Steady-state dynamics of Cajal body components in the <i>Xenopus</i> germinal vesicle

Handwerger, Korie E.; Murphy, Christine; Gall, Joseph G.

doi:10.1083/jcb.200212024

Cited by 73 publications

(76 citation statements)

References 41 publications

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“…These findings are consistent with numerous studies on the mobilities of nuclear proteins that have demonstrated rapid exchange between the nucleoplasm and speckles (Kruhlak et al, 2000;Phair and Misteli, 2000;Molenaar et al, 2004), the nucleolus (Phair and Misteli, 2000;Snaar et al, 2000;Chen and Huang, 2001), and Cajal bodies (Snaar et al, 2000;Handwerger et al, 2003;Dundr et al, 2004). We also show here that the SC35 protein is extremely dynamic in both within and outside speckles.…”

Section: Discussionsupporting

confidence: 80%

“…Our results do not rule out the presence of a very small slowmoving or immobile class of poly(A) RNA in the nucleus, but if it exists, our results indicate that it is Ͻ5% of the total poly(A) RNA and is present in similar amounts in both speckles and the nucleoplasm. Therefore, it is unlikely that poly(A) RNA serves as an immobile scaffolding to form the speckle; rather, it can move freely in and out of speckles in all directions and visit other speckles.These findings are consistent with numerous studies on the mobilities of nuclear proteins that have demonstrated rapid exchange between the nucleoplasm and speckles (Kruhlak et al, 2000;Phair and Misteli, 2000;Molenaar et al, 2004), the nucleolus (Phair and Misteli, 2000;Snaar et al, 2000;Chen and Huang, 2001), and Cajal bodies (Snaar et al, 2000;Handwerger et al, 2003;Dundr et al, 2004). We also show here that the SC35 protein is extremely dynamic in both within and outside speckles.…”

supporting

confidence: 80%

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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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Section: Discussionsupporting

confidence: 80%

supporting

confidence: 80%

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

Politz

Tuft

Prasanth

et al. 2006

MBoC

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“…Immunoelectron microscopy studies of CBs reveal thread-like, coilin-positive aggregates, suggesting that coilin is integral to CB structure 2 . Consistent with this, coilin resides longer in CBs than any other examined component in vivo 3,18 . Notably, depletion of coilin results in the dispersal of many CB components.…”

Section: 3 a R T I C L E Ssupporting

confidence: 77%

Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis

Strzelecka

Trowitzsch

Weber

et al. 2010

Nat Struct Mol Biol

152

164

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0 3 a r t i c l e sIn eukaryotes, organelles delimited by lipid bilayers support the organization of cellular functions, as exemplified by the segregation of chromatin into the cell nucleus. However, many subcellular compartments-nucleoli, Cajal and PML bodies, polar granules, stress granules, P bodies and others-lack membranes. How these structures contribute to cellular physiology is largely unknown 1 . The Cajal body (CB) is a 0.5-to 1-µm nuclear compartment initially described by Ramon y Cajal in silver-stained sections of vertebrate cerebral cortex 2 . Because the CB is conserved in evolution and is uniquely marked by the protein coilin, it has served as a general model for subnuclear structure and function 1,2 . Numerous factors essential for pre-mRNA splicing, histone mRNA 3′-end processing, telomere maintenance and rRNA processing are concentrated in CBs. Yet CB components exchange rapidly with the nucleoplasm, where most of these processes occur 3 . The challenge, therefore, has been to identify the function of CBs.Despite the localization of splicing machinery in CBs, they are not the sites of pre-mRNA splicing 4,5 . Splicing occurs throughout the nucleus and is catalyzed by the spliceosome, a macromolecular complex formed on pre-mRNA from five essential snRNPs 6 . Each spliceosomal snRNP comprises a unique 100-to 200-nucleotide snRNA (U1, U2, U4, U5 and U6) that is post-transcriptionally modified and associated with a number of snRNP-specific proteins 6,7 . Several roles for CBs in spliceosomal snRNP biogenesis have been proposed based on the localization of specific steps in snRNP assembly in the CBs of cultured cell lines. After transcription and export to the cytoplasm, U1, U2, U4 and U5 snRNAs receive a heteroheptameric ring of Sm proteins assembled by the SMN complex; subsequently, their 5′ ends are hypermethylated 8 . The Sm ring and trimethylguanosine (TMG) cap are signals for snRNP reimport into the nucleus, where these still-immature core snRNPs first concentrate in CBs 9-12 . CBs contain scaRNAs, which then guide site-specific modification of the snRNAs 13 . Importantly, intermediates in the final snRNP maturation steps, reflecting RNA structural rearrangements and the recruitment of snRNP-specific proteins, are highly concentrated in CBs 14-17 . These observations have led to the proposal that CBs are the sites of snRNP assembly, but an essential role for CBs in this process has not been shown.The coilin protein may hold the key to CB function. Immunoelectron microscopy studies of CBs reveal thread-like, coilin-positive aggregates, suggesting that coilin is integral to CB structure 2 . Consistent with this, coilin resides longer in CBs than any other examined component in vivo 3,18 . Notably, depletion of coilin results in the dispersal of many CB components. Without coilin, spliceosomal factors become nucleoplasmic, and other classes of CB-localized factors (for example, SMN, fibrillarin and small Cajal body-specific RNAs (scaRNAs)) continue to self-associate in discrete 'residual bodies' ...

