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Genes to Cells (2004) 9, 959-965. doi:10.1111/j.1365-2443.2004.00774.x
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SR proteins preferentially associate with mRNAs in the nucleus and facilitate their export to the cytoplasm

Kaoru Masuyama, Ichiro Taniguchi, Naoyuki Kataoka and Mutsuhito Ohno*

Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, and CREST, JST (Japan Science and Technology Agency), Kawaguchi, Saitama 332-0012, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Different classes of RNA are exported to the cytoplasm by distinct mechanisms. Each class of RNA forms distinct complexes with nuclear proteins prior to its export to the cytoplasm. In our attempt to obtain comprehensive information of protein factors that specifically associate with mRNAs in the nucleus, we performed in vivo UV-crosslinking analysis after microinjection of various RNAs into Xenopus oocyte nucleus. We found a group of proteins preferentially crosslinked to mRNAs. Immunoprecipitation experiments revealed that some of the crosslinked signals corresponded to SR (serine/arginine-rich) proteins, a family of essential RNA-binding proteins involved in pre-mRNA splicing. It was previously suggested that some members of SR protein family are involved in export of a specific intronless mRNA, histone H2A mRNA and some spliced mRNAs. However, it is still to be clarified if SR proteins are involved in export of general mRNAs, especially general intronless mRNAs that do not contain specific RNA export elements. When we microinjected an antibody against SR proteins into the nucleus, export of mRNAs was severely inhibited, regardless of whether the mRNAs were produced via pre-mRNA splicing or not, whereas export of other RNAs was not affected. These results unequivocally showed that SR proteins are involved in export of both general intronless and spliced mRNAs.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Separation of the eukaryotic cell into two major compartments the nucleus and the cytoplasm by the nuclear envelope benefits in organizing and maintaining its massive genomic DNA content but in turn demands transport of biomolecules between the two compartments. Proteins are made in the cytoplasm and many of them must be imported into the nucleus through the nuclear pore complexes (NPCs) that form aqueous channels spanning the nuclear envelope (Macara 2001; Lei & Silver 2002; Suntharalingam & Wente 2003). By contrast the majority of RNA molecules is exported to the cytoplasm through NPCs after their synthesis in the nucleus. There are many different RNA species that are exported to the cytoplasm including rRNAs, tRNAs, U snRNAs and mRNAs. It has been shown that different RNA species are exported by distinct mechanisms (Jarmolowski et al. 1994; Komeili & O'Shea 2001; Cullen 2003). Among them export of U snRNAs is one of the best characterized examples.

Major spliceosomal U snRNAs such as U1, U2, U4, and U5 are initially exported from the nucleus in metazoan (Mattaj 1988; Will & Lührmann 2001). U snRNA export is mediated by CRM1, a member of the importin ß transport receptor family (Fornerod et al. 1997; Fukada et al. 1997; Stade et al. 1997). CRM1 is also known as the export receptor for proteins carrying a leucine-rich nuclear export signal or NES (Fischer et al. 1995; Wen et al. 1995; Ossareh-Nazari et al. 1997). CRM1 binds directly to NES but indirectly to U snRNAs. Two adaptor proteins bridge interaction between CRM1 and U snRNAs. One is the heterodimeric Cap Binding Complex or CBC, which binds specifically to the essential export signal of U snRNAs, the 7-methyl Guanosine cap structure (Izaurralde et al. 1995). The other adaptor is PHAX that bridges interaction between CRM1 and the CBC/RNA complex (Ohno et al. 2000). PHAX has a leucine-rich NES to which CRM1 binds cooperatively with RanGTP. Thus these five proteins and a U snRNA assemble into the export complex in the nucleus and this complex subsequently transits to the cytoplasm (Ohno et al. 2000).

In contrast to U snRNA, cap structure is not essential for mRNA export (Hamm & Mattaj 1990; Jarmolowski et al. 1994). The major export receptor for mRNAs is a protein called TAP or NXF1 (and its related proteins) in vertebrates and Mex67p in yeast (Segref et al. 1997; Grüter et al. 1998; Katahira et al. 1999). NXF1 is one of the few non-importin ß family transport receptors known to date. Several RNA-binding proteins are also implicated in mRNA export (Dreyfuss et al. 2002; Stutz & Izaurralde 2003; Dimaano & Ullman 2004) and at least one of them, REF/Aly (Yral in yeast) mediates interaction of NXF1 with mRNAs (Strässer & Hurt 2000; Stutz et al. 2000; Zhou et al. 2000; Rodrigues et al. 2001). REF proteins however, were shown to be not essential in mRNA export in Drosophila and C. elegance suggesting functional redundancy or complexity of the adaptor proteins in those organisms (Gatfield & Izaurralde 2002; Longman et al. 2003).

