Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Oct;22(19):6871-82.
doi: 10.1128/MCB.22.19.6871-6882.2002.

Nuclear export and retention signals in the RS domain of SR proteins

Demian Cazalla  1 , Jun Zhu, Lisa Manche, Elisabeth Huber, Adrian R Krainer, Javier F Cáceres
Affiliations

Nuclear export and retention signals in the RS domain of SR proteins

Demian Cazalla et al. Mol Cell Biol. 2002 Oct.

Abstract

Splicing factors of the SR protein family share a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RS domain, which is extensively phosphorylated, promotes protein-protein interactions and directs subcellular localization and-in certain situations-nucleocytoplasmic shuttling of individual SR proteins. We analyzed mutant versions of human SF2/ASF in which the natural RS repeats were replaced by RD or RE repeats and compared the splicing and subcellular localization properties of these proteins to those of SF2/ASF lacking the entire RS domain or possessing a minimal RS domain consisting of 10 consecutive RS dipeptides (RS10). In vitro splicing of a pre-mRNA that requires an RS domain could take place when the mutant RD, RE, or RS10 domain replaced the natural domain. The RS10 version of SF2/ASF shuttled between the nucleus and the cytoplasm in the same manner as the wild-type protein, suggesting that a tract of consecutive RS dipeptides, in conjunction with the RRMs of SF2/ASF, is necessary and sufficient to direct nucleocytoplasmic shuttling. However, the SR protein SC35 has two long stretches of RS repeats, yet it is not a shuttling protein. We demonstrate the presence of a dominant nuclear retention signal in the RS domain of SC35.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
In vitro splicing activities of SF2/ASF proteins with variant RS domains. (A) Sequences of SF2/ASF wild-type (wt) and variant RS domains. All the Arg-Ser or Ser-Arg dipeptides in the RS domain of SF2/ASF were deleted (broken lines) to generate the ΔRS mutant protein. An additional mutant form in which the entire RS domain was deleted (RRM1/RRM2) behaved in a manner similar to that of the ΔRS mutant form. In addition, either Arg or Ser residues within RS or SR dipeptides were substituted with Thr, Gly, Lys, Asp, or Glu to generate RT, RG, GS, KS, RD, or RE mutant proteins. The simplified RS10 domain consists of 10 consecutive RS dipeptides. RS or SR dipeptides as well as the corresponding mutant dipeptides are indicated in bold. The ΔRS, RRM1/RRM2, RT, RG, GS, KS, and RS10 mutant proteins were previously described (4, 68). (B) In vitro splicing activities of the RS10, RE, and RD mutant proteins. Shown is the in vitro splicing of β-globin pre-mRNA (left panel) and IgM M1-M2 pre-mRNA (right panel) in HeLa cell nuclear extract (NE) (lanes 1), S100 extract alone (lanes 2), and S100 extract complemented with 4 and 8 pmol of recombinant SF2/ASF (wild type [WT]) (lanes 3 and 4, respectively), ΔRS (lanes 5 and 6, respectively), RS10 (lanes 7 and 8, respectively), RD (lanes 9 and 10, respectively), or RE (lanes 11 and 12, respectively).
FIG. 2.
FIG. 2.
Role of the RS domain of SF2/ASF in subcellular localization and distribution in nuclear speckles. (A) Immunoblot analysis of the phosphorylation state of SF2/ASF wild-type (WT) and variant proteins. The indicated T7-tagged constructs were transfected into HeLa cells, total cell lysates were incubated in the presence (+) or absence (−) of alkaline phosphatase (AP), and tagged SF2/ASF was detected by Western blotting. Wild-type SF2/ASF and the RS10 mutant protein were phosphorylated in vivo, as indicated by the increase in mobility observed upon treatment with alkaline phosphatase. In contrast, the ΔRS, RD, and RE mutant proteins were not detectably phosphorylated in vivo. (B) HeLa cells were transfected with plasmids encoding the indicated T7-tagged proteins and fixed at 24 h posttransfection. The localization of the expressed proteins was determined by indirect immunofluorescence analysis with anti-T7 monoclonal antibody and fluorescein isothiocyanate-conjugated secondary antibody. Bar, 5 μm.
