Evidence for channeled diffusion of pre-mRNAs during nuclear RNA transport in metazoans.

Zachar, Zuzana; Kramer, John; Mims, I P; Bingham, Paul M.

doi:10.1083/jcb.121.4.729

Cited by 141 publications

(96 citation statements)

References 60 publications

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“…This idea is supported by observations that certain mRNAs exit from one side of the nucleus (8) and that, in yeast, many transcriptionally active gene loci are located near the nuclear periphery (9). By contrast, a number of other studies have found that mRNP complexes move quite freely within the nucleus (10)(11)(12)(13)(14)(15)(16). This view is supported by studies of the distribution of newly synthesized Balbiani ring RNA in the salivary gland cells of insects (11), fluorescence recovery after photobleaching and fluorescence correlation spectroscopy studies of probes that bind to the poly(A) tails of mRNAs (12)(13)(14)(15), and from single-particle analysis of mRNP complexes bound to GFP-linked proteins (16).…”

supporting

confidence: 50%

“…The mRNA clusters were always present at the edge of a dense chromatin region. These chromatin-associated, immobile clusters of mRNA probably comprise nascent transcripts that are still attached to the gene via RNA polymerase, having not yet undergone the 3Ј-terminal processing events required for their release (10,25).…”

Section: Dispersal Of Mrnp Particles From the Sites Of Transcriptionmentioning

confidence: 99%

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Mechanism of mRNA transport in the nucleus

Vargas¹,

Raj²,

Marras³

et al. 2005

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

235

227

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The mechanism of transport of mRNA-protein (mRNP) complexes from transcription sites to nuclear pores has been the subject of many studies. Using molecular beacons to track single mRNA molecules in living cells, we have characterized the diffusion of mRNP complexes in the nucleus. The mRNP complexes move freely by Brownian diffusion at a rate that assures their dispersion throughout the nucleus before they exit into the cytoplasm, even when the transcription site is located near the nuclear periphery. The diffusion of mRNP complexes is restricted to the extranucleolar, interchromatin spaces. When mRNP complexes wander into dense chromatin, they tend to become stalled. Although the movement of mRNP complexes occurs without the expenditure of metabolic energy, ATP is required for the complexes to resume their motion after they become stalled. This finding provides an explanation for a number of observations in which mRNA transport appeared to be an enzymatically facilitated process.gene expression ͉ live cell imaging ͉ mRNA export ͉ nuclear viscosity A fter mRNAs are synthesized, processed, and become associated with a number of different proteins at the transcription site, they are released into the nucleoplasm (1). The mechanism by which these large mRNA-protein (mRNP) complexes then move through dense nucleoplasm to reach the nuclear pores has been the subject of intense study and speculation (2, 3). Early workers proposed that mRNP complexes are transferred along a chain of receptors until they reach a nuclear pore, expending metabolic energy in the process (4). This solid-state transport model is supported by observations made in fixed nuclei that show some transcripts distributed along tracks that originate from the locus of the parent gene (5, 6). A second theory, called the ''gene-gating'' hypothesis, proposes that active genes are situated near the nuclear periphery and that mRNAs exit the nucleus through the nearest pores (7). This idea is supported by observations that certain mRNAs exit from one side of the nucleus (8) and that, in yeast, many transcriptionally active gene loci are located near the nuclear periphery (9). By contrast, a number of other studies have found that mRNP complexes move quite freely within the nucleus (10-16). This view is supported by studies of the distribution of newly synthesized Balbiani ring RNA in the salivary gland cells of insects (11), fluorescence recovery after photobleaching and fluorescence correlation spectroscopy studies of probes that bind to the poly(A) tails of mRNAs (12-15), and from single-particle analysis of mRNP complexes bound to GFP-linked proteins (16).Although the latter studies found that mRNP complexes are able to diffuse within the nuclear matrix, there was a paradoxical active transport component to their motility, because both a reduction in temperature and ATP depletion curtailed the mobility of the complexes (14-16). To better understand the nature of mRNP mobility, we have developed a system of fluorogenic probes and mRNA constructs that allo...

