74A mRNA and protein accumulation. The peak of E74A protein induced by the late larval ecdysone pulse follows the peak of E74A mRNA by approximately 2 h. This delay is not seen in 10 h prepupae, when the next pulse of ecdysone induces the simultaneous expression of E74A mRNA and protein. We discuss possible mechanisms for post-transcriptional regulation of E74A expression and suggest that the unusually long and complex 5′ leader in the E74A mRNA may regulate its translation.

Ultrastructural in situ hybridization to nascent transcripts of highly transcribed rRNA genes in chromatin spreads

Chromosoma ◽  
1994 ◽  
Vol 103 (2) ◽  
pp. 122-128 ◽  
Author(s):  
Marina M. O'Reilly ◽  
Sarah L. French ◽  
Martha L. Sikes ◽  
Oscar L. Miller
Keyword(s):  
In Situ Hybridization ◽  
Rrna Genes ◽  
Nascent Transcripts

scholarly journals Small nuclear ribonucleoproteins and heterogeneous nuclear ribonucleoproteins in the amphibian germinal vesicle: loops, spheres, and snurposomes.

1991 ◽  
Vol 113 (3) ◽  
pp. 465-483 ◽  
Author(s):  
Z A Wu ◽  
C Murphy ◽  
H G Callan ◽  
J G Gall
Keyword(s):  
Germinal Vesicle ◽  
Spherical Shape ◽  
Nucleic Acid Hybridization ◽  
Immunofluorescent Staining ◽  
U1 Snrna ◽  
Heterogeneous Nuclear Ribonucleoproteins ◽  
Specific Antigens ◽  
Nascent Transcripts ◽  
Relationship Of

We have examined the distribution of snRNPs in the germinal vesicle (GV) of frogs and salamanders by immunofluorescent staining and in situ nucleic acid hybridization. The major snRNAs involved in pre-mRNA splicing (U1, U2, U4, U5, and U6) occur together in nearly all loops of the lampbrush chromosomes, and in hundreds to thousands of small granules (1-4 microns diameter) suspended in the nucleoplasm. The loops and granules also contain several antigens that are regularly associated with snRNAs or spliceosomes (the Sm antigen, U1- and U2-specific antigens, and the splicing factor SC35). A second type of granule, often distinguishable by morphology, contains only U1 snRNA and associated antigens. We propose the term "snurposome" to describe the granules that contain snRNPs ("snurps"). Those that contain only U1 snRNA are A snurposomes, whereas those that contain all the splicing snRNAs are B snurposomes. GVs contain a third type of snRNP granule, which we call the C snurposome. C snurposomes range in size from less than 1 micron to giant structures greater than 20 microns in diameter. Usually, although not invariably, they have B snurposomes on their surface. They may also contain from one to hundreds of inclusions. Because of their remarkably spherical shape, C snurposomes with their associated B snurposomes have long been referred to as spheres or sphere organelles. Most spheres are free in the nucleoplasm, but a few are attached to chromosomes at specific chromosome loci, the sphere organizers (SOs). The relationship of sphere organelles to other snRNP-containing structures in the GV is obscure. We show by immunofluorescent staining that the lampbrush loops and B snurposomes also react with antibodies against heterogeneous nuclear ribonucleoproteins (hnRNPs). Transcription units on the loops are uniformly stained by anti-hnRNP and anti-snRNP antibodies, suggesting that nascent transcripts are associated with hnRNPs and snRNPs along their entire length, perhaps in the form of a unitary hnRNP/snRNP particle. That B snurposomes contain so many components involved in pre-mRNA packaging and processing suggests that they may serve as sites for assembly and storage of hnRNP/snRNP complexes destined for transport to the nascent transcripts on the lampbrush chromosome loops.

scholarly journals Chromosomal Storage of the RNA-editing Enzyme ADAR1 in Xenopus Oocytes

2005 ◽  
Vol 16 (7) ◽  
pp. 3377-3386 ◽  
Author(s):  
Nina B. Sallacz ◽  
Michael F. Jantsch
Keyword(s):  
Rna Editing ◽  
Binding Sites ◽  
Rna Binding ◽  
Adenosine Deaminases ◽  
A Site ◽  
Nascent Transcripts ◽  
Editing Enzyme ◽  
Nuclear Injection ◽  
Processing Factors

