Immunocytochemical identification of nuclear structures containing snRNPs in isolated rat liver cells

The localization of U1 and U2 small nuclear RNAs (snRNAs) has been examined by in situ hybridization using 2'-O-alkyl oligonucleotide probes. We have found that these snRNAs, which are essential for pre-mRNA splicing, localize in a speckled distribution, in addition to being present in three of four foci, in HeLa cell nuclei. However, in cells of defined passage, such as Detroit 551 and WI-38 fibroblasts, these snRNAs are concentrated in nuclear speckles, and foci are not observed. The speckled distribution of U1 and U2 snRNAs is coincident with the speckled regions enriched in small nuclear ribonucleoprotein particle (snRNP) proteins and the essential non-snRNP splicing factor SC-35. The localization of these key components of the pre-mRNA splicing machinery to speckled nuclear regions suggests that these regions may be involved in pre-mRNA splicing.

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“…Based on this study and previous studies (23,24), snRNPs are associated with at least three different nuclear structures:…”

Section: Resultsmentioning

confidence: 59%

U1 and U2 small nuclear RNAs are present in nuclear speckles.

Huang

Spector

1992

Proc. Natl. Acad. Sci. U.S.A.

134

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“…The interchromatin granule clusters contain only low levels of rapidly labeled RNA [for review, see 39] and hnRNP proteins [33]. However, they do contain poly(A) (see above) and large amounts of different splicing factars [23,[33][34][35]40]. Although it is generally assumed that the interchromatin granule clusters do not represent splicing organelles, their true function in nuclear RNA metabolism remains unknown [see 30 for a recent discussion] .…”

Section: Resul Ts and Discussionmentioning

confidence: 99%

Immunodetection of Poly(A) Binding Protein II in the Cell Nucleus

Krause

Fakan

Weis

et al. 1994

Experimental Cell Research

116

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During the polyadenylation of pre-mRN A in vitro~ poly(A) binding protein 11 (PAB 11) bind. to tbe growing poly(A) taH, stimulating its extension. Tbe subcellular localization of PAß 11 was investigated witb an antibody affinity-purified from rabbit serum raised against the purified protein. Immunoßuorescence microscopy detected PAß 11 exclusively in tbe cell nueleus, botb in a widespread staining and in more intensely stained "speckles." PAß 11 was excluded from the nucleoli. ßy electron microscopy, PAß II was also found almost exclusively in the nucleus, predominantly in clusters of interchromatin granules, likely corresponding to the speckles observed by immunofluorescence microscopy, and in perichromatin fibrils, wbich represent nascent transeripts and probably tbe sites of pre-mRN A processing. In addition, electron microscopy also detected P AB 11 in nucleoli. Tbe distribution corresponds largely to that of other factors involved in the processing of premRNA and is thus in agreement witb the proposed role of the protein in polyadenylation.

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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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Immunocytochemical identification of nuclear structures containing snRNPs in isolated rat liver cells

Cited by 89 publications

References 23 publications

U1 and U2 small nuclear RNAs are present in nuclear speckles.

U1 and U2 small nuclear RNAs are present in nuclear speckles.

Immunodetection of Poly(A) Binding Protein II in the Cell Nucleus

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

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