A Human Sequence Homologue of Staufen Is an RNA-Binding Protein That Is Associated with Polysomes and Localizes to the Rough Endoplasmic Reticulum

Abstract

In the course of a two-hybrid screen with the NS1 protein of influenza virus, a human clone capable of coding for a protein with high homology to the Staufen protein from Drosophila melanogaster (dmStaufen) was identified. With these sequences used as a probe, cDNAs were isolated from a λ cDNA library. The encoded protein (hStaufen-like) contained four double-stranded RNA (dsRNA)-binding domains with 55% similarity and 38% identity to those of dmStaufen, including identity at all residues involved in RNA binding. A recombinant protein containing all dsRNA-binding domains was expressed in Escherichia coli as a His-tagged polypeptide. It showed dsRNA binding activity in vitro, with an apparent K_d of 10⁻⁹ M. Using a specific antibody, we detected in human cells a major form of the hStaufen-like protein with an apparent molecular mass of 60 to 65 kDa. The intracellular localization of hStaufen-like protein was investigated by immunofluorescence using a series of markers for the cell compartments. Colocalization was observed with the rough endoplasmic reticulum but not with endosomes, cytoskeleton, or Golgi apparatus. Furthermore, sedimentation analyses indicated that hStaufen-like protein associates with polysomes. These results are discussed in relation to the possible functions of the protein.

ACKNOWLEDGMENTS

We are indebted to A. Nieto, M. Kiebler, I. Mattaj, J. A. Melero, and T. Zürcher for critical comments on the manuscript. We thank F. Becker, A. Ephrussi, J. P. García-Ballesta, J. Krijnse-Locker, B. Moss, and J. Renard for providing biological materials. The technical assistance of J. Fernández is gratefully acknowledged.

P.F. and R.M.M. were fellows from Programa Nacional de Formación de Personal Investigador. This work was supported by Programa Sectorial de Promoción General del Conocimiento (grant PB94-1542).

The first two authors contributed equally to this work.