The hepatitis B virus posttranscriptional regulatory element (PRE) is an RNA element that increases the expression of unspliced mRNAs, apparently by facilitating their export from the nucleus. We have identified a cellular protein that binds to the PRE as the polypyrimidine tract binding protein (PTB), which shuttles rapidly between the nucleus and the cytoplasm. Mutants of the PRE with mutations in PTB binding sites show markedly decreased activity, while cells that stably overexpress PTB show increased PRE-dependent gene expression. Export of PTB from the nucleus, like PRE function, is blocked by a mutant form of Ran binding protein 1 but not by leptomycin B. Therefore, PTB is important for PRE activity and appears to function as an export factor for PRE-containing mRNAs.Eucaryotic mRNA transcription takes place in the nucleus, but translation occurs in the cytoplasm, necessitating the export of mRNA through nuclear pores. In mammalian cells, this export is strictly controlled, in that only fully spliced and processed mRNA is exported (22,27). Part of the control is at the level of retention of incompletely spliced mRNA, probably by splicing factors binding to splice sites. However, this retention mechanism cannot explain all of the available data. First, some genes give rise to alternatively spliced transcripts, in which some of the mature mRNAs still contain splice sites. Second, for at least some cellular genes (e.g., the ␤-globin gene), removal of all introns leads to a defect in the export of mRNA to the cytoplasm (4). Therefore, the presence of splice sites does not always preclude RNA export, while the absence of splice sites does not always lead to export. These data imply that at least some mRNAs contain cis-acting elements that can effect export independently of splicing.In recent years, the existence of such RNA export elements has been confirmed. The best-studied element is the Rev response element (RRE) of human immunodeficiency virus (HIV) and related lentiviruses (6,14). Like almost all retroviruses, HIV contains only one promoter that gives rise to a transcript that is alternatively spliced, resulting in the export of completely spliced, partially spliced, and unspliced mRNAs into the cytoplasm. HIV codes for a trans-acting protein product that modulates the relative amounts of completely spliced versus incompletely spliced and unspliced messages. This protein, called Rev, binds to the RRE in the nucleus and strongly enhances the export of RRE-containing, unspliced or incompletely spliced transcripts. It has become clear that Rev contains a leucine-rich nuclear export signal (NES) that allows it to form a trimolecular complex with two cellular proteins, Crm1 (exportin) and Ran (8,9,29,34). This complex, together with its RNA cargo, interacts with components of the nuclear pore in order to migrate into the cytoplasm. Rev is then stripped off the mRNA in the cytoplasm and is recycled back into the nucleus by virtue of a nuclear localization signal.Other retroviruses also contain RNA export elemen...

The hepatitis B virus posttranscriptional regulatory element (PRE) is an RNA cis-element that is required for high-level expression of viral surface gene transcripts and appears to function by activating mRNA export to the cytoplasm. We have previously shown that multiple fragments of the PRE bind to two cellular proteins of approximately 35 and 55 kDa in molecular mass and that this binding correlates with function. By a combination of column chromatographic techniques and SDS-polyacrylamide gel electrophoresis, we have been able to purify the smaller protein. Amino-terminal sequencing of the purified protein shows identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an RNA-binding glycolytic enzyme that has been implicated in the export of tRNA. Immunoprecipitation analysis reveals that GAPDH is indeed present in the protein-RNA complex resulting from incubation of crude nuclear extracts with a functional region of the PRE. Furthermore, binding of the cellular 35 kDa protein to the PRE fragment is blocked by NAPDH, as would be expected for RNA binding by GAPDH. Finally, purified commercial GAPDH also binds specifically to this RNA fragment. Therefore, GAPDH is one of the cellular proteins that binds to the PRE, and may be involved in the posttranscriptional regulation of hepatitis B virus gene expression.

Transcription factor IIIA (TFIIIA) is required for the activation of 5S RNA gene transcription as well as the storage of 5s RNA as a 7S ribonucleoprotein particle. Interaction with both nucleic acids is mediated through nine C2H2 zinc fingers. In order to determine amino acid regions necessary for nucleic acid interaction, a series of substitution mutants Xenopus laevis TFIIIA have been constructed and expressed as recombinant proteins in Escherichia coli. The mutant proteins were purified to homogeneity and analyzed for 5S RNA gene and 5S RNA binding activities using a nitrocellulose filter binding assay. All of the mutant TFIIIA proteins retained full 5S RNA binding activity. Substitution of fingers 2, 3, and 4-6 of TFIIIA with zinc finger sequences from other proteins significantly reduced the interaction of the protein with the 5S RNA gene. In contrast, substitution of finger 1 or finger 7 had little effect on the interaction of TFIIIA with the 5S RNA gene. The results of scanning substitution mutagenesis within the first three zinc fingers of TFIIIA suggested that DNA contacts made by the alpha-helical regions of finger 2 and particularly of finger 3 provide the majority of the free energy of the TFIIIA-DNA interaction. Basic amino acids found at the same position within the alpha-helices of fingers 2 and 3 of TFIIIA are required for high-affinity DNA binding activity. The identification of amino acid residues critical for the formation of a TFIIIA-DNA complex contributes to our understanding of zinc finger protein-nucleic acid interactions.

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