| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Howard Hughes Medical Institute and Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148, USA
2 Protein Interaction Laboratory, University of Southern Denmark, DK-5230 Odense M, Denmark
Reprint requests to: Gideon Dreyfuss, Howard Hughes Medical Institute and Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148, USA; e-mail: gdreyfuss{at}hhmi.upenn.edu; fax: (215) 573-2000.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Keywords: eIF4A3; exon junction complex; RNA helicase; splicing; translation
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
; Dreyfuss et al. 1993). Pre-mRNA processing in the nucleus includes addition of a cap structure, splicing, and polyadenylation, and is accompanied by changes in the composition and arrangements of proteins associated with the mRNA (Dreyfuss et al. 2002). Recently, pre-mRNA splicing was shown to be accompanied by a profound change in the protein composition of the mRNA (Luo and Reed 1999; Kataoka et al. 2000; Le Hir et al. 2000b). In particular, mRNAs generated by splicing acquire specific protein complexes that assemble 20 to 24 nt upstream of spliced junctions, termed the exon junction complex (EJC; Kataoka et al. 2000, 2001; Le Hir et al. 2000a, 2001; Zhou et al. 2000; Kim et al. 2001a; Luo et al. 2001).
The known proteins of the EJC, SRm160, RNPS1, UAP56, Aly/REF, Upf3, Y14, and magoh, function at many levels during mRNA metabolism. SRm160 and RNPS1 are activators of pre-mRNA splicing (Blencowe et al. 1998; Mayeda et al. 1999), whereas Aly/REF and UAP56 were shown to function in the nuclear export of mRNAs (Zhou et al. 2000; Gatfield et al. 2001). RNPS1 (Lykke-Andersen et al. 2001), hUpf3 (Lykke-Andersen et al. 2000), and Y14 (Gehring et al. 2003) were shown to play a role in nonsense-mediated decay (NMD) of mRNAs, a process that leads to degradation of mRNAs containing premature termination codons. Y14 along with its associated protein magoh were also shown to be important for oskar mRNA localization in Drosophila during oogenesis (Newmark and Boswell 1994; Hachet and Ephrussi 2001; Mohr et al. 2001). In addition, the EJC may be important for translation efficiency. The observation that the presence of an intron can enhance translation efficiency of some mRNAs (Matsumoto et al. 1998; Nott et al. 2003; Wiegand et al. 2003) and the finding that most EJC proteins bind spliced but not intronless mRNAs (Dreyfuss et al. 2002) suggests that the EJC may be involved in increasing translation efficiency of spliced mRNAs. Thus, the fate of processed mRNAs is partly influenced by the acquisition of the EJC. In addition to providing information about the overall structure of the gene from which the mRNA is produced, EJC proteins could determine the path through which mRNAs are processed from their precursors and possibly provide additional signals (Dreyfuss et al. 2002).
Among the components of the EJC, magoh and Y14 are of considerable interest because they persist on mRNAs after export from the nucleus to the cytoplasm, where they are removed by the translation machinery (Dostie and Dreyfuss 2002). Therefore, the identification of proteins that associate with Y14 and magoh or the complexes that contain them is of particular importance in studying the function of the EJC in postsplicing events. Here, we identify eIF4A3 as a novel component of the EJC. We show that eIF4A3, a member of the eIF4A DEAD-box helicase family of translation initiation factors, binds spliced but not intronless mRNAs. Furthermore, eIF4A3 associates with spliced mRNAs at the position of the EJC. We suggest that eIF4A3 may provide a link between splicing and translation in the cytoplasm.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
). To allow proper incorporation of the tagged proteins without disruption of the endogenous complexes, cell lines were established and characterized under conditions where low levels of the tagged proteins were expressed. Proteins that associate with Y14- and magoh-containing complexes were identified by immunoprecipitation with anti-flag antibody (M2) from both the cytoplasmic and nucleoplasmic fractions. Proteins bound to the anti-flag antibody beads were eluted with flag peptides, resolved by SDS-PAGE, and detected by silver staining. Proteins that associated with magoh- or Y14-containing complexes but not with hnRNP C1 complexes were isolated from the gel and identified by nanoelectrospray mass spectrometry. Two peptide sequences were identified for the 47kD protein band (Fig. 1). The first peptide sequence, GIYAYGFEKPSAIQQR, is found in eukaryotic initiation factors eIF4A1, eIF4A2, and eIF4A3, whereas the second peptide sequence, LDYGWHVV AGTPGR, is found only in eIF4A3 (Fig. 2). Therefore, these peptides uniquely identify eIF4A3 as part of the 47-kD protein band coimmunoprecipitated with magoh and Y14 complexes.
