U2AF Participates in the Binding of TAP (NXF1) to mRNA

Abstract

TAP/NXF1 is a conserved mRNA export receptor serving as a link between messenger ribonucleoproteins (mRNPs) and the nuclear pore complex. The mechanism by which TAP recognizes its export substrate is unclear. We show here that TAP is added to spliced mRNP in human cells. We identified a distinct region of TAP that targets it to mRNP. Using yeast two-hybrid screens and in vitro binding studies, we found that this region coincides with a direct binding site for U2AF35, the small subunit of the splicing factor U2AF. This interaction is evolutionarily conserved across metazoa, indicating its significance. We further found in human cells that the exogenously expressed large U2AF subunit, U2AF65, accumulates in spliced mRNP, leading to the recruitment of U2AF35 and TAP. Similarly to TAP, U2AF65 stimulated directly the nuclear export and expression of an mRNA that is otherwise retained in the nucleus. Together with our finding that U2AF is continuously exported from the nucleus, these data suggest that U2AF participates in nuclear export, by facilitating TAP's addition to its mRNA substrates.TAP (also called NXF1)/Mex67p proteins define a conserved mRNA export pathway in eukaryotes (1-4). The Saccharomyces cerevisiae Mex67p and Caenorhabditis elegans Ce-NXF-1 are essential for mRNA export (4, 5), and the vertebrate TAP mediates the export of mRNA, but not the other classes of RNA (3, 6, 7). The Schizosaccharomyces pombe spMex67p is nonessential, but its mRNA export function is required in the absence of the essential Rae1p (8,9). TAP belongs to the NXF family of proteins that share the ability to bind both RNA and the components of the nuclear pore complex (10). The proposed substrate binding domain of TAP (aa 1 1-372) comprises an RNA binding domain and leucine-rich repeat domain and exhibits general RNA binding and high affinity binding to the viral CTE RNA (2, 3, 11). This region is also required for the direct binding to REF (also called Aly) proteins (1, 12), which are the metazoan homologues of yeast mRNA export factor Yra1p (13).At all metabolic steps, mRNA is part of ribonucleoprotein complexes (mRNP). The primary transcripts associate with a subset of heterogeneous nuclear ribonucleoproteins (hnRNPs) (reviewed in Ref. 14) and with the splicing machinery (15, 16), leading to their nuclear retention before splicing. Splicing changes the mRNP composition by promoting the assembly of export-competent mRNP (17, 18) and by depositing "splicing signature" proteins onto ligated exons (19). This protein complex is composed of the splicing-associated factors SRm160, DEK, and RNP S1, as well as REF and the RNA-binding protein Y14 (19, 20). Hence, REF was proposed to provide a link to export, possibly by recruiting TAP (18,21,22). As shown in human cells, TAP is added efficiently to the endogenous postsplicing mRNP complexes (23). In contrast, TAP is not added to the complexes produced by splicing in vitro (18, 19) or in microinjected oocyte nuclei (2). In this work, we addressed the assembly of TAP with the endogenous mR...

Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

“…4A, lane 14). Amino acids 61-118 showed a strong RNA cross-linking activity consistent with earlier work (25) and amino acids 1-60 a weaker activity. Within amino acids 61-118 there are a large number of arginines potentially involved in RNA binding, and despite low overall conservation of this region across organisms, TAP orthologs possess a basic region at the N terminus (Fig.…”

“…Earlier work demonstrated the TAP RNP-type RNA-binding domain (RBD) was dispensable for activity in vivo (23), although it had not been shown that the isolated RBD (amino acids 118-198) bound RNA. Amino Acids 96-198, encompassing the RBD, bind RNA (24), yet a separate study showed that amino acids 61-121 were involved in RNA binding (25). We clarified which region of TAP is responsible for nonspecific RNA binding by using truncations, internal deletions, and mutations (Fig.…”

Mammalian pre-mRNA 3′ End Processing Factor CF Im68 Functions in mRNA Export

“…Shuttling RNA-binding proteins may contribute to the assembly of an export competent mRNP by recruiting mRNA export factors. Consistent with this view U2AF 35-kDa subunit (Zolotukhin et al, 2002), several shuttling SR proteins (Huang et al, 2003;Lai and Tarn, 2004), and now CF I m have been shown to interact with the mRNA export receptor NXF1. In addition, shuttling RNA-binding proteins may fulfill different roles in the nucleus and in the cytoplasm.…”

“…This raises the possibility that multiple and partially redundant adaptor proteins may be responsible for the recruitment of NXF1. Indeed, spliceosomal proteins, including U2AF35 (Zolotukhin et al, 2002) and some members of the SR family of splicing factors, were shown to interact with NXF1 and act as adaptors for NXF1-dependent export of poly(A) mRNAs (Huang and Steitz, 2001;Huang et al, 2003;Lai and Tarn, 2004;Hargous et al, 2006;Tintaru et al, 2007).…”

The Wilms’ tumor 1 (WT1) gene (+KTS isoform) functions with a CTE to enhance translation from an unspliced RNA with a retained intron

“…In fact, it has been well documented that RNAs that serve as substrates for CTE-mediated export initially interact with the splicing machinery even when they remain completely unspliced (Lu et al 1990;Hammarskjöld 1997). It has also been shown that U2AF65 can stimulate the nuclear export and expression of an unspliced reporter RNA retaining an intron and that exogenously expressed U2AF65 can recruit Tap to mRNP complexes (Zolotukhin et al 2002). These results thus suggest a role for splicing factors in the formation of an export-competent CTE-containing mRNP complex.…”