REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus

Abstract

The REF family of evolutionarily conserved heterogeneous ribonucleoprotein (hnRNP)-like proteins consists of one central RNPtype RNA binding domain flanked by Arg-Gly-rich regions of variable length. Members of this protein family bind directly to RNA and the mRNA export factor TAP͞Mex67p, and it has been suggested that they facilitate the recruitment of TAP͞Mex67p to cellular mRNPs. We show that the variable regions are necessary for binding of REFs to RNA and to TAP. Antibodies specific to REFs prevent their interaction with RNA in vitro. After microinjection into Xenopus oocytes, these antibodies inhibit mRNA nuclear export. This inhibition of export is observed whether or not the mRNAs are generated by splicing. The antibodies do not interfere with pre-mRNA splicing or with the nuclear export of constitutive transport element (CTE)-containing RNAs (directly mediated by TAP), so REF proteins must play a critical role in mRNA nuclear export, acting downstream of splicing and upstream of TAP͞ Mex67p. We also show that recombinant REFs stimulate directly the export of mRNAs that are otherwise exported inefficiently. Together, our data indicate that REFs are directly implicated in the export of mRNAs from the nucleus. More generally, we show that spliced and unspliced mRNAs use common export factors to reach the cytoplasm. Yra1p, a Saccharomyces cerevisiae member of the REF family, is an essential nuclear protein first identified from its RNAannealing activity (3). More recently, Yra1p was shown to be involved in the export of mRNA from the nucleus in yeast cells (2, 4). In mouse, REFs are encoded by at least three different genes (ref 1-3) and differ at multiple positions in the variable regions because of deletions and͞or amino acid changes (2). In contrast, all murine REF proteins are 98% identical in the RBD and 100% in the conserved boxes (2). The complexity of the murine subfamily is further increased by the expression of multiple splice variants (2). Murine REF1-II is generated by alternative splicing of REF1-I (also named Aly; see ref. 5) and lacks the N-terminal variable region (see Fig. 2 A), whereas murine REF2-I and REF2-II differ by one single amino acid insertion in REF2-I (Q198, ref. 2). REF1-I (Aly) was first identified as a protein interacting with LEF-1, a transcription factor that participates in the regulation of the T-cell receptor ␣ enhancer (5). In this context, it was proposed that Aly facilitates the interaction of multiple proteins in the T-cell receptor ␣ enhancer complex. In this study, we have defined the domains of REF interacting with RNA and TAP, and we have investigated the role of vertebrate REFs on the export of mRNAs from the nucleus. We show that the variable regions are required for binding of REFs to RNA and TAP. Antibodies specific to REFs, which prevent their binding to RNA in vitro, inhibit mRNA nuclear export when injected into Xenopus laevis oocytes. This export inhibition is observed whether or not the mRNAs have been generated by splicing. We show that microinjection ...

The spatial targeting and nuclear matrix binding domains of SRm160

“…More recently, SRm160 has been found on spliced mRNAs at sites 20-24 nt upstream from exon-exon junctions in an EJC also containing the mRNA export factors DEK, RNPS1, Y14, Aly͞ REF (16)(17)(18), and Magoh (19). Y14 and the mRNA export factor REF continuously shuttle between nucleus and the cytoplasm (40,41), whereas SRm160 is exclusively located in the nucleus (J.A.N., K. M. Wan, G. Krockmalnic, and S.W., unpublished data). The targeting of all N-terminal domains of SRm160 to the nuclear periphery suggests a possible role of the SRm160 N terminus in the subnuclear transport of spliced mRNA to specific sites near nuclear pores, where SRm160 would leave the complex.…”

The Evolutionarily Conserved Kaposi's Sarcoma-associated Herpesvirus ORF57 Protein Interacts with REF Protein and Acts as an RNA Export Factor

“…Plasmid pCMV128 containing the chloramphenicol acetyltransferase (CAT) coding region within an intron sequence was as described (57,58). Recombinant plasmids pGEXCS-REF1-II, pRSETB-REF1-II (and their mutants) expressing GST, and histidine (His) fusions, respectively, of REF1-II (40,44), pCMV-HA-Rev, and pCMV-HA-RevM10 expressing HIV-1 Rev and export deficient mutant RevM10 proteins (48) were kindly provided by Dr. E. Izaurralde. Plasmids pCMV-HARev and pCMV-HA-RevM10 (48) contain an N-terminal hemagglutinin (HA) tag for detection of Rev, RevM10, and RevM10-ORF57 fusion proteins.…”

“…TAP proteins represent a class of nuclear transport receptors that, via an essential C-terminal domain, directly interact with nucleoporin components of the NPC (49 -51). The interaction of TAP with cellular RNAs is likely mediated by adaptor proteins such as REF, through protein-protein interactions (38,40,44). However, recent studies of Drosophila melanogaster cells and Caenorhabditis elegans indicate that while REF and other exon junction complex components contribute to mRNA export (52,53), they appear to be dispensable, with additional adaptors bridging the interaction between TAP and cellular mRNAs (53).…”

“…REF recruits the export receptor TAP protein to cellular mRNPs (38,40) forming part of the exon junction complex recruited to mRNAs during splicing (37,43). Independent of splicing, REF can also associate with intronless mRNAs, influenced by both mRNA sequence and length (44).…”

RNA splicing capability of live neuronal dendrites

“…Although it is clear that these cytoplasmic RNA-binding proteins (RBPs) have critical roles in the postsplicing regulation of the intracellular transport, stability, nonsense-mediated decay (NMD), and translation of cellular mRNAs (9,10), it is not known whether they retain the ability to assemble pre-mRNA splicingcompetent complexes in the dendritic milieu. Unspliced or incompletely spliced pre-mRNAs are often sequestered in the nucleus, yet removal of introns by splicing, in many cases, is not essential for mRNA export from the nucleus (11)(12)(13)(14)(15)(16). It is believed that some viral mRNAs and alternatively spliced mRNAs are likely exported to the cytosol as intron-retaining transcripts (4).…”