SR proteins are a family of abundant non-snRNP splicing factors that have been implicated in pre-mRNA splicing (Graveley 2000; Hastings & Krainer 2001; Cáceres & Kornblihtt 2002). However, since some SR proteins can shuttle between the nucleus and cytoplasm, a role of SR proteins in mRNA transport has been suggested in addition to their well-documented role in splicing (Cáceres et al. 1998). Subsequently, two members of the SR protein family, SRp20 and 9G8 were shown to promote export of a specific intronless histone H2A mRNA (Huang & Carmichael 1997; Huang & Steitz 2001). These two SR proteins were shown to bind to a specific RNA sequence in histone H2A mRNA termed intronless transport element and to recruit NXF1 to the mRNA (Huang et al. 2003). Huang et al. (2003) also suggested that these two SR proteins have more general role in mRNA export. Microinjection of a N-terminal fragment of 9G8 protein that could sequester NXF1 inhibited export of the intronless histone mRNA and spliced AdML mRNA. However, it is possible that the N-terminal fragment of 9G8 inhibits mRNA export simply because it sequesters NXF1. It is still to be clarified if 9G8 and other SR proteins have a direct role in mRNA export. Furthermore, it needs to be investigated if SR proteins have a role in export of general intronless mRNAs that do not possess specific RNA export elements.

In the course of attempts to identify proteins that associate with export-competent RNAs in the nucleus, we found that SR proteins preferentially associated with mRNA in Xenopus oocyte nucleus. An antibody against SR proteins inhibited export of both general intronless and spliced mRNAs in Xenopus oocytes, suggesting strongly an important general role of SR proteins in mRNA export in vertebrates.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
UV-crosslinking analysis in Xenopus oocyte nucleus

Although many protein factors that may associate with U snRNAs and/or mRNAs prior to and during their export to the cytoplasm have already been identified, comprehensive understanding of proteomic information of export RNPs is still far from being accomplished. Towards this goal, we developed an in vivo UV-crosslinking assay. In brief 32P-labelled RNAs were microinjected into the nuclei of Xenopus oocytes and the injected nuclei were manually isolated at 60 min post injection. The isolated nuclei were immediately irradiated with UV-light of 254 nm that should induce covalent protein-RNA crosslinking. The irradiated nuclear sample was subsequently treated with RNase to destroy RNA moieties. The crosslinked 32P-labelled proteins were fractionated by SDS-PAGE and visualized by autoradiography.

When this assay was performed with U1{Delta}Sm snRNA and DHFR mRNA, several bands of crosslinked proteins were visible (Fig. 1A, lanes 1 and 3). A band of approximately 55 kDa (p55) was preferentially seen with U1 RNA whereas bands ranging from 43 kDa to 33 kDa (p43~p33) were much stronger with DHFR mRNA than with U1. In addition, a 26 kDa band (p26) was detected exclusively with mRNA in this condition. A faint 21 kDa band (p21) was reproducibly seen with both RNAs. We also employed another RNA, U1-ftz RNA (Fig. 1A, lane 2). This RNA is a derivative of U1 snRNA in which a 300nt sequence from fushitarazu (ftz) cDNA was inserted into U1 snRNA (Ohno et al. 2002). This elongated U1 snRNA was shown to utilize the mRNA export mechanism instead of the U snRNA export mechanism (Ohno et al. 2002), thereby serving good control to the experiment. The crosslinking pattern of U1-ftz RNA was indistinguishable to that of DHFR mRNA (compare lanes 2 and 3) suggesting that the crosslinking pattern of these two RNAs is applicable to general mRNAs.



