Serine Phosphorylation of SR Proteins Is Required for Their Recruitment to Sites of Transcription In Vivo

Misteli, Tom; Cáceres, Javier F.; Clement, Jade Q.; Krainer, Adrian R.; Wilkinson, Miles; Spector, David L.

doi:10.1083/jcb.143.2.297

Cited by 241 publications

(216 citation statements)

References 59 publications

(127 reference statements)

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“…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, 1999;Shav-Tal et al, 2004). Instead, it seems that splicing components typically move away from speckles to sites of gene expression where splicing occurs cotranscriptionally (Huang and Spector, 1996b;Misteli et al, 1997Misteli et al, , 1998Eils et al, 2000). These findings have therefore made it difficult to understand why poly(A) RNA is associated with speckles.…”

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confidence: 73%

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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confidence: 73%

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

Politz

Tuft

Prasanth

et al. 2006

MBoC

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“…For example, splicing factors could be released from the speckles, where they are stored. It has been demonstrated that CLKs regulate the intranuclear localization of SR proteins (Duncan et al, 1998;Nayler et al, 1998b) and that phosphorylation of the RS domain is necessary for targeting SR proteins to sites of transcription (Misteli, 2000;Misteli et al, 1998). To test this hypothesis, we compared the effect of several proteins that are phosphorylated by CLKs in vitro with the CLK effect on exon 10 regulation.…”

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confidence: 99%

Regulation of Alternative Splicing of Human Tau Exon 10 by Phosphorylation of Splicing Factors

Hartmann

Rujescu

Giannakouŕos

et al. 2001

Molecular and Cellular Neuroscience

103

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“…This process appears to be controlled by protein phosphorylation Misteli, 1999); serine-arginine (SR) proteins, a major constituent of IGs (Mintz et al, 1999), are phosphoproteins containing a C-terminal serine-arginine rich RS domain and N-terminal RNA recognition motifs (RRMs; for review, see . Experiments monitoring GFP-tagged SF2/ASF SR protein in living cells have shown that phosphorylation controls the movement of SR proteins from speckles to sites of active transcription (Misteli et al, , 1998. Similarly, in vitro and in vivo studies using several kinases indicate that phosphorylation of SR proteins releases them from IGs (for review, see Stojdl and Bell, 1999), whereas phosphatases have an opposite effect (Misteli and Spector, 1996).…”

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“…Similarly, in vitro and in vivo studies using several kinases indicate that phosphorylation of SR proteins releases them from IGs (for review, see Stojdl and Bell, 1999), whereas phosphatases have an opposite effect (Misteli and Spector, 1996). Therefore, it has been proposed Misteli et al, 1998;Stojdl and Bell, 1999) that the recruitment of splicing factors from speckles occurs in two steps. First, SR proteins are physically released from speckles via RS domain phosphorylation.…”

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Nuclear Relocalization of the Pre-mRNA Splicing Factor PSF during Apoptosis Involves Hyperphosphorylation, Masking of Antigenic Epitopes, and Changes in Protein Interactions

Shav‐Tal

Cohen

Lapter

et al. 2001

MBoC

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The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1-70K and SR proteins and therefore may acquire new functions. INTRODUCTIONSplicing factors are found within the nucleus both in a diffuse form and in distinct domains termed interchromatin granules (IG) or "speckles" (Spector, 1993). These functional compartments are spatially dynamic with regard to movement and composition (Eils et al., 2000). Splicing factors within IGs are highly mobile and are constantly associating and dissociating from their compartments (Phair and Misteli, 2000). The mAb, B92, which recognizes the polypyrimidine tract binding protein (PTB)-associated splicing factor (PSF), produces typical specked nuclear pattern in immunostaining. These PSF speckles disappear during granulocytic differentiation (Lee et al., 1996;Shav-Tal et al., 2000). We recently showed that this disappearance is associated with partial degradation (Shav-Tal et al., 2000) and by the formation of alternative nuclear structures (Shav-Tal et al., 2001). The present study indicates that PSF speckles disappear also through a different mechanism, involving conformational changes that cause breakdown of PSF speckles and relocalization of the molecule in the nucleus. The functional nature of speckles is a matter of some controversy (for review, see Park and De Boni, 1999). These structures have been suggested to play a role in storage and recycling of splicing factors (Jimenez-Garcia and Spector, 1993), whereas a portion may act as reservoirs for the recruitment to active sites of transcription (Huang and Spector, 1996;Zeng et al., 1997). This view does not predict a direct involvement of speckles in pre-mRNA splicing. On the other hand, it has been shown that nascent mRNA transcripts and transcription sites are closely associated with speckles, that transcription occurs at the surface of speckles (Clemson and Lawrence, 1996), and that microinjected pre-mRNAs have high affinity to speckles (Wang et al., 1991). It has thus been proposed that speckles can act coordinately in transcription and splicing and that pre-mRNA splicing can take place bo...

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Serine Phosphorylation of SR Proteins Is Required for Their Recruitment to Sites of Transcription In Vivo

Cited by 241 publications

References 59 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

Regulation of Alternative Splicing of Human Tau Exon 10 by Phosphorylation of Splicing Factors

Nuclear Relocalization of the Pre-mRNA Splicing Factor PSF during Apoptosis Involves Hyperphosphorylation, Masking of Antigenic Epitopes, and Changes in Protein Interactions

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