|
|
). Because eIF4A3 shares 67%–68% identity and is of similar size to eIF4A1 and eIF4A2 (Fig. 2
), we tested the specificity of 3F1 on recombinant eIF4A3, eIF4A2, and eIF4A1. As shown in Figure 3B, 3F1 specifically recognized eIF4A3 by Western blotting. In addition, 3F1 specifically immunoprecipitated in vitro translated eIF4A3 but not in vitro translated eIF4A1 or eIF4A2 (Fig. 3C). Immunofluorescence microscopy on HeLa cells using 3F1 indicates that eIF4A3 primarly localizes to the nucleoplasm (Fig. 4). A similar localization pattern was observed for transiently transfected myc-eIF4A3. These results are in agreement with a previous study showing that eIF4A3 is a nuclear protein (Holzmann et al. 2000). In contrast, transiently transfected myc-eIF4A2 and myc-eIF4A1 localized to the cytoplasm. Although eIF4A3 predominantly localized to the nucleus by immunofluorescence, significant amounts were found in the cytoplasm by cell fractionation (Fig. 5, bottom panel).
|
|
|
shows that eIF4A3 efficiently coimmunoprecipitates with magoh and Y14 from the nucleoplasm but not with hnRNP C1. The preferential association of eIF4A3 with nuclear magoh and Y14 was not due to the presence of higher levels of the flag-tagged proteins in the nucleus, because higher amounts of flag-magoh and flag-Y14 were immunoprecipitated from the cytoplasmic fraction as compared to the nucleoplasmic fraction (Fig. 5). Also, the preferential coimmunoprecipitation of eIF4A3 with nuclear magoh and Y14 was not due to higher nuclear levels of eIF4A3 because considerable amounts of eIF4A3 were also found in the cytoplasm (Fig. 5, cytoplasmic input lane). Thus, these results demonstrate that eIF4A3 associates in vivo with magoh- and Y14-containing complexes and suggest that this association takes place predominantly in the nucleus. Because eIF4A3 is highly similar in sequence to eIF4A1 and eIF4A2, it was possible that these proteins might also be associated with Y14 complexes. To determine whether eIF4A1 and eIF4A2 are associated with Y14-containing complexes, myc-eIF4A1, myc-eIF4A2, and myc-eIF4A3 were transiently transfected into either flag-Y14 or flag-hnRNP C1 cell lines. Flag-Y14 and flag-hnRNP C1 were immunoprecipitated as described above and analyzed by Western blotting with anti-myc and anti-flag antibodies. In agreement with the results presented for the endogenous protein, myc-eIF4A3 was coimmunoprecipitated with nuclear flag-Y14 but not with flag-hnRNP C1. In contrast, myc-eIF4A2 was not coimmunoprecipitated with nuclear or cytoplasmic flag-Y14, whereas similar to myc-eIF4A3, low levels of myc-eIF4A1 coimmunoprecipitated with cytoplasmic flag-Y14 (data not shown). Together, these results demonstrate that eIF4A3 specifically associates with Y14-containing complexes in the nucleus and suggest that this interaction persists into the cytoplasm.
