An amino acid sequence motif sufficient for subnuclear localization of an arginine/serine-rich splicing factor.

Hedley, Mary Lynne; Amrein, Hubert; Maniatis, Tom

doi:10.1073/pnas.92.25.11524

Cited by 135 publications

(139 citation statements)

References 48 publications

(43 reference statements)

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“…Both sequences are characterized by an unusual high content of arginine, proline, and serine: 88% for SRm160 300 -350 and 64% for SRm160 . SR domains have been reported to be sufficient for speckle localization for a subset of SR proteins (26)(27)(28). Although the SRm160 speckle targeting sequences contain some SR repeats, these are less clustered, more disperse, and scattered in smaller domains.…”

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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“…As shown in Figure 1a, this region includes a nuclear localization signal (amino acids 821 ± 827, NLS) and overlaps with a nuclear speckle retention signal (amino acids 860 ± 967, SRS; Hedley et al, 1995) and with a PEST sequence (amino acids 839 ± 934). This region, which is rich in proline, glutamic acid, serine and threonine, confers susceptibility to rapid intracellular proteolysis (Rogers et al, 1986).…”

Section: Two-hybrid Screening For the Isolation Of The Hmgi(y)-interamentioning

confidence: 99%

High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth

et al. 2001

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The HMGI proteins (HMGI, HMGY and HMGI-C) have an important role in the chromatin organization and interact with di erent transcriptional factors. The HMGI genes are expressed at very low levels in normal adult tissues, whereas they are very abundant during embryonic development and in several experimental and human tumours. In order to isolate proteins interacting with the HMGI(Y) proteins, a yeast two-hybrid screening was performed using the HMGI(Y) protein as bait. This analysis led to the isolation of homeodomaininteracting protein kinase-2 (HIPK2), a serine/threonine nuclear kinase. HIPK2 co-immunoprecipitates with the HMGI(Y) protein in 293T cells. The interaction between HIPK2 and HMGI(Y) occurs through the PEST domain of HIPK2 and it is direct because in vitro translated HIPK2 binds HMGI(Y). We also show that HIPK2 is able to phosphorylate the HMGI(Y) protein by an in vitro kinase assay. In order to understand a possible role of HIPK2 gene in cell growth we performed a colony assay which showed an impressive HIPK2 inhibitory e ect on normal thyroid cells. Flow cytometric analysis would indicate the block of cell growth at the G2/M phase of the cell cycle. Since normal thyroid cells do not express detectable HMGI(Y) protein levels, we assume that the HIPK2 inhibitory e ect is independent from the interaction with the HMGI(Y) protein. Oncogene (2001) 20, 6132 ± 6141.

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“…However, taking into account that (i) SR proteins are trans-acting factors that play an important role in splicing process through binding to splicing enhancers and mediating protein-protein interaction (44); (ii) SR proteins are targeted to stores with the appearance of speckles through their RS domain (45,46); and (iii) that they are released from them upon phosphorylation (47), the more likely hypothesis is that SC35 is released from speckles upon phosphorylation by GSK-3. However, SC35 phosphorylation by GSK-3 seems to require prephosphorylation by priming kinases.…”

Section: Gsk-3 Tau Splicing and Sc35 Phosphorylationmentioning

confidence: 99%

Glycogen Synthase Kinase-3 Plays a Crucial Role in Tau Exon 10 Splicing and Intranuclear Distribution of SC35

Hernández

Pérez

Lucas

et al. 2004

Journal of Biological Chemistry

128

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Tauopathies, including Alzheimer's disease, are neurodegenerative disorders in which tau protein accumulates as a consequence of alterations in its metabolism. At least three different types of alterations have been described; in some cases, an aberrant mRNA splicing of tau exon 10 occurs; in other cases, the disorder is a consequence of missense mutations and, in most cases, aberrant tau hyperphosphorylation takes place. Glycogen synthase kinase-3 (GSK-3) has emerged as a key kinase that is able to interact with several proteins involved in the ethiology of Alzheimer's disease and other tauopathies. Here, we have evaluated whether GSK-3 is also able to modulate tau-mRNA splicing. Our data demonstrate that GSK-3 inhibition in cultured neurons affects tau splicing resulting in an increase in tau mRNA containing exon 10. Pre-mRNA splicing is catalyzed by a multimolecular complex including members of the serine/arginine-rich (SR) family of splicing factors. Immunofluorescence studies showed that after GSK-3 inhibition, SC35, a member of the SR family, is redistributed and enriched in nuclear speckles and colocalizes with the kinase. Furthermore, immunoprecipitated SC35 is phosphorylated by recombinant GSK-3␤. Phosphorylation of a peptide from the SR domain by GSK-3 revealed that the peptide needs to be prephosphorylated, suggesting the involvement of a priming kinase. Our results demonstrate that GSK-3 plays a crucial role in tau exon 10 splicing, raising the possibility that GSK3 could contribute to tauopathies via aberrant tau splicing.

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An amino acid sequence motif sufficient for subnuclear localization of an arginine/serine-rich splicing factor.

Cited by 135 publications

References 48 publications

The spatial targeting and nuclear matrix binding domains of SRm160

The spatial targeting and nuclear matrix binding domains of SRm160

High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth

Glycogen Synthase Kinase-3 Plays a Crucial Role in Tau Exon 10 Splicing and Intranuclear Distribution of SC35

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