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  		Figure 1  UV-crosslinking analysis in Xenopus oocyte nuclei. (A) 32P-labelled U1{Delta}Sm RNA (U1), U1-ftz RNA (U1-ftz), or DHFR mRNA was microinjected into Xenopus oocyte nuclei. After 1 h, the injected nuclei were isolated and irradiated with UV light. The irradiated samples were subsequently treated with RNases and the UV-crosslinked proteins were fractionated by 12% SDS-PAGE followed by autoradiography. The same cpm of each sample was loaded on to the corresponding lane. The positions of molecular size markers are shown on the left, and the tentative designations of the crosslinked proteins on the right. (B) The protein sample that had been crosslinked to U1-ftz RNA was subject to immunoprecipitation (IP) with either anti-SR (SR), anti-SRp20 (SRp20), anti-PHAX (PHAX), or Control (Cont.) antibody, and the precipitated proteins were analysed as in A. Lane 1 was loaded with 12.5% of the IP input. Lanes 6 and 7 are longer exposure of lanes 3 and 5, respectively.light. The irradiated samples were subsequently treated with RNases and the UV-crosslinked proteins were fractionated by 12% SDS-PAGE followed by autoradiography. The same cpm of each sample was loaded on to the corresponding lane. The positions of molecular size markers are shown on the left, and the tentative designations of the crosslinked proteins on the right. (B) The protein sample that had been crosslinked to U1-ftz RNA was subject to immunoprecipitation (IP) with either anti-SR (SR), anti-SRp20 (SRp20), anti-PHAX (PHAX), or Control (Cont.) antibody, and the precipitated proteins were analysed as in A. Lane 1 was loaded with 12.5% of the IP input. Lanes 6 and 7 are longer exposure of lanes 3 and 5, respectively.

 
Association of SR proteins with mRNAs in the nucleus

To elucidate the identity of the crosslinked products, we examined whether the crosslinked bands could be immunoprecipitated with antibodies against various known RNA-binding proteins, including different hnRNP proteins, SR proteins, etc. We will focus on some of the antibodies in the present study and the detail of the entire analysis will be described elsewhere.

First we utilized two antibodies against SR proteins. The first antibody was anti-SR monoclonal antibody that was originally raised and characterized in Mark Roth's lab (Tuma et al. 1993). This antibody was reported to recognize many if not all SR proteins (Neugebauer & Roth 1997). The second antibody was well-characterized anti-SRp20 antibody that recognizes specifically the 20 kDa SR protein (Neugebauer & Roth 1997). When the anti-SR antibody was used in immunoprecipitation experiments with the crosslinked proteins with U1-ftz, p43~p33 and p26 were precipitated (Fig. 1B, lane 2). By contrast when anti-SRp20 antibody was used, only p26 was precipitated (Fig. 1B, lanes 3, and 6 for longer exposure). Anti-PHAX antibody (Ohno et al. 2000) gave no clear specific precipitates (Fig. 1B, lane 4). These results indicated that p43~p33 and p26 were Xenopus SR proteins and p26 was Xenopus SRp20, although Xenopus SR proteins have not been well characterized so far. The 43-33 kDa bands that were crosslinked relatively weakly to U1 snRNA (Fig. 1A, lane 1) were also precipitated with anti-SR antibody (data not shown) indicating that they were also Xenopus SR proteins.

To confirm that SR proteins are associated with export-competent mRNAs in the nucleus, we examined if mRNAs could be preferentially immunoprecipitated with the anti-SR antibody from the nuclear fraction. A mixture of 32P-labelled RNAs containing three different mRNAs (DHFR mRNA, U1-ftz RNA, ß-globin mRNA) and two U snRNAs (U1 and U5 snRNAs) was microinjected into the nucleus of Xenopus oocytes. After 60 min, the nuclear fraction was prepared and immunoprecipitation with anti-SR antibody was performed (Fig. 2A,B for quantification). All three mRNAs were efficiently precipitated with anti-SR antibody in response to the different amounts of the antibody (Fig. 2A, lanes 2, 3), whereas two U snRNAs were precipitated much less efficiently, confirming the conclusion that SR proteins are preferentially associated with export-competent mRNAs in Xenopus oocyte nucleus.



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  		Figure 2  Preferential association of SR proteins with mRNAs in the nucleus. (A) A mixture of 32P-labelled DHFR mRNA, U1-ftz RNA (U1-ftz), ß-globin mRNA, U1{Delta}Sm (U1) and U5{Delta}Sm (U5) snRNAs was injected into Xenopus oocyte nuclei. After 1 h, the nuclear fraction of the injected oocytes was subject to immunoprecipitation (IP) with either low dose (low; 1 µg/IP reaction) or high dose (high; 4 µg/IP reaction) of anti-SR antibody (SR), or control antibody (Cont.). RNA was recovered from the precipitated material and analysed by 8% denaturing PAGE. Lane 1 was loaded with 10% of the IP input. (B) Quantification of A.