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“…Because their concentration is high in CBs, they are detectable there by in situ hybridization, whereas their low concentration in the nucleoplasm puts them below the usual level of detection. This hypothesis simply extends to scaRNAs what is well known for other CB components, particularly from FRAP (fluorescence recovery after photobleaching) experiments (Handwerger et al, 2003;Deryush-eva and Gall, 2004;Dundr et al, 2004). Even coilin, the canonical CB marker, is widely distributed in the nucleoplasm, although it is concentrated in CBs (Bellini and Gall, 1998;Matera, 1998).…”

Section: Scarnas In the Nucleoplasm?mentioning

confidence: 99%

“…These nuclei retain their physiological activity for hours (Lund and Paine, 1990;Paine et al, 1992;Handwerger et al, 2003;Deryusheva and Gall, 2004). Furthermore, U2 snRNA injected into such nuclei is properly modified (Yu et al, 2001).…”

Section: In Vitro Modification Assaysmentioning

confidence: 99%

Small Cajal Body–specific RNAs ofDrosophilaFunction in the Absence of Cajal Bodies

Deryusheva

Gall

2009

MBoC

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During their biogenesis small nuclear RNAs (snRNAs) undergo multiple covalent modifications that require guide RNAs to direct methylase and pseudouridylase enzymes to the appropriate nucleotides. Because of their localization in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific RNAs (scaRNAs). Using a fluorescent primer extension technique, we mapped the modified nucleotides in Drosophila U1, U2, U4, and U5 snRNAs. By fluorescent in situ hybridization (FISH) we showed that seven Drosophila scaRNAs are concentrated in easily detectable CBs. We used two assays based on Xenopus oocyte nuclei to demonstrate that three of these Drosophila scaRNAs do, in fact, function as guide RNAs. In flies null for the CB marker protein coilin, CBs are absent and there are no localized FISH signals for the scaRNAs. Nevertheless, biochemical experiments show that scaRNAs are present at normal levels and snRNAs are properly modified. Our experiments demonstrate that several scaRNAs are concentrated as expected in the CBs of wild-type Drosophila, but they function equally well in the nucleoplasm of mutant flies that lack CBs. We propose that the snRNA modification machinery is not limited to CBs, but is dispersed throughout the nucleoplasm of cells in general. INTRODUCTIONThe two major ribosomal RNAs (rRNAs) and the spliceosomal small nuclear RNAs (snRNAs) U1, U2, U4, U5, and U6 contain many posttranscriptionally modified nucleotides, which are crucial for RNA-RNA and RNA-protein interactions, as well as spliceosome function (Yu et al., 1998; Yu, 2004, 2007). The most abundant modifications in both rRNAs and snRNAs are 2Ј-O-methylation and pseudouridylation, which are directed by small box C/D and box H/ACA guide RNAs, respectively. Each guide RNA molecule is associated with a set of four core proteins: box C/D RNAs form RNP particles with fibrillarin (the methyltransferase), Nop56, Nop58, and a 15.5-kDa protein, whereas box H/ACA RNAs associate with NAP57/dyskerin (the pseudouridine synthase), GAR1, NHP2, and NOP10 proteins (reviewed in Yu et al., 2004;Matera et al., 2007). Most guide RNAs are concentrated in the nucleolus, where they are involved in posttranscriptional modifications of rRNA. Because of their localization, they are referred to as small nucleolar RNAs (snoRNAs). However, the guide RNAs that mediate modification of the snRNAs are preferentially found in another nuclear organelle, the Cajal body (CB). These guide RNAs are called small CB-specific RNAs (scaRNAs).The first scaRNA to be studied in detail was U85 scaRNA (Jády and Kiss, 2001;Darzacq et al., 2002). U85 is a remarkable double guide RNA that contains both box C/D and box H/ACA motifs. In a set of detailed experiments it was shown that human U85 scaRNA guides both 2Ј-O-methylation at C45 and pseudouridylation at U46 in U5 snRNA. Modification takes place in the nucleus after import of the U5 snRNA from the cytoplasm . CB localization of U85 scaRNA was originally demonstrated by fluorescent in situ hybridization (FISH) in mammalian and ...

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Steady-state dynamics of Cajal body components in the Xenopus germinal vesicle

Cited by 73 publications

References 41 publications

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

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

Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis

Small Cajal Body–specific RNAs ofDrosophilaFunction in the Absence of Cajal Bodies

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