Nuclear RNA Export

The identification of CRM1 as a protein export factor was initiated by the finding that nuclear export of unspliced HIV-1 RNA depends on binding of the viral protein Rev to CRM1 via a leucine-rich NES (Neville et al. 1997) (Fig. 2). Further studies demonstrated that although CRM1 mediates nuclear export of HIV-1 mRNA, it is not responsible for the export of bulk cellular mRNA (Cullen 2003). Instead, CRM1 acts as a RNA-export receptor for the export of rRNA, Usn RNAs and several specific mRNAs (e.g., c-Fos, Cyclin D1, CD83), which is, however, mediated via RNA-binding adapter proteins that interact with CRM1 (reviewed in Hutten and Kehlenbach 2007) (Fig. 2).

In contrast to complex lentiviruses like HIV-1 encoding Rev-type RNA-binding proteins, incompletely or unspliced RNAs from type D retroviruses are exported due to the presence of a ds-acting RNA sequence named constitutive transport element (CTE) (Bray et al. 1994). Investigation of the CTE-mediated export mechanism allowed for the discovery of the major mRNA export receptor, named TAP/NXF1 (in yeast termed Mex67p), which interacts with the CTE element and thus facilitates nuclear export of CTE-containing transcripts (Kang and Cullen 1999) (Fig. 2). Later on, it was demonstrated that TAP interacts with p15, and that this heterodimer is responsible for bulk metazoan mRNA export to the cytoplasm via direct interaction with the nuclear pore (Katahira et al. 1999, 2002). Although TAP is able to bind directly to CTE-containing viral mRNA, additional factors are needed to bridge the interaction between TAP-p15 and metazoan mRNA since metazoan RNA does not contain CTE-like RNA secondary structures (Liker et al. 2000). The production of mature mRNA in eukaryotes involves a complex series of nuclear processing reactions that occur co-transcriptionally and include the addition of the 5' cap, removal

Fig. 2 Nuclear mRNA export pathways. Shown is a schematic overview of several nuclear mRNA export pathways used by retroviruses or by metazoan cells. Retroviral nuclear RNA export: unspliced mRNAs encoded by the simple retrovirus MPMV are exported via direct binding of the constitutive transport element (CTE) RNA target to the cellular mRNA export receptor TAP-p15; unspliced RNA encoded by HIV-1 binds via the Rev-responsive element (RRE) RNA target to the viral factor Rev. Rev then interacts with the cellular protein CRM1 through its leucine-rich NES, thus mediating nuclear export of unspliced HIV-1 mRNA. Metazoan nuclear RNA export: ribosomal RNAs, small nuclear RNAs (U snRNAs) as well as some specific mRNAs are exported from the nucleus via the karyopherin CRM1; however, adaptor proteins binding to CRM1 via a leucine-rich NES are required to mediate these interactions. Bulk cellular mRNA export occurs via the TAP-p15 export receptor: during splicing of vertebrate mRNA, a complex of proteins, the exon junction complex (EJC) that contains UAP56 and REF is deposited on spliced mRNAs upstream of exon-exon junctions. REF proteins present in EJCs subsequently recruit the export receptor TAP-p15. Association of TAP-p15 with the mRNPs displaces UAP56

Fig. 2 Nuclear mRNA export pathways. Shown is a schematic overview of several nuclear mRNA export pathways used by retroviruses or by metazoan cells. Retroviral nuclear RNA export: unspliced mRNAs encoded by the simple retrovirus MPMV are exported via direct binding of the constitutive transport element (CTE) RNA target to the cellular mRNA export receptor TAP-p15; unspliced RNA encoded by HIV-1 binds via the Rev-responsive element (RRE) RNA target to the viral factor Rev. Rev then interacts with the cellular protein CRM1 through its leucine-rich NES, thus mediating nuclear export of unspliced HIV-1 mRNA. Metazoan nuclear RNA export: ribosomal RNAs, small nuclear RNAs (U snRNAs) as well as some specific mRNAs are exported from the nucleus via the karyopherin CRM1; however, adaptor proteins binding to CRM1 via a leucine-rich NES are required to mediate these interactions. Bulk cellular mRNA export occurs via the TAP-p15 export receptor: during splicing of vertebrate mRNA, a complex of proteins, the exon junction complex (EJC) that contains UAP56 and REF is deposited on spliced mRNAs upstream of exon-exon junctions. REF proteins present in EJCs subsequently recruit the export receptor TAP-p15. Association of TAP-p15 with the mRNPs displaces UAP56