 
Anti-SR antibody inhibits mRNA export

Since SR proteins are preferentially associated with mRNAs in the nucleus, we were prompted to examine whether SR proteins are functionally important for mRNA export. For this purpose, a mixture of 32P-labelled RNAs containing DHFR mRNA, ftz mRNA, U1{Delta}Sm, U6{Delta}ss RNAs and tRNAPhe (Fig. 3A) was microinjected into the nuclei of Xenopus oocytes. Immediately after nuclear injection, all the RNAs were nuclear (Fig. 3A, lanes 1, 2). After 1.5 h, all the RNAs had been exported partially to the cytoplasm except for nonexported U6 RNA control (lanes 3, 4). To determine if SR proteins are important for mRNA export, the anti-SR antibody was co-injected with the RNA mixture (Fig. 3A, lanes 5, 6). Co-injection of anti-SR antibody strongly inhibited export of both DHFR mRNA and ftz mRNA, whereas export of U1 snRNA and tRNA was not affected. The effect of export inhibition was dependent on the dose of the antibody (data not shown). By contrast, the control antibody had no effect on RNA export (Fig. 3A, lanes 7, 8).



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  		Figure 3  Anti-SR antibody inhibits mRNA export. (A) A mixture of 32P-labelled DHFR mRNA, ftz mRNA and ß-globin mRNAs, U1{Delta}Sm (U1) and U6{Delta}ss (U6) snRNAs, and tRNAPhe (tRNA) was injected into Xenopus oocyte nuclei either with anti-SR antibody (1 mg/mL; SR; lanes 5 and 6), or control antibody (Cont.; lanes 7 and 8), or without antibodies (buffer; lanes 1–4). RNA from nuclear (N) and cytoplasmic (C) fractions was extracted immediately (0 h) or 1.5 h (1.5 h) after injection and analysed by 8% denaturing PAGE. (B) The same as in A except that a ftz pre-mRNA (pre-ftz) was employed in place of ftz mRNA and that tRNA was omitted. The bands corresponding to the spliced product (spliced-ftz) and the lariat intron (intron) were also indicated. (C) The same as in A except that a ß-globin pre-mRNA (pre-ß) was employed in place of DHFR mRNA and that tRNA was omitted. The bands corresponding to the spliced product (spliced-ß) and the lariat intron (intron) were indicated.

 
When we employed a ftz pre-mRNA that contains a single intron, export of the spliced ftz mRNA was also severely inhibited although splicing appeared unaffected (Fig. 3B). Similar results were obtained with another pre-mRNA substrate, a ß-globin pre-mRNA, although in this case splicing appeared to be affected to some extent (Fig. 3C). These results strongly suggested that SR proteins are generally important for export of both intronless and spliced mRNAs.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we could unequivocally show that SR proteins have an important role in general mRNA export. However, it is still to be clarified whether bulk SR proteins are required for mRNA export or only specific subsets are important. It was previously reported that there are two types of SR proteins that shuttle or do not shuttle between the nucleus and cytoplasm (Cáceres et al. 1998). It is possible that only the shuttling species are important for mRNA export as was originally hypothesized (Cáceres et al. 1998). One of the shuttling members SRp20 is the best candidate since it is highly specific to mRNAs in our UV-crosslinking assay. However, microinjection of the anti-SRp20 antibody did not inhibit general mRNA export in Xenopus oocytes (data not shown). This may suggest a redundant role of SR proteins in general mRNA export.

What is the role of SR proteins in general mRNA export? It has been proposed that SR proteins serve export adaptors that bridge the interaction between mRNAs and NXF1/TAP (Huang et al. 2003). However, REF/Aly is already implicated in the role as such an adaptor protein. What then is the relationship between REF/Aly and SR proteins? As was already proposed, REF and SR proteins might be alternative redundant adaptors (Huang et al. 2003; Gilbert & Guthrie 2004; Lai & Tarn 2004; Huang et al. 2004). However, the fact that mRNA export inhibition by anti-SR antibody is close to completion (see Fig. 3) suggests more crucial roles of SR proteins in the process. REF and SR proteins might work together in a protein complex. Alternatively, these two proteins may function sequentially in the course of assembly of the mRNA export complex. In these scenarios we would propose that SR proteins might be the ones that bind mRNA more directly since SR proteins are supposed to bind to exons (Chiara et al. 1996; Cartegni et al. 2002) and are more efficiently crosslinked to mRNAs (Fig. 1). Since SR proteins are pre-mRNA splicing factors, another important question is how SR proteins coordinate pre-mRNA splicing and mRNA export processes. Solving these open questions should lead to more complete understanding of mRNA export mechanisms.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Plasmids

U1{Delta}Sm, U5{Delta}Sm, U6{Delta}ss, tRNAPhe and U1-ftz RNA constructs have been previously described (Hamm et al. 1987; Hamm & Mattaj 1989; Jarmolowski & Mattaj 1993; Arts et al. 1998 and Ohno et al. 2002, respectively).