ADARs (adenosine deaminases that act on RNA) are RNA-editing enzymes that convert adenosines to inosines in structured or double-stranded RNAs. Expression and intracellular distribution of ADAR1 is controlled by a plethora of mechanisms suggesting that enzyme activity has to be tightly regulated. Mammalian ADAR1 is a shuttling protein, whereas Xenopus ADAR1 is exclusively nuclear. In oocytes, Xenopus ADAR1 associates with most nascent transcripts but is strongly enriched at a specific site on chromosome 3, termed the special loop. Enrichment at this site requires the presence of RNAs but is independent of ongoing transcription. Here we show that RNAs transcribed elsewhere in the genome accumulate at the special loop even in the absence of transcription. In situ hybridization experiments, however, indicate the absence of known editing substrates from this site. In the absence of transcription also other RNA binding and processing factors accumulate at the special loop, suggesting that ADAR1 is stored or assembled at the special loop in an RNA-containing complex. Nuclear injection of RNAs providing binding sites for ADAR1 dissociates the enzyme from the special loop, supporting the notion that the special loop represents a site where ADAR1 is stored, possibly for later use during development.

scholarly journals Ultrastructural Analysis of Transcription and Splicing in the Cell Nucleus after Bromo-UTP Microinjection

1999 ◽  
Vol 10 (1) ◽  
pp. 211-223 ◽  
Author(s):  
Dusan Cmarko ◽  
Pernette J. Verschure ◽  
Terence E. Martin ◽  
Michael E. Dahmus ◽  
Sabine Krause ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ultrastructural Level ◽  
Structural Domain ◽  
Mrna Transcription ◽  
Rna Transcription ◽  
Core Proteins ◽  
Transcription Sites ◽  
Nascent Transcripts

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

scholarly journals Transcription-dependent colocalization of the U1, U2, U4/U6, and U5 snRNPs in coiled bodies

1992 ◽  
Vol 117 (1) ◽  
pp. 1-14 ◽  
Author(s):  
M Carmo-Fonseca ◽  
R Pepperkok ◽  
MT Carvalho ◽  
AI Lamond
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Splicing Factor ◽  
Transcription Inhibitors ◽  
Coiled Bodies ◽  
Protein Components ◽  
Nascent Transcripts ◽  
Labeling Method ◽  
Alpha Amanitin

We have recently shown that discrete foci are present in the nuclei of mammalian cells in which each of the U1, U2, U4/U6, and U5 snRNPs involved in pre-mRNA splicing, and the non-snRNP-splicing factor U2AF, are concentrated (Carmo-Fonseca, M., D. Tollervey, R. Pepperkok, S. Barabino, A. Merdes, C. Brunner, P. D. Zamore, M. R. Green, E. Hurt, and A. I. Lamond. 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:195-206; Carmo-Fonseca, M., R. Pepperkok, B. S. Sproat, W. Ansorge, M. S. Swanson, and A. I. Lamond. 1991 EMBO (Eur. Mol. Biol. Organ.) J. 10:1863-1873). Here, we identify these snRNP-rich organelles as coiled bodies. snRNPs no longer concentrate in coiled bodies after cells are treated with the transcription inhibitors alpha-amanitin or actinomycin D. snRNP association with coiled bodies is also disrupted by heat shock. This indicates that the association of snRNPs with coiled bodies may be connected with the metabolism of nascent transcripts. A novel labeling method is described which shows both the RNA and protein components of individual snRNPs colocalizing in situ. Using this procedure all spliceosomal snRNPs are seen distributed in a nonhomogeneous pattern throughout the nucleoplasm, excluding nucleoli. They are most concentrated in coiled bodies, but in addition are present in "speckled" structures which are distinct from coiled bodies and which contain the non-snRNP splicing factor SC-35. U1 snRNP shows a more widespread nucleoplasmic staining, outside of coiled bodies and "speckled" structures, relative to the other snRNPs. The association of snRNPs with "speckles" is disrupted by heat shock but enhanced when cells are treated with alpha-amanitin.

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