of introns and addition of the 3' poly(A) tail (Bentley 2002; Maniatis and Reed 2002). The removal of introns results in the deposition of a protein complex, termed exon junction complex (EJC), on the RNA molecule immediately upstream of the splice site (Le Hir et al. 2000). One of the components of the EJC is the DExD/H box protein UAP56 (named Sub2p in Saccharomyces cerevisiae), a putative RNA helicase, which is thought to couple mRNA splicing with nuclear export (Reed and Hurt 2002). As a next step, UAP56 recruits the adapter protein Aly/REF (Yra1p in S. cerevisiae) to the mRNA, and Aly/REF subsequently interacts with the het-erodimeric TAP-p15, leading to the efficient export of the mRNA through the nuclear pore complex (Luo et al. 2001; Strasser and Hurt 2001) (Fig. 2). Interestingly, Sub2p is required for nuclear export of both intron-containing as well as intronless mRNAs, suggesting that the association of the helicase with the mRNA molecule is not necessarily coupled with the splicing reaction (Strasser and Hurt 2001). Indeed, several studies showed that UAP56 recruitment to the mRNA can also occur via co-transcriptional mechanisms (Zenklusen et al. 2002; Kiesler et al. 2002; Strasser et al. 2002). Furthermore, while REF proteins were found to be dispensable for bulk mRNA export, inactivation of UAP56 leads to a nuclear retention of poly(A) mRNA, indicating that this protein plays a central role for mRNA export (Gatfield and Izaurralde 2002; Gatfield et al. 2001; Kapadia et al. 2006).

Interaction of the Human Cytomegalovirus Protein pUL69 with the mRNA Export Factor UAP56

The human cytomegalovirus protein encoded by the open reading frame UL69 belongs to a family of regulatory factors that is conserved among all herpesviruses and includes the proteins ICP27 of herpes simplex type I (HSVI), EB2 of Epstein-Barr virus (EBV), and the ORFs 57 of Kaposi's sarcoma-associated herpesvirus (KSHV) and of Herpesvirus saimiri (HVS) (for reviews see Lischka and Stamminger 2006; Sandri-Goldin 2004; Sandri-Goldin 2001). Although the amino acid identity among these proteins is not very high, ranging from 17% to 36%, they share a region showing a higher conservation of approximately 40% sequence identity. This conserved region can be found at the C-terminus of the a- and y-herpesvirus proteins, whereas it corresponds to the central part of the p-herpesviral proteins since they have a unique C-terminal domain (Winkler et al. 2000) (see Fig. 3) . Recently, we demonstrated that this homology region folds into a globular core domain according to secondary structure predictions and is responsible for a shared

Nuclear Export Signal

Fig. 3 Domain organization of the HCMV UL69 protein in comparison to the HSV-1 protein ICP27 showing the positions of important functional regions. The sequence of the UAP56-binding motif within pUL69 is depicted, as is the sequence of the leptomycin B-insensitive NES; underlined amino acid residues are critical for the function of the respective motifs. NES nuclear export signal, NLS nuclear localization signal, R1, R2, RS arginine-rich regions, RBD RNA-binding domain, KH1-3 putative KH RNA-binding motifs, ICP27 homology domain of pUL69 with high homology to ICP27, SID self-interaction domain, REF UAP56, hSPT6 binding sites of the respective cellular factors

Fig. 3 Domain organization of the HCMV UL69 protein in comparison to the HSV-1 protein ICP27 showing the positions of important functional regions. The sequence of the UAP56-binding motif within pUL69 is depicted, as is the sequence of the leptomycin B-insensitive NES; underlined amino acid residues are critical for the function of the respective motifs. NES nuclear export signal, NLS nuclear localization signal, R1, R2, RS arginine-rich regions, RBD RNA-binding domain, KH1-3 putative KH RNA-binding motifs, ICP27 homology domain of pUL69 with high homology to ICP27, SID self-interaction domain, REF UAP56, hSPT6 binding sites of the respective cellular factors property of all members of this protein family, namely the propensity to self-interact and thus to form multimeric protein complexes (Lischka et al. 2007).

A second shared feature of all characterized members of this protein family is a function as posttranscriptional regulators. For instance, the prototype of this protein family, ICP27 of HSVI, has been shown to redistribute small nuclear ribonucleoprotein particles (snRNPs), to inhibit cellular splicing, to bind to intronless viral RNAs, and to shuttle between the nucleus and the cytoplasm, thus acting as a viral mRNA export factor (reviewed by Sandri-Goldin 2004; Sandri-Goldin 2001; Smith et al. 2005). The latter properties are of particular importance for herpesviruses since the majority of viral transcripts are intronless and thus do not interact with the splicing machinery, leading to inefficient nuclear export of viral mRNA. Further investigation of the RNA export mechanism revealed that ICP27 binds viral RNA through an N-terminal RGG box RNA-binding motif (Mears and Rice 1996; Sandri-Goldin 1998). Additionally, ICP27 was shown to interact with the adaptor protein Aly/REF, thereby recruiting intronless viral RNAs to the cellular TAP-p15 mRNA export receptor (Chen et al. 2002; Koffa et al. 2001) (see Fig. 4). An interaction with Aly/REF could also be demonstrated for the y-herpesviral proteins EB2 of EBV and ORF 57 of KSHV or HVS, suggesting that several ICP27 homologous proteins may use a common mechanism for the nuclear export of viral intronless mRNAs (Hiriart et al. 2003; Malik et al. 2004; Williams et al. 2005).

