Abstract
Traffic between the nucleus and cytoplasm takes place through a macromolecular structure termed the nuclear pore complex. To understand how the vital process of nucleocytoplasmic transport occurs, the contribution of individual pore proteins must be elucidated. One such protein, the nucleoporin Nup153, is localized to the nuclear basket of the pore complex and has been shown to be a central component of the nuclear transport machinery. Perturbation of Nup153 function was demonstrated previously to block the export of several classes of RNA cargo. Moreover, these studies also showed that Nup153 can stably associate with RNA in vitro. In this study, we have mapped a domain within Nup153, encompassing amino acids 250-400 in human Nup153, that is responsible for RNA association. After cloning this region of Xenopus Nup153, we performed a cross-species analysis. Despite variation in sequence conservation between Drosophila, Xenopus, and human, this domain of Nup153 displayed robust RNA binding activity in each case, indicating that this property is a hallmark feature of Nup153 and pointing toward a subset of amino acid residues that are key to conferring this ability. We have further determined that a recombinant fragment of Nup153 can bind directly to RNA and that this fragment can interact with endogenous RNA targets. Our findings identify a functionally conserved domain in Nup153 and suggest a role for RNA binding in Nup153 function at the nuclear pore.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF434196
Further progress in delineating pore architecture and in analyzing the functional contribution made by individual nuclear pore proteins to the transport machinery is essential to a comprehensive understanding of the nucleocytoplasmic transport process
Xenopus egg extract, which provides a vast supply of soluble nuclear pore complex (NPC) components, was incubated with poly(G) immobilized on agarose beads
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF434196. Following washes in heparin sulfate-containing buffer to reduce nonspecific binding to the beads, the poly(G)-associated proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot first using antibodies specific for several nuclear pore proteins.
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