The constructs for DHFR mRNA (Kambach & Mattaj 1992), ß-globin pre-mRNA & ß-globin mRNA (Krainer et al. 1984; Mayeda & Ohshima 1990), ftz mRNA and ftz pre-mRNA (Rio 1988) have been previously described.

In vitro transcription and RNA export analysis

In vitro transcription was performed according to the standard protocol from the manufacturer with T7 or SP6 RNA polymerase (Promega). The transcribed ß-globin pre-mRNA contains 1st exon and a part of 2nd exon. The ftz pre-mRNA contains a part of 5' exon and a portion of 3' exon as previously described (Rio 1988). RNA injection analysis in Xenopus oocytes was performed as previously described (Jarmolowski et al. 1994).

Antibodies

Anti-SR and anti-SRp20 mouse monoclonal antibodies were purchased from Zymed. Rabbit anti-PHAX antiserum was a kind gift from Dr Iain Mattaj, EMBL, Heidelberg, Germany.

In vivo UV-crosslinking analysis

32P-labelled RNAs (~3 x 109cpm/mL) were microinjected into the Xenopus oocyte nuclei and the injected nuclei were manually isolated in RSB100 buffer (10 mM Tris [pH 7.4], 100 mM NaCl, 2.5 mM MgCl2) containing 10% glycerol (RSB100G) at 60 min post-injection. The isolated nuclei in cold RSB100G buffer were immediately irradiated with 254 nm UV-light (FUNA-UV-LINKER FS-800; Funakoshi, Tokyo, Japan) at a distance of ~5 cm for 10 min. The irradiated nuclear sample was treated with RNase A (0.5 mg/mL; Nacalai Tesque) and RNase T1 (3000 U/mL; Roche Biochemicals) at 37 °C for 15 min and the crosslinked proteins were analysed by SDS-PAGE.

Immunoprecipitation of UV-crosslinked proteins

Different antibodies were individually bound to Protein A-Sepharose beads (Amersham Biosciences) on a rotating platform at 4 °C for 1 h. In case of monoclonal antibodies, rabbit anti-mouse IgG antibody was also used as a bridging antibody. The crosslinked protein sample was incubated with the antibody-bound beads in RSB100 buffer containing 0.1% NP-40 (RSB100N) supplemented with complete protease inhibitor cocktail (Roche) at 4 °C for 1 h. The beads were subsequently washed four times with RSB100N buffer and once with RSB100 buffer, and the bound material was eluted by boiling the beads in the SDS-PAGE sample buffer. The eluted proteins were analysed by SDS-PAGE.

Immunoprecipitation of injected RNAs

After RNA injection into Xenopus oocyte nuclei, injected nuclei were isolated and resuspend in RSB100N buffer supplemented with complete protease inhibitor cocktail and 1000 U/mL RNasin (Promega). The mixture was centrifuged at 17 800 g for 15 min at 4 °C in a microfuge, and the resultant supernatant was incubated with the antibody-bound beads at 4 °C for 1 h. After washing five times with RSB100N buffer, the beads were incubated in HomoMix (50 mM Tris [pH 7.4], 5 mM EDTA, 1.5% SDS, 300 mM NaCl, 1.5 mg/mL Proteinase K) for 30 min at 50 °C to elute RNA in the sup. RNA was recovered from the sup by Phenol/Chloroform extraction and ethanol precipitation. The recovered RNA was analysed by PAGE containing 7 M urea.


   Acknowledgements
 
We thank Dr Iain Mattaj for generously providing anti-PHAX antibody and plasmids. We also thank members of our laboratory for suggestions and discussion throughout the course of this work and criticisms on this manuscript. This work was supported by CREST, JST and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. K.M. and I. T. were supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology.


   Footnotes
 
Communicated by: Yoshikazu Nakamura

K. Masuyama and I. Taniguchi contributed equally to this work.

* Correspondence: E-mail: hitoohno{at}virus.kyoto-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 21 April 2004
Accepted: 7 July 2004




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