Initial studies on the HCMV homolog of ICP27, the UL69 protein, revealed several differences between these two regulatory factors:

1. In contrast to the immediate early expression of ICP27, pUL69 could be detected during the early and late phase of the replication cycle and is incorporated as a tegument protein into viral particles (Winkler et al. 1994; Winkler and Stamminger 1996);

2. pUL69 did not repress expression depending on the presence of an intron within a reporter gene but revealed a rather pleiotropic activation of various promoters upstream of the luciferase reporter (Sandri-Goldin and Mendoza 1992; Winkler et al. 1994);

3. No redistribution of snRNPs is induced by pUL69 (Winkler et al. 2000);

4. pUL69 could not complement the growth of an ICP27 deletion mutant of HSVI, further emphasizing the existence of functional differences (Winkler et al. 1994).

In an attempt to unravel the mechanism of pUL69-mediated transactivation, we searched for cellular interaction partners of this viral protein by yeast two hybrid screenings, which revealed at least two proteins with a potential role for mRNA biogenesis and processing. One of the identified cellular proteins corresponded to the transcription elongation factor hSPT6 (Endoh et al. 2004; Kaplan et al. 2000); we were able to demonstrate that this interaction occurs within the conserved homology region of pUL69 and is functionally required for pUL69-mediated trans-activation (Winkler et al. 2000). Interestingly, hSpt6 has recently been shown to bind both to the C-terminal domain of RNA polymerase II and to a novel factor

Terminal Domain Rna Polymerase

Fig. 4 Models of ICP27- and pUL69-mediated viral mRNA export. ICP27 of HSVI binds directly to viral intronless transcripts. Via its interaction with the adaptor protein REF, the RNA is recruited to the cellular TAP-p15 mRNA export receptor, thus forming an export-competent mRNP. The UL69 protein of HCMV interacts both with the cellular transcription elongation factor hSPT6 and the mRNA export factor UAP56. Thus, pUL69 may facilitate mRNA export by optimizing the co-transcriptional loading of RNA export factors to nascent mRNA

Fig. 4 Models of ICP27- and pUL69-mediated viral mRNA export. ICP27 of HSVI binds directly to viral intronless transcripts. Via its interaction with the adaptor protein REF, the RNA is recruited to the cellular TAP-p15 mRNA export receptor, thus forming an export-competent mRNP. The UL69 protein of HCMV interacts both with the cellular transcription elongation factor hSPT6 and the mRNA export factor UAP56. Thus, pUL69 may facilitate mRNA export by optimizing the co-transcriptional loading of RNA export factors to nascent mRNA

named hlwsl, the depletion of which induces nuclear retention of poly(A) RNA. This observation suggests that hSPT6 integrates transcription elongation with downstream mRNA export (Yoh et al. 2007). However, with respect to the documented interaction of ICP27 with Aly/REF, it was even more interesting to identify the putative DExH/D box RNA helicase URH49 as a binding protein of pUL69 by yeast two-hybrid screening. URH49 is highly related to the mRNA export protein UAP56, which is located upstream of Aly/REF in the cellular mRNA export pathway (see Fig. 2) (Pryor et al. 2004). In both yeast two-hybrid and co-immunopre-cipitation experiments, we demonstrated that pUL69 interacts not only with URH49, but also with UAP56 (Lischka et al. 2006b). Although details on the biological function of URH49 are unknown so far, initial data support the assumption that both DExH/D box proteins exert largely overlapping functions in the processing and export of mammalian mRNAs (Kapadia et al. 2006; Pryor et al. 2004). In this context, it should be noted that UAP56, alternatively termed BAT1, has been described as a multifunctional cellular protein: in addition to its role in recruiting Aly/REF to mature mRNAs, it plays a well-documented role in pre-mRNA splicing, where it facilitates the association of U2 snRNP with the splice branch point, presumably by dissociating U2AF65 from the polypyrimdine track downstream of the splice branchpoint (Fleckner et al. 1997). Furthermore, several studies propose that the UAP56/BAT1 gene, which is situated in the central region of the major histocompatibility complex, acts as an anti-inflammatory gene, and polymorphism in its promoter region may predispose for specific inflammatory disorders (Allcock et al. 1999, 2001; Ramasawmy et al. 2006). However, it is presently unclear whether this potential antiinflammatory activity of UAP56 is related to its function in mRNA processing and export.

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