Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription.

Neugebauer, Karla M.; Roth, Mark B.

doi:10.1101/gad.11.9.1148

Cited by 133 publications

(132 citation statements)

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“…RNA Polymerase II is a component of the transcription machinery 50 and also colocalizes with pre-mRNA splicing factors. 51 In HeLa cells expressing the EGFP RI-3A þ fusion, RNA Polymerase II did not colocalize with murine AF4 (Figure 3e). …”

Section: Af4 Does Not Colocalize With Known Nuclear Proteinsmentioning

confidence: 99%

MLL fusion partners AF4 and AF9 interact at subnuclear foci

et al. 2003

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The MLL gene is involved in translocations associated with both acute lymphoblastic and acute myelogenous leukemia. These translocations fuse MLL with one of over 30 partner genes. Collectively, the MLL partner genes do not share a common structural motif or biochemical function. We have identified a protein interaction between the two most common MLL fusion partners AF4 and AF9. This interaction is restricted to discrete nuclear foci we have named 'AF4 bodies'. The AF4 body is non-nucleolar and is not coincident with any known nuclear structures we have examined. The AF4-AF9 interaction is maintained by the MLL-AF4 fusion protein, and expression of the MLL-AF4 fusion can alter the subnuclear localization of AF9. In view of other research indicating that other MLL fusion partners also interact with one another, these results suggest that MLL fusion partners may participate in a web of protein interactions with a common functional goal. The disruption of this web of interactions by fusion with MLL may be important to leukemogenesis.

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Section: Af4 Does Not Colocalize With Known Nuclear Proteinsmentioning

confidence: 99%

MLL fusion partners AF4 and AF9 interact at subnuclear foci

et al. 2003

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“…Sites of these accumulations likely represent the sites of active (cotranscriptional) splicing (Neugebauer and Roth, 1997;Misteli and Spector, 1998;Misteli, 2000). However, in growing mammalian cells, many factors of the splicing apparatus are specifically enriched in the speckles (Spector, 1990;Spector et al, 1991;Bregman et al, 1995;Misteli, 2000).…”

confidence: 99%

“…The splicing of pre-mRNAs may be a cotranscriptional event (Beyer and Osheim, 1988;Neugebauer and Roth, 1997;Custodio et al, 1999). However, not all pre-mRNA sequences are processed cotranscriptionally and posttranscriptional splicing does occur (Zachar et al, 1993;Baurén and Wieslander, 1994;Wuarin and Schibler, 1994).…”

Section: Introductionmentioning

confidence: 99%

“…Spliceosomes are generated by the constitutive assembly of U1, U2, U5, U4/U6 small nuclear ribonucleoprotein particles (snRNPs) and various non-snRNP factors on pre-mRNAs in the cascade of sequence-specific steps (Steitz et al, 1988;Lamm and Lamond, 1993;Moore et al, 1993;Newman, 1994;Krämer, 1996). The formation of the functional spliceosome is thus preceded by the formation of a number of prespliceosomal complexes, termed E, A, and B complexes containing, together with unspliced pre-mRNA, defined combinations of snRNP particles and non-snRNP factors (Steitz et al, 1988;Lamm and Lamond, 1993;Moore et al, 1993;Newman, 1994;Krämer, 1996).The splicing of pre-mRNAs may be a cotranscriptional event (Beyer and Osheim, 1988;Neugebauer and Roth, 1997;Custodio et al, 1999). However, not all pre-mRNA sequences are processed cotranscriptionally and posttranscriptional splicing does occur (Zachar et al, 1993;Baurén and Wieslander, 1994;Wuarin and Schibler, 1994).…”

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confidence: 99%

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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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“…When these sites are viewed in the electron microscope, most of them are found to represent interchromatin granule clusters (Fakan et al, 1984. However, RNA polymerase II transcription sites are distributed throughout the nucleus, indicating that although some of the RNA present in interchromatin granules/speckles is nascent, transcription is not restricted to this compartment, and much of the poly(A) RNA there has likely been transcribed previously and/or at distant sites (Fakan and Nobis, 1978;Wansink et al, 1993, Zeng et al, 1997, Neugebauer and Roth, 1997, Cmarko et al, 1999Guillot et al, 2004). Indeed, although transcripts that are being rapidly produced or that contain many introns are sometimes observed in speckles at the light microscopy level (Johnson et al, 2000;Shopland et al, 2003), the majority of studies over the years has indicated that most speckles are not primary sites of pre-mRNA splicing (for reviews, see Mattaj, 1994;Huang and Spector, 1996a;Neugebauer and Roth, 1997;Lamond and Spector, 2003).…”

Section: Introductionmentioning

confidence: 99%

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

Politz

Tuft

Prasanth

et al. 2006

MBoC

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Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22°C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy. INTRODUCTIONNuclear speckles are morphologically distinct regions of the nucleoplasm that contain pre-mRNA splicing components as well as poly(A) RNA (Carter et al., 1993;Zhang et al., 1994;Lamond and Spector, 2003). They are operationally defined by their immunostaining with a variety of pre-mRNA splicing factor antibodies, and they also show in situ hybridization signal using probes for poly(A). When these sites are viewed in the electron microscope, most of them are found to represent interchromatin granule clusters (Fakan et al., 1984. However, RNA polymerase II transcription sites are distributed throughout the nucleus, indicating that although some of the RNA present in interchromatin granules/speckles is nascent, transcription is not restricted to this compartment, and much of the poly(A) RNA there has likely been transcribed previously and/or at distant sites (Fakan and Nobis, 1978;Wansink et al., 1993, Zeng et al., 1997, Neugebauer and Roth, 1997, Cmarko et al., 1999Guillot et al., 2004). Indeed, although transcripts that are being rapidly produced or that contain many introns are sometimes observed in speckles at the light microscopy level (Johnson et al., 2000;Shopland et al., 2003), the majority of studies over the years has indicated that most speckles are not primary sites of pre-mRNA splicing (for reviews, see Mattaj, 1994;Huang and Spector, 1996a;Neugebauer and Roth, 1997;Lamond and Spector, 2003). In two cases, a single species of pre-mRNA has been followed from its transcription site to the nuclear pore, and in neither case did the RNA seem to accumulate in nuclear subcompartments (Singh et al.,...

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Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription.

Cited by 133 publications

References 53 publications

MLL fusion partners AF4 and AF9 interact at subnuclear foci

MLL fusion partners AF4 and AF9 interact at subnuclear foci

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

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