eIF4A3 binds mRNAs produced by splicing but not intronless mRNA
Y14 and magoh were previously shown to be components of postsplicing, pretransport mRNPs and to preferentially associate with spliced mRNAs (Kataoka et al. 2000, 2001; Le Hir et al. 2000a). Because eIF4A3 associates with nuclear magoh- and Y14-containing complexes (Fig. 5), we asked whether eIF4A3 might also predominantly bind spliced mRNAs. To test this, we examined the binding of eIF4A3 to splicing products of a previously characterized pre-mRNA derived from the chicken 
-crystallin (CDC1) gene (Ohno et al. 1987). Splicing reactions were immunoprecipitated with antibodies against hnRNP C (4F4), Y14 (4C4), eIF4A3 (3F1), or with the nonimmune antibody SP2/0 as control, and associated RNAs were analyzed on a polyacrylamide denaturing gel. Similar to Y14, eIF4A3 preferentially associated with spliced but not intronless mRNAs, whereas pre-mRNAs were predominantly coimmunoprecipitated with hnRNP C1 (Fig. 6). The association of eIF4A3 with spliced mRNAs is a general characteristic of this protein and not unique to the CDC RNA, as similar results were obtained with a splicing substrate derived from the adenovirus major late promotor (Ad2; data not shown; Pellizzoni et al. 1998). These results demonstrate that eIF4A3 associates with spliced mRNAs.
|
). Because of this high degree of sequence similarity, we asked whether eIF4A1 and eIF4A2 could also bind spliced mRNAs. To answer this question, splicing reactions made from total extracts of HeLa cells transiently transfected with flag-eIF4A1, flag-eIF4A2, or flag-eIF4A3 were immunoprecipitated with anti-flag antibodies and processed as described above (Fig. 6). In this assay, flag-eIF4A3 was predominantly bound to spliced mRNAs (Fig. 7, lane 4) but flag-eIF4A2 and flag-eIF4A1 were not (Fig. 7A, lanes 8,12). Because the amount of flag-eIF4A3, flag-eIF4A2, and flag-eIF4A1 proteins in each splicing extract was similar (Fig. 7B), the difference in binding to spliced mRNAs reflects an intrinsic characteristic of eIF4A3. Thus, eIF4A3 is the only eIF4A family member that has the capacity to bind spliced mRNAs in an in vitro splicing assay.
|
; Kataoka et al. 2001; Kim et al. 2001b). In this assay, in vitro splicing reactions using Ad2 pre-mRNA were incubated with recombinant RNase H and DNA oligonucleotides, which are complementary to mRNA sequences flanking the EJC site (A10 and A5 on Fig. 8). The reactions were then used in immunoprecipitation experiments with the indicated antibodies (Fig. 8A). As illustrated in Figure 8B, several fragments were generated by the sequence-specific digestion of the Ad2 mRNAs with the oligonucleotides. The shortest fragment (F2) contains the 20-nt region upstream of the exon–exon junction and corresponds to the region where the EJC proteins are bound. Figure 8A shows that similar to Y14, eIF4A3 associates with the F2 fragment from the in vitro splicing reaction. Anti-cap antibody (H20), used as a control, did not immunoprecipitate the F2 fragment. Similar results were obtained from a mapping experiment performed with the CDC pre-mRNA (data not shown). These findings indicate that eIF4A3, like Y14, magoh, and other EJC components, binds at the position of the exon junction complex and is therefore a bona fide EJC protein.
|
, eIF4A3 efficiently associates with the mRNA export factor TAP and with Aly/REF. In this assay, GST-eIF4A3 did not significantly bind to magoh or Y14 (binding less than 2%), suggesting that eIF4A3, as a component of the EJC, associates with Y14 and magoh indirectly through its interaction with TAP and Aly/REF. The ability of GST-eIF4A3 to bind efficiently to in vitro translated TAP and Aly/REF was not shared by GST-eIF4A1 (Fig. 9).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
). However, nothing is known about the function of eIF4A3 as part of the EJC. eIF4A3 was recently reported to be present in the B and C spliceosomal complexes whereas two of the EJC proteins, Y14 and magoh, were only found in the latter (Jurica et al. 2002; Jurica and Moore 2003). Thus, it is possible that in the nucleus, eIF4A3 functions to promote unwinding of secondary structures within pre-mRNA substrates to stimulate splicing and/or facilitate assembly of EJCs on spliced mRNAs.