FIG. 3.
FIG. 3.
Analysis of nucleocytoplasmic shuttling of SF2/ASF and RS10 by transient expression in interspecies heterokaryons. Wild-type (WT) SF2/ASF and the RS10 mutant protein were transiently expressed in HeLa cells. At 24 h posttransfection, the cells were treated with cycloheximide and subsequently fused with mouse NIH 3T3 cells in the presence of polyethylene glycol to form heterokaryons. The cells were further incubated for 2 h in the presence of cycloheximide, followed by fixation. (Left panels) The localization of the epitope-tagged proteins in the heterokaryons was determined by indirect immunofluorescence analysis with anti-T7 monoclonal antibody (mAbT7) and fluorescein isothiocyanate-conjugated secondary antibody. (Middle panels) The cells were simultaneously incubated with DAPI for differential staining of human and mouse nuclei within heterokaryons. (Right panels) Phase-contrast images of the same heterokaryons. Arrows indicate the mouse nuclei within human-mouse heterokaryons.
FIG. 4.
FIG. 4.
NRS in the RS domain of SC35. (A) Sequences of the SF2/ASF and SC35 RS domains. The long tracts of RS dipeptides are indicated in grey, whereas RS dipeptides outside of these tracts are indicated in bold. (B) Analysis of nucleocytoplasmic shuttling of chimeric forms of hnRNP A1 by transient expression in interspecies heterokaryons. Wild-type hnRNP A1 and chimeric proteins were transiently expressed in HeLa cells, which were then fused with NIH 3T3 cells and analyzed as described in the legend to Fig. 3. Arrows indicate the mouse nuclei within human-mouse heterokaryons. (C) Diagram showing the structures of chimeric proteins in which SC35, the RS domain of SC35, or the RS domain of SF2/ASF was fused to a shuttling protein—either hnRNP A1 or SF2/ASF. The nuclear export properties of these chimeric proteins were analyzed by interspecies heterokaryon assays (panel B) and by transcriptional inhibition assays (data not shown), and the results are summarized. Act D, actinomycin D.
FIG. 4.
FIG. 4.
NRS in the RS domain of SC35. (A) Sequences of the SF2/ASF and SC35 RS domains. The long tracts of RS dipeptides are indicated in grey, whereas RS dipeptides outside of these tracts are indicated in bold. (B) Analysis of nucleocytoplasmic shuttling of chimeric forms of hnRNP A1 by transient expression in interspecies heterokaryons. Wild-type hnRNP A1 and chimeric proteins were transiently expressed in HeLa cells, which were then fused with NIH 3T3 cells and analyzed as described in the legend to Fig. 3. Arrows indicate the mouse nuclei within human-mouse heterokaryons. (C) Diagram showing the structures of chimeric proteins in which SC35, the RS domain of SC35, or the RS domain of SF2/ASF was fused to a shuttling protein—either hnRNP A1 or SF2/ASF. The nuclear export properties of these chimeric proteins were analyzed by interspecies heterokaryon assays (panel B) and by transcriptional inhibition assays (data not shown), and the results are summarized. Act D, actinomycin D.
FIG. 4.
FIG. 4.
NRS in the RS domain of SC35. (A) Sequences of the SF2/ASF and SC35 RS domains. The long tracts of RS dipeptides are indicated in grey, whereas RS dipeptides outside of these tracts are indicated in bold. (B) Analysis of nucleocytoplasmic shuttling of chimeric forms of hnRNP A1 by transient expression in interspecies heterokaryons. Wild-type hnRNP A1 and chimeric proteins were transiently expressed in HeLa cells, which were then fused with NIH 3T3 cells and analyzed as described in the legend to Fig. 3. Arrows indicate the mouse nuclei within human-mouse heterokaryons. (C) Diagram showing the structures of chimeric proteins in which SC35, the RS domain of SC35, or the RS domain of SF2/ASF was fused to a shuttling protein—either hnRNP A1 or SF2/ASF. The nuclear export properties of these chimeric proteins were analyzed by interspecies heterokaryon assays (panel B) and by transcriptional inhibition assays (data not shown), and the results are summarized. Act D, actinomycin D.
