From transcription to translation, mRNA is complexed with heterogeneous nuclear ribonucleoproteins (hnRNP proteins) that mediate mRNA processing, export from the nucleus, and delivery into the cytoplasm. Although the mechanism is unknown, export of mature mRNA from the nucleus is a critical regulatory step in gene expression. Analyses of hnRNP proteins have shown that many of these proteins are required for this essential cellular process. In this study, we characterize the Saccharomyces cerevisiae Nab2 protein, which was first identified as a poly(A) RNA-binding protein (Anderson, J. T., Wilson, S. M., Datar, K. V., and Swanson, M. S. (1993) Mol. Cell. Biol. 13, 2730 -2741). Our work indicates that poly(A) RNA export from the nucleus is dependent upon a functional Nab2 protein; correspondingly, export of Nab2p from the nucleus is dependent upon ongoing RNA polymerase II transcription. Furthermore, we show that Nab2p is modified within its RGG domain by the type I protein-arginine methyltransferase, Hmt1p. Our experiments demonstrate that arginine methylation is required for the export of Nab2p from the nucleus and therefore establish an in vivo effect of this modification. Overall, these experiments provide evidence that Nab2p is an hnRNP protein that is required for poly(A) RNA export and whose export from the nucleus is regulated by Hmt1p.The evolutionary divergence of prokaryotes and eukaryotes also marks the evolution of compartmentalization of the genetic material in the form of a membrane-bound nucleus. In eukaryotes, movement of macromolecules (i.e. proteins and RNA) between the nucleus and the cytoplasm occurs through the nuclear pore complex, which is embedded within the nuclear envelope (1, 2). The presence of the nucleus also compartmentalizes the nuclear transcriptional machinery from the cytoplasmic translational machinery. mRNA serves as the linker molecule between these two cellular processes.Active genes are first transcribed to pre-mRNA via RNA polymerase II and then processed within the nucleus to form mature mRNA transcripts (3). These processing events occur co-transcriptionally and include the addition of a 5Ј 7-methylguanosine cap (4), the splicing of introns (5), and cleavage of the 3Ј end followed by polyadenylation (4, 6). Fully processed transcripts are then exported from the nucleus to the cytoplasm where they can be translated into functional proteins at ribosomes (7,8). Export of mRNA from the nucleus serves as an essential checkpoint in the regulation of gene expression. However, the detailed mechanism of the export of mature mRNA transcripts from the nucleus is poorly understood.From transcription to translation, mRNA is bound by various heterogeneous nuclear ribonucleoproteins (hnRNPs) 1 that serve to regulate the mRNA life cycle (9). Approximately 30 human hnRNP proteins have been identified that have been implicated in various stages of mRNA processing and export (10). Some of these hnRNP proteins shuttle between the nucleus and the cytoplasm (11). They are first imported int...

Messenger RNA transcripts are coated from cap to tail with a dynamic combination of RNA binding proteins that process, package, and ultimately regulate the fate of mature transcripts. One class of RNA binding proteins essential for multiple aspects of mRNA metabolism consists of the poly(A) binding proteins. Previous studies have concentrated on the canonical RNA recognition motif-containing poly(A) binding proteins as the sole family of poly(A)-specific RNA binding proteins. In this study, we present evidence for a previously uncharacterized poly(A) recognition motif consisting of tandem CCCH zinc fingers. We have probed the nucleic acid binding properties of a yeast protein, Nab2, that contains this zinc finger motif. Results of this study reveal that the seven tandem CCCH zinc fingers of Nab2 specifically bind to polyadenosine RNA with high affinity. Furthermore, we demonstrate that a human protein, ZC3H14, which contains CCCH zinc fingers homologous to those found in Nab2, also specifically binds polyadenosine RNA. Thus, we propose that these proteins are members of an evolutionarily conserved family of poly(A) RNA binding proteins that recognize poly(A) RNA through a fundamentally different mechanism than previously characterized RNA recognition motif-containing poly(A) binding proteins.CCCH zinc finger ͉ poly(A) binding protein ͉ RNA binding

