Abstract.A monoclonal antibody that reacts with proteins in the nuclear pore complex of rat liver (Snow, C. M., A. Senior, and L. Gerace. 1987. J. Cell Biol. 104:1143-1156) has been shown to cross react with similar components in Xenopus oocytes, as determined by immunofluorescence microscopy and immunoblotting. We have microinjected the antibody into oocytes to study the possible role of these polypeptides in nucleocytoplasmic transport. The antibody inhibits import of a large nuclear protein, nucleoplasmin, in a time-and concentration-dependent manner.It also inhibits export of 5S ribosomal RNA and mature tRNA, but has no effect on transcription or intranuclear tRNA processing. The antibody does not affect the rate of diffusion into the nucleus of two small proteins, myoglobin and ovalbumin, indicating that antibody binding does not result in occlusion of the channel for diffusion. This suggests that inhibition of protein and RNA transport occurs by binding of the antibody at or near components of the pore that participate in mediated transport. MOLECULAR traffic between the nuclear and cytoplasmic compartments of the eukaryotic cell takes place through pore complexes that perforate the nuclear envelope (for reviews see references 11, 14, and 23). The pore complex is a supramolecular protein assembly that forms an aqueous channel through the double-nuclear membrane at points where the inner and outer membranes are fused (23, 51). As seen by electron microscopy, the pore complex contains two eightfold symmetrical rings with outer diameters of 120 nm, one on each face of the envelope, which surround a central channel (24, 51). Radial spokes appear to project inward from the walls of the channel, where a central particle can often be found (51). Since transport appears to take place through the center of this channel (21), the central particle may contain material in the process of being transported.The nuclear pore complex behaves as a molecular sieve that allows the passive diffusion of solutes at a rate inversely proportional to molecular size, up to a limit of 9-11 nm diam (2, 41-44). Thus, globular proteins of less than "020 kD will readily diffuse across the envelope, while proteins of more than ,065 kD are effectively excluded (2). Because many nuclear proteins are larger than the limit for diffusion, or would diffuse too slowly across the envelope to account for their physiological rates of appearance in the nucleus (3, 20), transport of most nuclear proteins is thought to be achieved by a mediated mechanism. Carol Featherstone's and Larry Gerace's current address is Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.Several nuclear proteins have been shown to contain short stretches of amino acids that confer a nuclear location on the protein (for examples see references 27,31,32,34,46,53). This suggests that nuclear proteins might interact with one or several receptors for these sequences, most probably located in the pore complex. Export of RNA also appears to...

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A zinc finger library with degenerate α-helices was displayed on the surface of bacteriophage and proteins that bind human immunodeficiency virus type-1 (HIV-1) Rev response element stem loop IIB (RRE-IIB) RNA or 5S rRNA were isolated. DNA encoding affinity selected zinc fingers was shuffled by recombination in vitro to isolate proteins with higher RNA binding affinity. Proteins constructed in this way bind RNA specifically both in vitro and in vivo. These results demonstrate that RNA substrate specificity of zinc fingers can be changed through mutation of α-helices to construct novel RNA binding proteins.Zinc fingers constitute the most frequently recognized class of nucleic acid binding motif in the human genome 1 . C 2 H 2 zinc fingers (Fig 1a), though commonly considered a DNA binding motif, bind specifically and avidly to both DNA and RNA 2-5 . Structural conservation and modular interaction of this class of zinc finger has been exploited to design sequence specific transcriptional regulators for potential applications in gene therapy 6-9 . A relatively unexplored avenue with potential for molecular intervention is disruption of essential RNA-protein interactions with sequence-specific RNA binding proteins. We recently determined that zinc finger interaction with RNA is similar to DNA in that binding requires a few critical amino acids in the finger α-helix 10 , suggesting zinc fingers could provide a stable framework for designing RNA binding proteins. To test this hypothesis we displayed a degenerate zinc finger library on bacteriophage and selected for zinc finger peptides with affinity for a specific RNA.A zinc finger library for phage display was constructed from two zinc fingers each based on the β-sheet of RNA binding zinc finger 4 from transcription factor IIIA (TFIIIA), which is essential for high affinity RNA binding by TFIIIA-derived protein fragments and therefore unlikely to contain amino acids detrimental to RNA interaction. Two histidines (+7 and +11 with respect to the start of the α−helix) required as zinc ligands, together with a phenylalanine (-3) and leucine (+4) essential for zinc finger structure were retained (Fig. 1a). Amino acids at nine positions, between -2 and +10 of the zinc finger α-helix, were randomized to code for a subset of amino acids present in α-helices of potential zinc finger proteins (see Methods and Fig. 1b). Additionally, one amino acid position in the linker between fingers was randomized to permit alternate finger orientations 11 . The library was displayed on the surface of bacteriophage fd as a fusion with coat protein III 7,12,13 and phage that bound exclusively to one of two unrelated 'selector' RNAs were isolated. 5S rRNA was used as a selector, because it is a natural substrate for zinc finger proteins TFIIIA and p43 4,14 . RRE-IIB RNA is the high affinity binding site of HIV-1 Rev protein and a critical regulatory element for HIV-1 late gene expression 15 . No zinc finger proteins are known to bind the Rev response element and its interaction with Rev prote...

Zinc fingers in transcription factor IIIA (TFIIIA) contribute differentially to RNA and DNA binding affinity. We investigated whether the same putative ␣-helix amino acids in TFIIIA zinc fingers are essential for both RNA and DNA binding. In published structures, zinc fingers make DNA base contacts through amino acids ؊1, ؉2, ؉3, and ؉6 of the recognition helix. Alanine substitution at these four positions were made in TFIIIA RNA binding zinc fingers, tz4 -7 and DNA binding zinc fingers, tz1-3. Substitution in zinc fingers 4 or 6 of tz4 -7 reduced RNA affinity 77-and 38-fold, respectively, whereas substitution in zinc fingers 5 or 7 had little effect. DNA binding affinity of tz1-3 was eliminated by alanine substitution in any one zinc finger. We determined which amino acids supported RNA binding by phage display of a library of zinc finger 4 mutants. Lysine at helix position ؊1 of zinc finger 4 was conserved in all selected tz4 -7 fusions. Point mutation of Lys ؊1 to alanine in zinc finger 4 reduced tz4 -7 RNA affinity 30-fold. We propose that RNA binding by TFIIIA shows similarity to DNA binding in the use of the recognition helix. Helix positions ؊1 and ؉2 may have particular significance for RNA binding.