The observation that eIF4A3 efficiently interacts with TAP and Aly/REF in vitro (Fig. 9) suggests that eIF4A3 might play a role in nuclear mRNA export. TAP, Aly/REF, and UAP56 were previously shown to be important for mRNA export (Zhou et al. 2000; Gatfield et al. 2001). The nucleocytoplasmic transport of mRNAs through nuclear pore complexes (NPCs) is an active, energy-dependent process during which considerable mRNP remodeling, including the removal of several hnRNP and EJC proteins, is thought to occur. Because eIF4A3 is an ATP-dependent RNA helicase, it might contribute to the remodeling and translocation of mRNPs during transit through NPCs. Another DEAD-box RNA helicase, Dbp5, has been implicated in similar processes (Snay-Hodge et al. 1998; Zhao et al. 2002).
Although the step at which eIF4A3 is displaced from the mRNP is unknown, it is possible that it persists on mRNAs into the cytoplasm, as low levels of eIF4A3 were found to associate with Y14-containing complexes in the cytoplasm (Fig. 5). Considering its reported activity as a translation inhibitor (Li et al. 1999), eIF4A3 might inhibit premature or aberrant translation in both the nucleus and the cytoplasm. eIF4A3 was shown to interact with the translation initiation factor eIF4G, although more weakly than the positive effectors of translation, eIF4A1 and eIF4A2. Thus, in addition to directly inhibiting translation initiation along the length of mRNAs, eIF4A3 may also indirectly stimulate translation in the cytoplasm by increasing the local concentration of eIF4G and other initiation factors onto spliced mRNPs. In this model, eIF4A3 would remain associated with mRNPs until it is removed by the ribosome during the first round of translation. This concept also suggests a role for eIF4A3 in NMD. Alternatively, eIF4A3 might be replaced by eIF4A1 or eIF4A2 on the mRNP to stimulate the first round of translation. The discovery of eIF4A3 as a component of EJCs will provide further insight into the role of the EJC in influencing postsplicing processes including translation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Generation of stable cell lines
HeLa Tet-On cells (Clontech) that constitutively express the tetracycline transactivator were cotransfected by the calcium phosphate method with the pTRE plasmids encoding either flag-hnRNP C1, flag-magoh, or flag-Y14 and with a pTK-Hyg plasmid carrying the hygromycin resistance gene for selection. Stable clones were obtained by double selection in the presence of G418 (100 µg/mL) and hygromycin (200 µg/mL). Individual clones were isolated and analyzed by Western blotting for the expression of flag-hnRNP C1, flag-magoh, or flag-Y14 using the anti-flag antibody M2 (Sigma) after induction with doxycycline (5 µg/mL) for 24 h. Stable HeLa Tet-On (parental cell), flag-hnRNPCl, flag-magoh, and flag-Y14 cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (complete DMEM), G418 (100 µg/mL), and hygromycin (200 µg/mL). HeLa Tet-On cells (control) were cultured in complete DMEM in the presence of G418 (100 µg/mL).
Affinity purification of flag-hnRNP C1, flag-magoh, and flag-Y14 complexes
Flag-hnRNP C1, flag-magoh, flag-Y14, and HeLa Tet-On cells (control) were grown as described above in the presence of doxycycline (1.25 µg/mL) for 24 h. Subcellular fractionation of the induced cell lines was performed as previously described (Siomi et al. 1997). Briefly, cytoplasmic extracts were prepared by resuspending cells in RSB 150 (10 mM Tris-HCl at pH 7.4, 2.5 mM MgCl2, 150 mM NaCl) containing 0.05% digitonin, protease inhibitors (Complete; Roche) and 1 mM DTT. Cells were disrupted by passing through a 27 gauge needle five times, and centrifuged at 3000g for 1 min. The supernatant was clarified by centrifugation at 27,000g for 10 min and filtered through a 0.45 µm filter. The remaining pellets (after the 3000g spin), enriched with nuclei, were resuspended in RSB 150 containing 0.5% Triton X-100, protease inhibitors (Complete; Roche) and 1 mM DTT. The nuclear fraction was briefly sonicated on ice, centrifuged 27,000g for 10 min, and filtered through a 0.45 µm filter. Cytoplasmic and nucleoplasmic extracts were incubated with anti-flag antibody beads for 1 h at 4°C for binding. After binding, supernatants were discarded, and beads were washed five times with lysis buffer containing 0.5% Triton X-100, protease inhibitors (Complete; Roche) and 1 mM DTT. Bound complexes were eluted from the anti-flag beads with 5–10 bed volumes of RBS 100 (10 mM Tris-HCl at pH 7.4, 2.5 mM MgCl2, 100 mM NaCl) containing 300 ng/µL 3X-flag peptide (Sigma) for 1 h at 4°C.