Mature poly(A) RNA transcripts are exported from the nucleus in complex with heterogeneous nuclear ribonucleoproteins (hnRNPs). Nab2p is an essential Saccharomyces cerevisiae hnRNP protein that interacts with poly(A) RNA and shuttles between the nucleus and cytoplasm. Functional Nab2p is required for export of poly(A) RNA from the nucleus. The Nab2 protein consists of the following four domains: a unique N-terminal domain, a glutamine-rich domain, an arginine-glycine (RGG) domain, and a zinc finger domain. We generated Nab2p deletion mutants to analyze the contribution of each domain to the in vivo function of Nab2p. We first tested whether the deletion mutants could replace the essential NAB2 gene. We then examined the impact of these mutations on Nab2p localization, poly(A) RNA localization, and association of Nab2p with poly(A) RNA. Our analyses revealed that the N-terminal domain is required for nuclear export of both poly(A) RNA and Nab2p. We confirm that the RGG domain is important for Nab2p import in vivo. Finally, the zinc finger domain is critical for the interaction between Nab2p and poly(A) RNA in vivo. Our data support a model where Nab2p associates with poly(A) RNA in the nucleus through the zinc finger domain and facilitates the export of the poly(A) RNA through protein interactions mediated by the N-terminal domain.The eukaryotic cell is divided into functional compartments that separate important cellular processes. This compartmentalization prevents cross-talk between these cellular processes in an inappropriate or unregulated manner. For example, the membrane-bound nucleus serves at least two critical purposes: to sequester and protect the genetic material and to separate the nuclear processes of transcription and pre-mRNA processing from protein translation in the cytoplasm. As a result of this sequestration, the eukaryotic cell has evolved highly regulated mechanisms to actively transport macromolecules into and out of the nucleus (1, 2). Proteins that function within the nucleus are imported through large proteinaceous nuclear pore complexes that are embedded within the nuclear envelope (3). Similarly, macromolecules, including mRNP 1 complexes, consisting of mRNA and hnRNP proteins, are exported from the nucleus through nuclear pores (4).Mature, fully processed mRNAs are selectively exported from the nucleus (4 -6). Thus, nuclear export of poly(A) RNA is extremely complex because it depends on numerous pre-mRNA processing events that must be completed within the nucleus prior to export (6). Nascent transcripts are co-transcriptionally modified by proteins that mediate 5Ј-capping (7), 3Ј-cleavage, polyadenylation (7,8), and splicing (9). During pre-mRNA processing, the maturing mRNAs associate with many heterogeneous nuclear ribonucleoproteins (hnRNPs) (4), which may serve as markers for the completion of pre-mRNA processing events (5). Successful completion of these processing steps results in a mature mRNA species that is ready to be exported from the nucleus to the cytoplasm where it can ...

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While modified vaccinia virus Ankara (MVA) is currently in clinical development as a safe vaccine against smallpox and heterologous infectious diseases, its immunogenicity is likely limited due to the inability of the virus to replicate productively in mammalian hosts. In light of recent data demonstrating that vaccinia viruses, including MVA, preferentially infect antigen-presenting cells (APCs) that play crucial roles in generating antiviral immunity, we hypothesized that expression of specific cytokines and chemokines that mediate APC recruitment and activation from recombinant MVA (rMVA) vectors would enhance the immunogenicity of these vectors. To test this hypothesis, we generated rMVAs that express murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), human CCL20/human macrophage inflammatory protein 3␣ (hCCL20/ hMIP-3␣), or human fms-like tyrosine kinase 3 ligand (hFlt3-L), factors predicted to increase levels of dendritic cells (DCs), to recruit DCs to sites of immunization, or to promote maturation of DCs in vivo, respectively. These rMVAs also coexpress the well-characterized, immunodominant lymphocytic choriomeningitis virus nucleoprotein (NP) antigen that enabled sensitive and quantitative assessment of antigen-specific CD8 ؉ T-cell responses following immunization of BALB/c mice. Our results demonstrate that immunization of mice with rMVAs expressing mGM-CSF or hCCL20, but not hFlt3-L, results in two-to fourfold increases of cellular immune responses directed against vector-encoded antigens and 6-to 17-fold enhancements of MVA-specific antibody titers, compared to those responses elicited by nonadjuvanted rMVA. Of note, cytokine augmentation of cellular immune responses occurs when rMVAs are given as primary immunizations but not when they are used as booster immunizations, suggesting that these APC-modulating proteins, when used as poxvirus-encoded adjuvants, are more effective at stimulating naïve T-cell responses than in promoting recall of preexisting memory T-cell responses. Our results demonstrate that a strategy to express specific genetic adjuvants from rMVA vectors can be successfully applied to enhance the immunogenicity of MVA-based vaccines.