An RNA Recognition Motif (RRM) Is Required for the Localization of PTB-Associated Splicing Factor (PSF) to Subnuclear Speckles

Dye, Billy T.; Patton, James G.

doi:10.1006/excr.2000.5097

Cited by 101 publications

(109 citation statements)

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“…Examples are the RNA recognition motif (RRM) in the polypyrimidine track binding (PTB)-associated splicing factor (PSF) (29), the ''ForkHead-associated'' domain of the protein phosphatase-1 regulator NIPP1 (30), and the TP repeat domain of the splicing protein SF3b (155) (31). In contrast to other SR proteins, SRm160 does not contain an RRM.…”

Section: Discussionmentioning

confidence: 99%

The spatial targeting and nuclear matrix binding domains of SRm160

Wagner

Chiosea

Nickerson

2003

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

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The Ser-Arg (SR)-related protein SRm160 is a coactivator of premRNA splicing. It bridges splicing factors located at the 5 splice site, branch site, and 3 splice site. Recently, SRm160 has also been shown to be involved in mRNA export as part of an exon-junction complex. SRm160 is highly concentrated in splicing speckles but is also present in long branched intranuclear tracks connecting splicing speckles with sites at the nuclear lamina. In this study we identified domains of SRm160 important for spatial targeting within the nucleus and for binding to the nuclear matrix. Using a series of FLAG-and enhanced GFP-conjugated deletion mutants we found two contiguous sequences that independently target SRm160 to nuclear matrix sites at splicing speckled domains: amino acids 300 -350 and 351-688. Constructs containing amino acids 300 -350 were also targeted to sites peripheral to speckled domains where most mRNA originate subsequent to splicing. Sequences from the N-terminal domain localized proteins to the nuclear lamina near sites where mRNA leaves the nucleus.RNA splicing ͉ RNA export ͉ speckled domains ͉ SRm300 S ince the discovery of RNA splicing (1), its mechanism has been elucidated by the clever use of in vitro assays (2). Simple precursor RNAs, usually with one small intron, are added to a nuclear extract. After the addition of ATP, spliceosomal complexes form, and introns are removed slowly. In marked contrast, native RNA splicing in cells is far more rapid and efficient, capable of processing more complicated substrates. Precursor RNAs as large as 80,780 bases with as many as 175 introns (3) are rapidly spliced, often in complicated but precise alternative patterns. The very rapid splicing seen in vivo likely reflects, in part, the accurate positioning of splicing substrates and factors by the highly ordered architecture of the nucleus. Most RNA splicing factors are concentrated in subnuclear structures that appear as speckled domains when visualized by immunofluorescence microscopy (4). When seen by electron microscopy, these correspond to interchromatin granule clusters (5) that are surrounded by regions rich in the perichromatin fibrils that contain many new transcripts (5, 6). A majority of these transcripts are spliced at or near speckled domains (7), and mechanisms have been described for recruiting splicing factors from these domains to newly activated genes (8,9).Evidence that the nuclear matrix has a critical role in RNA splicing has emerged from studies examining cells expressing a ␤-globin pre-mRNA splicing construct (10,11). This precursor remains associated with the nuclear matrix after its isolation and is spliced rapidly after addition of the ATP (11). In contrast to conventional in vitro splicing reactions, splicing in situ on nuclear matrix preparations occurs without a lag period, indicating that spliceosomal commitment complexes are preassembled and fully functional.Two strong candidates for factors that might couple splicing components are Ser-Arg (SR)-related matrix protein of 160 kDa (...

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Section: Discussionmentioning

confidence: 99%

The spatial targeting and nuclear matrix binding domains of SRm160

Wagner

Chiosea

Nickerson

2003

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

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“…We therefore used human GFP-PSF (Dye and Patton, 2001) that localized in speckles in interphase nuclei and also produced a diffuse nucleoplasmic staining (Figure 4,C and D). In mitotic cells part of the GFP-PSF protein was concentrated in several rounded structures, whereas some was diffusely distributed throughout the cytoplasm (Figure 4,E and F).…”

Section: Overexpressed Gfp-tagged Psf Localizes In Apoptotic Bodies Imentioning

confidence: 99%

“…Figure 7C shows that both full-length GFP-PSF (amino acids 1-707) and endogenous PSF are detected by the B92 antibody in Western blots, and retarded migration of both proteins is observed in nocodazole-arrested cells (metaphase). Because phosphorylation is thought to control the localization of splicing factors in speckles, we checked whether the removal of RRM2 of PSF, which is responsible for PSF targeting to speckles (Dye and Patton, 2001;Figure 7, D and F), is the region hyperphosphorylated during mitosis. However, although GFP-PSF ⌬RRM2 does not localize in speckles, it was also hyperphosphorylated during metaphase ( Figure 7F).…”

Section: Psf Is Hyperphosphorylated During Apoptosismentioning

confidence: 99%

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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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“…It copurifies with both early (H, A, B) and late (C) spliceosomal complexes, the U4/U6.U5 tri-snRNP, U5 snRNP, and is found in nuclear speckles, colocalizing with many splicing factors. 14,[16][17][18][19][20][21][22][23] It is comprised of an N-terminal glycine-rich domain, a proline/glutamine (P/Q) rich domain, two RNA recognition motifs (RRMs), and a C-terminal region with two nuclear localization signals. A closely related protein with a similar domain structure, p54 nrb (and its mouse homolog nonO) 24,25 has been found to associate with PSF.…”

Section: Introductionmentioning

confidence: 99%

The Splicing Factor PSF is Part of a Large Complex That Assembles in the Absence of pre-mRNA and Contains All 5 snRNPs

Peng¹,

Hawkins²,

Link³

et al. 2006

RNA Biology

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Previously published online as a RNA Biology E-publication: http://www.landesbioscience.com/journals/rnabiology/abstract.php?id=3017 KEY WORDS Research PaperThe Splicing Factor PSF is Part of a Large Complex that Assembles in the Absence of pre-mRNA and Contains All 5 snRNPs ABSTRACT PSF (PTB-associated splicing factor) is a large nuclear protein that has been implicated in numerous processes including transcription and RNA splicing. It has been shown to directly associate with U5 snRNA and has also been found within numerous purified splicing complexes. Here, we show that when HeLa nuclear extracts are adjusted to splicing conditions, PSF is found as part of a large complex that contains all five snRNPs and most known splicing factors. Formation of the complex does not require addition of exogenous pre-mRNA substrate and occurs at 4˚C but is salt sensitive. Sedimentation experiments and identification of individual components by mass spectrometry revealed association with multiple nuclear factors, most of which overlap with spliceosome components.

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An RNA Recognition Motif (RRM) Is Required for the Localization of PTB-Associated Splicing Factor (PSF) to Subnuclear Speckles

Cited by 101 publications

References 79 publications

The spatial targeting and nuclear matrix binding domains of SRm160

The spatial targeting and nuclear matrix binding domains of SRm160

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

The Splicing Factor PSF is Part of a Large Complex That Assembles in the Absence of pre-mRNA and Contains All 5 snRNPs

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