Antibodies, Western blotting and immunofluorescence microscopy
Mouse anti-eIF4A3 monoclonal antibody 3F1 was prepared by immunizing BALB/c mice with full-length recombinant GST-eIF4A3 as previously described (Kataoka et al. 2000). Nonimmune mouse IgG, SP2/0 (Kataoka et al. 2000), and anti-Y14 antibody 4C4 (Kim et al. 2001b) were previously described. The antimonomethylguanosine cap structure antibody H20 (Bochnig et al. 1987) was a generous gift from Dr. Reinhard Lührmann (Max Planck Institute of Biophysical Chemistry, Department of Cellular Biochemistry, Göttingen, Germany). Anti-flag monoclonal antibody M2 and anti-myc monoclonal antibody 9E10 were purchased from Sigma and Santa Cruz, respectively. Peroxidase-coupled anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories. Western blotting and immunofluorescence microscopy were performed as previously described (Siomi et al. 1997).
Protein microsequencing by mass spectrometry
Protein bands were excised from a silver-stained polyacrylamide gel (Novex mini gel, Invitrogen) and in gel digested with trypsin as described previously (Shevchenko et al. 1996). Tryptic peptides in the supernatant were analyzed by nano-electrospray tandem mass spectrometry using a QSTAR (MDS Sciex) equipped with a nano-electrospray ion source (MDS Proteomics) as previously described (Wilm et al. 1996). Peptides were identified with comprehensive protein and expressed sequence tag databases using the Protein and Peptide Software Suite (MDS Proteomics). No limitation was imposed on protein molecular weight and species of origin.
In vitro splicing, RNase H digestion, and immunoprecipitation
Template preparation, in vitro transcription, and in vitro splicing reactions (with minor modification) were performed as previously described (Kataoka et al. 2001; Kataoka and Dreyfuss, 2003). Briefly, HeLa cells or HeLa cells transfected with flag-pcDNA3 (control), flag-eIF4A3, flag-eIF4A2, or flag-eIF4A1 were resuspended in Buffer D' (20 mM HEPES-KOH at pH 7.9, 100 mM KCL, 0.2 mM EDTA). The cells were disrupted by sonication on ice and centrifuged at 27,000g for 15 min. The supernatant (Total Hela cell splicing extract) was stored at -80°C until used in splicing reactions. A 40-µL splicing reaction contained 16 µL of HeLa total cell splicing extracts and 4 µL of 10x SP buffer (Pellizzoni et al. 1998). RNase H digestion with A5 and A10 oligonucleotides and immunoprecipitation were performed as previously described (Hanamura et al. 1998; Kim et al. 2001b).
In vitro protein binding experiment
All [35S]methionine-labeled proteins were produced in vitro using a TNT-coupled rabbit reticulocyte extract system (Promega) according to the manufacturer’s protocol. All proteins shown in Figure 9 were expressed from pET28a with a His tag except for RNPS1 and SRm160, which were expressed from pcDNA3 with a flag-tag. Ten microliters of the in vitro translated products were added to 5 µg of GST-fusion proteins coupled to glutathione beads in the binding buffer (10 mM Tris-HCl at pH 7.4, 2.5 mM MgCl2, 100 mM NaCl, 0.5 mM DTT, 0.1% NP40) and mixed for 1 h at 4°C. After binding, beads were washed five times with the same buffer and bound proteins eluted with SDS-PAGE sample buffer and resolved by SDS-PAGE and detected by autoradiography.
|
ACKNOWLEDGMENTS |
|---|
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
|
Footnotes |
|---|
Received November 13, 2003; accepted November 19, 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Blencowe, B.J., Issner, R., Nickerson, J.A., and Sharp, P.A. 1998. A coactivator of pre-mRNA splicing. Genes & Dev. 12: 996–1009.