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supporting

confidence: 50%

Section: Dispersal Of Mrnp Particles From the Sites Of Transcriptionmentioning

confidence: 99%

Mechanism of mRNA transport in the nucleus

Vargas¹,

Raj²,

Marras³

et al. 2005

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

235

227

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“…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). Importantly, isolated pre-mRNAs from mammalian cells may contain both introns and poly(A) tails.…”

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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“…This localization has been somewhat perplexing, because speckles were also shown to contain stable, polyadenylated RNA species distinct in their behavior from most pre-mRNAs (for review, see Fakan 1994;Mattaj 1994). When sites of transcription and splicing have been examined using fluorescence in situ hybridization techniques, genes and the highest concentrations of unspliced pre-mRNA are coincident at the light microscopic level (Zachar et al 1993;Zhang et al 1994); however, endogenous intron-containing pre-mRNAs are not always concentrated within speckles (Zhang et al 1994;Xing et al 1995). This has led to the suggestion that speckles contain the highest concentrations of splicing factors--perhaps serving as storage or assembly sites--and that lower concentrations may be present where splicing occurs (Fakan et al 1984;Fakan 1994;Xing et al 1995;Huang and Spector 1996).…”

mentioning

confidence: 99%

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

Neugebauer¹,

Roth²

1997

Genes Dev.

132

118

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If pre-mRNA splicing begins during RNA synthesis, then transcriptionally active genes may be expected to contain high concentrations of pre-mRNA splicing factors. However, previous studies have localized splicing factors to a network of "speckles," which is distinct from individual sites of gene transcription where pre-mRNA is spliced. Speckles have been detected with antibodies specific for splicing snRNPs and members of the SR family of splicing factors. Here we report that dilution of these probes results in the visualization of hundreds of sites throughout the HeLa cell nucleus, the size and distribution of which are consistent with transcription units viewed with light microscopy. Importantly, these sites of highest SR protein concentration frequently coincide in three-dimensional space with active sites of RNA polymerase II transcription. A newly developed reagent specific for a single member of the SR family, SRp20, detects a subset (-20%) of these sites, suggesting the gene-specific accumulation of these splicing regulators, which have distinct functions in pre-mRNA splicing. These observations question the view that the nucleus and its functions are highly compartmentalized; instead, they support a model in which the localization of these and possibly other gene regulators is determined primarily by their function.[Key Words: Pre-mRNA splicing; RNA polymerase II; SR proteins; nuclear organization] Received December 6, 1996; revised version accepted March 18, 1997.As genes are transcribed, a diverse set of proteins and ribonucleoprotein particles (RNPs) associate with the nascent RNA. The binding of particular factors is likely to determine how the transcript will be processed to mature RNA. The observation that pre-rRNA processing and pre-mRNA splicing often occur at or very near sites of RNA synthesis (Penman et al. 1966;Rosbash and Singer 1993;Mattaj 1994) leads to the prediction that the factors regulating RNA processing may be similarly concentrated. The factors involved in pre-rRNA processing have been shown to be localized to the nucleolus, the site of rDNA transcription (Carmo-Fonseca et al. 1991;Jimenez-Garcia et al. 1994;Matera et al. 1994). In contrast, factors essential for the splicing of mRNA precursors in mammalian cell nuclei have been observed in large structures, termed "speckles," which are morphologically distinct from sites of RNA polymerase II (Pol II) transcription (Fu and Maniatis 1990;Spector 1990;Wansink et al. 1993;Zhang et al. 1994). As a first step toward resolving this enigma, we have re-examined the distribution of pre-mRNA splicing factors with respect to RNA polymerase II transcription, focusing on members of the SR protein family. 3Corresponding author. E-MAIL neugebau@embl-heidelberg.de; FAX 49 6221 387 518.Pre-mRNA splicing takes place within the spliceosome, a multicomponent complex of small nuclear ribonucleoprotein particles (snRNPs) and a number of nonsnRNP protein factors (Moore et al. 1993). The nine SR proteins constitute a family of nuclear phosphoproteins esse...

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Evidence for channeled diffusion of pre-mRNAs during nuclear RNA transport in metazoans.

Cited by 141 publications

References 60 publications

Mechanism of mRNA transport in the nucleus

Mechanism of mRNA transport in the nucleus

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

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

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