Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells

Politz, Joan C. Ritland; Tuft, Richard A.; Pederson, Thoru; Singer, Robert H.

doi:10.1016/s0960-9822(99)80136-5

Cited by 174 publications

(155 citation statements)

References 28 publications

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“…Indeed, recent measurements of the movement of caged¯uorescein-labelled oligo(dT) hybridized to poly(A) RNA in the nucleus, demonstrated that after a photolytic unmasking of the probe, the oligo(dT)-poly(A) RNA hybrids moved at solution rates. In addition, the same apparent diffusion coef®cient was measured for the movement at both 23 8C and 37 8C (Politz et al 1999). It follows that newly synthesized polyadenylated RNAs would diffuse randomly in the nucleoplasm until they reach the NPCs.…”

Section: Discussionmentioning

confidence: 61%

Rev‐dependent association of the intron‐containing HIV‐1 gag mRNA with the nuclear actin bundles and the inhibition of its nucleocytoplasmic transport by latrunculin‐B

Kimura¹,

Hashimoto²,

Yamamoto

et al. 2000

Genes to Cells

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Section: Discussionmentioning

confidence: 61%

Rev‐dependent association of the intron‐containing HIV‐1 gag mRNA with the nuclear actin bundles and the inhibition of its nucleocytoplasmic transport by latrunculin‐B

Kimura¹,

Hashimoto²,

Yamamoto

et al. 2000

Genes to Cells

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Section: Discussionmentioning

confidence: 95%

“…Much of the fluorescence label was found adjacent to the speckles, usually in the form of distinct dots. These results indicate that the movement of pre-mRNA within the interchromatin space toward the speckles was possibly a diffusion process (Politz et al, 1999), but the targeting inside the speckles themselves was temperature-dependent. The results of the experiments in which we microinjected the Ad1 RNA labeled with red fluorochrome and after 15 min of incubation at 37°C microinjected the same cells with the Ad1 RNA labeled with green fluorochrome and incubated for 30 min at 4°C supported this view ( Figure 2D).…”

mentioning

confidence: 83%

“…These findings point to the possible existence of the energydependent step, which is irreversible. Because microinjected RNAs accumulate around the speckles even at 4°C, we infer that the movement of RNAs consists of two steps: the movement of pre-mRNAs toward the speckles is probably the diffusion process (Politz et al, 1999) and their targeting within the speckles is likely an energy-dependent process.The results with mutant pre-mRNAs indicate that the (pre)spliceosomal assembly takes place inside the nuclear speckles. Targeting to, and accumulation within, the speckles depends on the nucleotide sequence of the microinjected RNAs.…”

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confidence: 95%

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Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

Melčák

Kopský

et al. 2001

MBoC

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Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. They have been shown to serve neighboring active genes as a reservoir of these factors. In this study, we show that, in HeLa cells, the (pre)spliceosomal assembly on precursor mRNA (pre-mRNA) is associated with the speckles. For this purpose, we used microinjection of splicing competent and mutant adenovirus pre-mRNAs with differential splicing factor binding, which form different (pre)spliceosomal complexes and followed their sites of accumulation. Splicing competent pre-mRNAs are rapidly targeted into the speckles, but the targeting is temperature-dependent. The polypyrimidine tract sequence is required for targeting, but, in itself, is not sufficient. The downstream flanking sequences are particularly important for the targeting of the mutant pre-mRNAs into the speckles. In supportive experiments, the behavior of the speckles was followed after the microinjection of antisense deoxyoligoribonucleotides complementary to the specific domains of snRNAs. Under these latter conditions prespliceosomal complexes are formed on endogenous pre-mRNAs. We conclude that the (pre)spliceosomal complexes on microinjected pre-mRNA are formed inside the speckles. Their targeting into and accumulation in the speckles is a result of the cumulative loading of splicing factors to the pre-mRNA and the complexes formed give rise to the speckled pattern observed. INTRODUCTIONNuclear speckles are enriched in splicing factors and in the factors of the transcription machinery (Spector, 1990;Krause et al., 1994;Bregman et al., 1995;Larsson et al., 1995). Even though there is a consensus that these compartments play a role in RNA metabolism, their exact function is presently unknown. When growing mammalian cells are labeled with antibodies to splicing components, 20 to 50 shining nuclear domains, i.e., nuclear speckles, also termed SC35 domains (protein SC35 being an important serine/arginine (SR)-rich splicing factor [Fu and Maniatis, 1990]), splicing factor compartments, or just speckles, are usually observed (Perraud et al., 1979;Spector et al., 1983;Spector, 1990;Misteli, 2000). In some cell types, they occupy as much as 20% of the nuclear volume. At the electron microscope level, they consist of morphologically well-defined interchromatin granule clusters and of domains of perichromatin fibrils, some of which are believed to represent precursor-mRNAs (premRNAs) (Fakan and Puvion, 1980;Spector et al., 1983;Puvion et al., 1984;Spector, 1990;Fakan, 1994;Raška, 1995;Melčák et al., 2000).Most mammalian pre-mRNAs contain introns and typically have to be spliced before being transported to the cytoplasm. It has been shown biochemically that spliceosome formation and splicing may be cotranscriptional events (Wuarin and Schibler, 1994). More recent results indicate that transcription and splicing are coupled with interactions of certain factors in both processes. They take part in the large macromolecular c...

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“…However, Tel15q TERRA clusters display D between 0.15 and 1 × 10 −3 µm 2 /sec (mean D = 0.02 µm 2 /sec), with similar diffusion coefficients observed in both clones (Figure 2E). Compared to previously imaged nuclear mRNAs [34,35], the size and dynamics of TERRA foci suggest that the small particles correspond to single Tel15q TERRA molecules, while the large foci represent clusters of several Tel15q TERRA molecules (supplementary movie 1 and 2).…”

Section: Resultsmentioning

confidence: 76%

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells

et al. 2018

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Telomeres cap the ends of eukaryotic chromosomes, protecting them from degradation and erroneous recombination events which may lead to genome instability. Telomeres are transcribed giving rise to telomeric repeat-containing RNAs, called TERRA. The TERRA long noncoding RNAs have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. While TERRA RNAs are predominantly nuclear and localize at telomeres, little is known about the dynamics and function of TERRA molecules expressed from individual telomeres. Herein, we developed an assay to image endogenous TERRA molecules expressed from a single telomere in living human cancer cells. We show that single-telomere TERRA can be detected as TERRA RNA single particles which freely diffuse within the nucleus. Furthermore, TERRA molecules aggregate forming TERRA clusters. Three-dimensional size distribution and single particle tracking analyses revealed distinct sizes and dynamics for TERRA RNA single particles and clusters. Simultaneous time lapse confocal imaging of TERRA particles and telomeres showed that TERRA clusters transiently co-localize with telomeres. Finally, we used chemically modified antisense oligonucleotides to deplete TERRA molecules expressed from a single telomere. Single-telomere TERRA depletion resulted in increased DNA damage at telomeres and elsewhere in the genome. These results suggest that single-telomere TERRA transcripts participate in the maintenance of genomic integrity in human cancer cells.

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Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells

Abstract: Poly(A) RNA can move freely throughout the interchromatin space of the nucleus with properties characteristic of diffusion.

Cited by 174 publications

References 28 publications

Rev‐dependent association of the intron‐containing HIV‐1 gag mRNA with the nuclear actin bundles and the inhibition of its nucleocytoplasmic transport by latrunculin‐B

Rev‐dependent association of the intron‐containing HIV‐1 gag mRNA with the nuclear actin bundles and the inhibition of its nucleocytoplasmic transport by latrunculin‐B

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells

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