Bochnig, P., Reuter, R., Bringmann, P., and Lührmann, R. 1987. A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact, class U, small nuclear ribonucleoproteins as well as with 7-methylguanosine-capped RNAs. Eur. J. Biochem. 168: 461–467.[Medline]
Choi, Y.D. and Dreyfuss, G. 1984. Isolation of the heterogeneous nuclear RNA-ribonucleoprotein complex (hnRNP): A unique supramolecular assembly. Proc. Natl. Acad. Sci. 81: 7471–7475.
Dostie, J. and Dreyfuss, G. 2002. Translation is required to remove Y14 from mRNAs in the cytoplasm. Curr. Biol. 12: 1060–1067.[CrossRef][Medline]
Dreyfuss, G., Matunis, M.J., Pinol-Roma, S., and Burd, C.G. 1993. hnRNP proteins and the biogenesis of mRNA. Annu. Rev. Biochem. 62: 289–321.[CrossRef][Medline]
Dreyfuss, G., Kim, V.N., and Kataoka, N. 2002. Messenger-RNA-binding proteins and the messages they carry. Nat. Rev. Mol. Cell. Biol. 3: 195–205.[CrossRef][Medline]
Gatfield, D., Le Hir, H., Schmitt, C., Braun, I.C., Kocher, T., Wilm, M., and Izaurralde, E. 2001. The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila. Curr. Biol. 11: 1716–1721.[CrossRef][Medline]
Gehring, N.H., Neu-Yilik, G., Schell, T., Hentze, M.W., and Kulozik, A.E. 2003. Y14 and hUpf3b form an NMD-activating complex. Mol. Cell. 11: 939–949.[CrossRef][Medline]
Hachet, O. and Ephrussi, A. 2001. Drosophila Y14 shuttles to the posterior of the oocyte and is required for oskar mRNA transport. Curr. Biol. 11: 1666–1674.[CrossRef][Medline]
Hanamura, A., Caceres, J.F., Mayeda, A., Franza Jr., B.R., and Krainer, A.R. 1998. Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors. RNA 4: 430–444.[Abstract]
Holzmann, K., Gerner, C., Poltl, A., Schafer, R., Obrist, P., Ensinger, C., Grimm, R., and Sauermann, G. 2000. A human common nuclear matrix protein homologous to eukaryotic translation initiation factor 4A. Biochem. Biophys. Res. Commun. 267: 339–344.[CrossRef][Medline]
Jurica, M.S. and Moore, M.J. 2003. Pre-mRNA splicing: Awash in a sea of proteins. Mol. Cell. 12: 5–14.[CrossRef][Medline]
Jurica, M.S., Licklider, L.J., Gygi, S.R., Grigorieff, N., and Moore, M.J. 2002. Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. RNA 8: 426–439.[Abstract]
Kataoka, N. and Dreyfuss, G. 2003. A simple whole-cell lysate system for in vitro splicing reveals a stepwise-assembly of the exon–exon junction complex. J. Biol. Chem. (in press).
Kataoka, N., Yong, J., Kim, V.N., Velazquez, F., Perkinson, R.A., Wang, F., and Dreyfuss, G. 2000. Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. Mol. Cell 6: 673–682.[CrossRef][Medline]
Kataoka, N., Diem, M.D., Kim, V.N., Yong, J., and Dreyfuss, G. 2001. Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon–exon junction complex. EMBO J. 20: 6424–6433.[CrossRef][Medline]
Kim, V.N., Kataoka, N., and Dreyfuss, G. 2001a. Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon–exon junction complex. Science 293: 1832–1836.