FIG. 5.
FIG. 5.
C-terminal deletions in the RS domain of SC35 relieve nuclear retention. (A) Sequences of the SC35 RS domain and of two C-terminal deletions. The long tracts of RS dipeptides are indicated in grey, whereas RS dipeptides outside of these tracts are indicated in bold. (B) Analysis of nucleocytoplasmic shuttling of SC35 deletion mutant proteins by transient expression in interspecies heterokaryons. Wild-type SC35 and deletion proteins were transiently expressed in HeLa cells, which were then fused with NIH 3T3 cells and analyzed as described in the legend to Fig. 3. Arrows indicate the mouse nuclei within human-mouse heterokaryons. (C) Sequence of the NRS present in the RS domain of SC35.
FIG. 5.
FIG. 5.
C-terminal deletions in the RS domain of SC35 relieve nuclear retention. (A) Sequences of the SC35 RS domain and of two C-terminal deletions. The long tracts of RS dipeptides are indicated in grey, whereas RS dipeptides outside of these tracts are indicated in bold. (B) Analysis of nucleocytoplasmic shuttling of SC35 deletion mutant proteins by transient expression in interspecies heterokaryons. Wild-type SC35 and deletion proteins were transiently expressed in HeLa cells, which were then fused with NIH 3T3 cells and analyzed as described in the legend to Fig. 3. Arrows indicate the mouse nuclei within human-mouse heterokaryons. (C) Sequence of the NRS present in the RS domain of SC35.
FIG. 5.
FIG. 5.
C-terminal deletions in the RS domain of SC35 relieve nuclear retention. (A) Sequences of the SC35 RS domain and of two C-terminal deletions. The long tracts of RS dipeptides are indicated in grey, whereas RS dipeptides outside of these tracts are indicated in bold. (B) Analysis of nucleocytoplasmic shuttling of SC35 deletion mutant proteins by transient expression in interspecies heterokaryons. Wild-type SC35 and deletion proteins were transiently expressed in HeLa cells, which were then fused with NIH 3T3 cells and analyzed as described in the legend to Fig. 3. Arrows indicate the mouse nuclei within human-mouse heterokaryons. (C) Sequence of the NRS present in the RS domain of SC35.
FIG. 6.
FIG. 6.
The NRS sequence identified in the RS domain of SC35 is transferable. (A) Diagram showing the structure of a chimeric protein in which the NRS identified in the RS domain of SC35 was fused to SF2/ASF. (B) Analysis of nucleocytoplasmic shuttling by transient expression in interspecies heterokaryons. Wild-type SF2/ASF and the chimeric protein SF2/NRS-SC35 were transiently expressed in HeLa cells, which were then fused with NIH 3T3 cells and analyzed as described in the legend to Fig. 3. Arrows indicate the mouse nuclei within human-mouse heterokaryons.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Allemand, E., R. Gattoni, H. M. Bourbon, J. Stévenin, J. F. Cáceres, J. Soret, and J. Tazi. 2001. Distinctive features of Drosophila alternative splicing factor RS domain: implication for specific phosphorylation, shuttling, and splicing activation. Mol. Cell. Biol. 21:1345-1359. - PMC - PubMed
    1. Birney, E., S. Kumar, and A. R. Krainer. 1993. Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res. 21:5803-5816. - PMC - PubMed
    1. Blencowe, B. J. 2000. Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem. Sci. 25:106-110. - PubMed
    1. Cáceres, J. F., and A. R. Krainer. 1993. Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains. EMBO J. 12:4715-4726. - PMC - PubMed
    1. Cáceres, J. F., and A. R. Krainer. 1997. Mammalian pre-mRNA splicing factors, p. 174-212. In A. R. Krainer (ed.), Eukaryotic mRNA processing. IRL Press, Oxford, United Kingdom.

Publication types

MeSH terms