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 lambda 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 Kd 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.

FIG. 1

Identification of the human sequence homologue of the dmStaufen gene. (A) Structure of hStaufen-like protein, including the four RNA-binding domains (hatched boxes) and the thSTL protein fragment encoded in clone C, aligned with the structure of dmStaufen protein. (B) Comparison of the protein sequences of dmStaufen and the protein predicted from λ clones obtained by using the thSTL insert present in clone C. Boxed residues show the positions conserved among dsRNA-binding domains present in protein members of the dmStaufen family (49).

FIG. 2

Characterization of hStaufen-like mRNA by Northern blot hybridization. Poly(A)⁺ RNA from human cell lines (A) or human organs (B) was separated by denaturing agarose gel electrophoresis and probed with a thSTL-specific probe or a β-actin probe as described in Materials and Methods. Size of molecular weight markers are indicated in kilobases to the left.

FIG. 3

Characterization of hStaufen-like protein by Western blotting. (A) Purified His-STL and His-HST proteins were analyzed by Western blotting as indicated in Materials and Methods, using anti-STL serum, preimmune serum, or anti-STL serum depleted with purified His-HST protein bound to Ni²⁺-NTA resin. (B) Total extracts from HeLa or 293 cells were analyzed as indicated above. Sizes of molecular weight markers are indicated in kilodaltons to the left. Arrows indicate the relevant protein bands.

FIG. 4

RNA-binding properties of hStaufen-like protein. Fixed amounts of labeled RNA probe (10,000 cpm) were incubated with increasing amounts of purified His-HST protein. The protein-bound probe was determined by filtration on a nitrocellulose filter. The results are presented as percentage of maximal retained probe and are averages and standard deviations of two to four independent experiments. The insert shows the purified His-HST protein used in the RNA-binding assays as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining. Closed circles, His-HST protein/bicoid probe; open circles, His-HST protein/poly(U) probe; triangles, BSA/bicoid probe.

FIG. 5

Immunofluorescence analysis of hStaufen-like in transfected HeLa cells. (A and B) Cultures of HeLa cells transfected with plasmid pCMV-STL, fixed, and processed for immunofluorescence as indicated in Materials and Methods, using anti-STL serum (A) and anti-T7 tag monoclonal antibody (B). (C) Overlay of the preceding images. (D) Anti-STL serum depleted with purified His-HST protein bound to Ni²⁺-NTA resin. (E) Anti-T7 tag monoclonal antibody. (F) Overlay of the preceding images. In these analyses, staining with anti-STL serum and with anti-STL serum depleted with purified His-HST protein were carried out under identical conditions, including dilutions of the sera and exposure times.

FIG. 6

Colocalization of hStaufen-like with rough endoplasmic reticulum. (A and B) Cultures of HeLa cells fixed and processed for immunofluorescence as indicated in Materials and Methods and the legend to Fig. 5, using anti-STL serum (A) and anti-Bip monoclonal antibody (B). (C) Overlay of the preceding images. (D) Anti-STL serum. (E) Anti-ribophorin I monoclonal antibody. (F) Overlay of the preceding images.

FIG. 7

Double-immunofluorescence studies with hStaufen-like and cellular markers. (A and B) cultures of HeLa cells fixed and processed for immunofluorescence as indicated in Materials and Methods and the legend to Fig. 5, using anti-STL serum (A) and anti-lamp2 monoclonal antibody (B). (C) Overlay of the preceding images. (D) Anti-STL serum. (E) Antitubulin monoclonal antibody. (F) Overlay of the preceding images. (G) Anti-STL serum. (H) Anti-mannII monoclonal antibody. (I) Overlay of the preceding images.

FIG. 8

Association of hStaufen-like protein with polysomes. Soluble extracts of HeLa cells were centrifuged in a sucrose gradient as described in Materials and Methods. (A) Untreated extracts. The fractions were analyzed by Western blotting using anti-STL serum. In addition, aliquots of each fraction were used to isolate RNA to carry out dot blot hybridization with an rDNA probe as indicated in Materials and Methods. (B) Extracts from cultures treated with puromycin. The cultures were treated with puromycin (100 μg/ml) for 60 min prior to preparation of cytoplasmic extracts. (C) Extracts treated with EDTA. The extracts prepared as described above were treated with 25 mM EDTA and separated as indicated except that the sucrose gradient was adjusted to 25 mM EDTA. The fractions were analyzed as described for panel A. Sizes of molecular weight markers are indicated in kilodaltons to the right.