Kim, V.N., Yong, J., Kataoka, N., Abel, L., Diem, M.D., and Dreyfuss, G. 2001b. The Y14 protein communicates to the cytoplasm the position of exon–exon junctions. EMBO J. 20: 2062–2068.[CrossRef][Medline]
Le Hir, H., Izaurralde, E., Maquat, L.E., and Moore, M.J. 2000a. The spliceosome deposits multiple proteins 20–24 nucleotides upstream of mRNA exon–exon junctions. EMBO J. 19: 6860–6869.[CrossRef][Medline]
Le Hir, H., Moore, M.J., and Maquat, L.E. 2000b. Pre-mRNA splicing alters mRNP composition: Evidence for stable association of proteins at exon–exon junctions. Genes & Dev. 14: 1098–1108.
Le Hir, H., Gatfield, D., Braun, I.C., Forler, D., and Izaurralde, E. 2001. The protein Mago provides a link between splicing and mRNA localization. EMBO Rep. 2: 1119–1124.[CrossRef][Medline]
Li, Q., Imataka, H., Morino, S., Rogers Jr., G.W., Richter-Cook, N.J., Merrick, W.C., and Sonenberg, N. 1999. Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII. Mol. Cell. Biol. 19: 7336–7346.
Luo, M.J. and Reed, R. 1999. Splicing is required for rapid and efficient mRNA export in metazoans. Proc. Natl. Acad. Sci. 96: 14937–14942.
Luo, M.L., Zhou, Z., Magni, K., Christoforides, C., Rappsilber, J., Mann, M., and Reed, R. 2001. Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly. Nature 413: 644–647.[CrossRef][Medline]
Lykke-Andersen, J., Shu, M.D., and Steitz, J.A. 2000. Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon. Cell 103: 1121–1131.[CrossRef][Medline]
———. 2001. Communication of the position of exon–exon junctions to the mRNA surveillance machinery by the protein RNPS1. Science 293: 1836–1839.
Matsumoto, K., Wassarman, K.M., and Wolffe, A.P. 1998. Nuclear history of a pre-mRNA determines the translational activity of cytoplasmic mRNA. EMBO J. 17: 2107–2121.[CrossRef][Medline]
Mayeda, A., Badolato, J., Kobayashi, R., Zhang, M.Q., Gardiner, E.M., and Krainer, A.R. 1999. Purification and characterization of human RNPS1: A general activator of pre-mRNA splicing. EMBO J. 18: 4560–4570.[CrossRef][Medline]
Mohr, S.E., Dillon, S.T., and Boswell, R.E. 2001. The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis. Genes & Dev. 15: 2886–2899.
Newmark, P.A. and Boswell, R.E. 1994. The mago nashi locus encodes an essential product required for germ plasm assembly in Drosophila. Development 120: 1303–1313.[Abstract]
Nott, A., Meislin, S.H., and Moore, M.J. 2003. A quantitative analysis of intron effects on mammalian gene expression. RNA 9: 607–617.
Ohno, M., Sakamoto, H., and Shimura, Y. 1987. Preferential excision of the 5' proximal intron from mRNA precursors with two introns as mediated by the cap structure. Proc. Natl. Acad. Sci. 84: 5187–5191.
Pellizzoni, L., Kataoka, N., Charroux, B., and Dreyfuss, G. 1998. A novel function for SMN, the spinal muscular atrophy disease gene product, in pre-mRNA splicing. Cell 95: 615–624.[CrossRef][Medline]
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. 1996. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68: 850–858.[Medline]
Siomi, M.C., Eder, P.S., Kataoka, N., Wan, L., Liu, Q., and Dreyfuss, G. 1997. Transportin-mediated nuclear import of heterogeneous nuclear RNP proteins. J. Cell. Biol. 138: 1181–1192.
Snay-Hodge, C.A., Colot, H.V., Goldstein, A.L., and Cole, C.N. 1998. Dbp5p/Rat8p is a yeast nuclear pore-associated DEAD-box protein essential for RNA export. EMBO J. 17: 2663–2676.[CrossRef][Medline]
Wiegand, H.L., Lu, S., and Cullen, B.R. 2003. Exon junction complexes mediate the enhancing effect of splicing on mRNA expression. Proc. Natl. Acad. Sci. 100: 11327–11332.
Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., and Mann, M. 1996. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379: 466–469.[CrossRef][Medline]
Zhao, J., Jin, S.B., Bjorkroth, B., Wieslander, L., and Daneholt, B. 2002. The mRNA export factor Dbp5 is associated with Balbiani ring mRNP from gene to cytoplasm. EMBO J. 21: 1177–1187.[CrossRef][Medline]
Zhou, Z., Luo, M.J., Straesser, K., Katahira, J., Hurt, E., and Reed, R. 2000. The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans. Nature 407: 401–405.[CrossRef][Medline]
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  R. R. Almon, E. Yang, W. Lai, I. P. Androulakis, D. C. DuBois, and W. J. Jusko Circadian Variations in Rat Liver Gene Expression: Relationships to Drug Actions J. Pharmacol. Exp. Ther., September 1, 2008; 326(3): 700 - 716. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. P. Kota, S. R. Wagner, E. Huerta, J. M. Underwood, and J. A. Nickerson Binding of ATP to UAP56 is necessary for mRNA export J. Cell Sci., May 1, 2008; 121(9): 1526 - 1537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. H. Apponi, S. M. Kelly, M. T. Harreman, A. N. Lehner, A. H. Corbett, and S. R. Valentini An Interaction between Two RNA Binding Proteins, Nab2 and Pub1, Links mRNA Processing/Export and mRNA Stability Mol. Cell. Biol., September 15, 2007; 27(18): 6569 - 6579. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Isken and L. E. Maquat Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function Genes & Dev., August 1, 2007; 21(15): 1833 - 3856. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Linder Dead-box proteins: a family affair--active and passive players in RNP-remodeling Nucleic Acids Res., September 10, 2006; 34(15): 4168 - 4180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B. Kunz, G. Neu-Yilik, M. W. Hentze, A. E. Kulozik, and N. H. Gehring Functions of hUpf3a and hUpf3b in nonsense-mediated mRNA decay and translation RNA, June 1, 2006; 12(6): 1015 - 1022. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. SHIBUYA, T. O. TANGE, M. E. STROUPE, and M. J. MOORE Mutational analysis of human eIF4AIII identifies regions necessary for exon junction complex formation and nonsense-mediated mRNA decay. RNA, March 1, 2006; 12(3): 360 - 374. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Kashima, A. Yamashita, N. Izumi, N. Kataoka, R. Morishita, S. Hoshino, M. Ohno, G. Dreyfuss, and S. Ohno Binding of a novel SMG-1-Upf1-eRF1-eRF3 complex (SURF) to the exon junction complex triggers Upf1 phosphorylation and nonsense-mediated mRNA decay Genes & Dev., February 1, 2006; 20(3): 355 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Dhalia, N. Marinsek, C. R. S. Reis, R. Katz, J. R. C. Muniz, N. Standart, M. Carrington, and O. P. de Melo Neto The two eIF4A helicases in Trypanosoma brucei are functionally distinct. Nucleic Acids Res., January 1, 2006; 34(9): 2495 - 2507. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. WEIL and K. L. BEEMON A 3' UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus RNA, January 1, 2006; 12(1): 102 - 110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. O. TANGE, T. SHIBUYA, M. S. JURICA, and M. J. MOORE Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core RNA, December 1, 2005; 11(12): 1869 - 1883. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Feng, D. N. Everly Jr., and G. S. Read mRNA Decay during Herpes Simplex Virus (HSV) Infections: Protein-Protein Interactions Involving the HSV Virion Host Shutoff Protein and Translation Factors eIF4H and eIF4A J. Virol., August 1, 2005; 79(15): 9651 - 9664. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-E. Bordeleau, J. Matthews, J. M. Wojnar, L. Lindqvist, O. Novac, E. Jankowsky, N. Sonenberg, P. Northcote, P. Teesdale-Spittle, and J. Pelletier Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation PNAS, July 26, 2005; 102(30): 10460 - 10465. [Abstract] [Full Text] [PDF]
|
-hnRNP C1), SP2/0 (control), 4C4 (
