Mammalian staufen is a double-stranded-RNA- and tubulin-binding protein which localizes to the rough endoplasmic reticulum - PubMed (original) (raw)

Mammalian staufen is a double-stranded-RNA- and tubulin-binding protein which localizes to the rough endoplasmic reticulum

L Wickham et al. Mol Cell Biol. 1999 Mar.

Abstract

Staufen (Stau) is a double-stranded RNA (dsRNA)-binding protein involved in mRNA transport and localization in Drosophila. To understand the molecular mechanisms of mRNA transport in mammals, we cloned human (hStau) and mouse (mStau) staufen cDNAs. In humans, four transcripts arise by differential splicing of the Stau gene and code for two proteins with different N-terminal extremities. In vitro, hStau and mStau bind dsRNA via each of two full-length dsRNA-binding domains and tubulin via a region similar to the microtubule-binding domain of MAP-1B, suggesting that Stau cross-links cytoskeletal and RNA components. Immunofluorescent double labeling of transfected mammalian cells revealed that Stau is localized to the rough endoplasmic reticulum (RER), implicating this RNA-binding protein in mRNA targeting to the RER, perhaps via a multistep process involving microtubules. These results are the first demonstration of the association of an RNA-binding protein in addition to ribosomal proteins, with the RER, implicating this class of proteins in the transport of RNA to its site of translation.

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Figures

FIG. 1

FIG. 1

Amino acid sequences of the hStau cDNAs. Shown is alignment of the cDNAs and PCR fragments with the translation of the putative protein sequences. Numbers refer to the sequence of the short cDNA. Positions of the four consensus dsRBDs (RBD1 to RBD4) and of the TBD are indicated between brackets above the sequence.

FIG. 2

FIG. 2

Characterization of the hStau mRNA and proteins. (A) Northern blot analysis of hStau expression in human tissues. A human multiple-tissue Northern blot (Clontech) was hybridized with the 1.2-kbp _Bam_HI fragment of hStau cDNA. Lane 1, brain; lane 2, pancreas; lane 3, heart; lane 4, skeletal muscles; lane 5, liver; lane 6, placenta; lane 7, lung; lane 8, kidney. (B) Western blot experiment with anti-hStau antibodies. Lane 1, HeLa cell extracts; lane 2, HEK 293 cell extracts. (C) HEK 293 cells were transfected with cDNAs coding for either the short (lane 2) or the long (lane 3) hStau isoform, lysed, and analyzed by Western blotting using the anti-hStau antibodies. Mock-transfected cells are shown in lane 1. (D) Schematic representation of the Drosophila (accession no. M69111), human and mouse (Hum/Mus), and C. elegans (accession no. U67949) Stau proteins. The human protein P1 has an insertion of 81 amino acids at its N-terminal extremity compared to protein P2. Large open and black boxes represent the full-length and short dsRBDs, respectively. Small boxes and lines are regions of high and low sequence similarity, respectively. The hatched boxes indicate the position of the region which is similar to the MAP1B microtubule-binding domain. The percentage of identity between the domains of the human and invertebrate proteins is indicated.

FIG. 3

FIG. 3

RNA-binding assay. Bacterially expressed His-hStau (A, lanes S) and His-neutral endopeptidase (A, lane N) fusion proteins or bacterially expressed MBP-mStau (B and C, lanes S) or MBP-aminopeptidase (B and C, lanes A) fusion proteins were electrophoresed on a polyacrylamide gel, transferred to nitrocellulose, and incubated with 32P-labeled nucleic acids, in the presence or absence of cold competitors, as indicated below each gel. After extensive washing, binding was detected by autoradiography. A representative Coomassie blue staining of the blots is shown on the left (A and B). Arrows, positions of overexpressed Stau; arrowheads, positions of overexpressed control proteins. Lanes M, molecular weight markers.

FIG. 4

FIG. 4

RNA-binding assay in solution. Dilutions of the purified His-hStau fusion protein were incubated with fixed amounts of labeled RNAs, and the RNA-protein complexes were filtered through nitrocellulose membranes. (A) RNA-binding affinity to dsRNAs. Triangles, 3′ UTR of bicoid RNA; squares, poly(rI)-poly(rC). The results are presented as a percentage of maximal retained probe and are the averages of three independent experiments done in duplicate. Inset, Coomassie blue staining of Ni-NTA-purified His/hStau after separation by SDS-PAGE. (B) RNA-binding affinity to RNAs. Squares, poly(rI)-poly(rC); triangles, poly(rI); inverted triangles, poly(rC); diamonds, BSA with poly(rI)-poly(rC), used as a control. The results are presented as a percentage of bound radioactivity and represent a single experiment done in duplicate. The same results were obtained in two other independent experiments.

FIG. 5

FIG. 5

Tubulin-binding assay. Bacterially expressed MBP-hStau (lanes S) or MBP-aminopeptidase (lanes A) fusion proteins were electrophoresed on SDS-polyacrylamide gels, transferred to nitrocellulose, and incubated with tubulin or actin. After extensive washing, tubulin and actin were detected with monoclonal antitubulin and antiactin antibodies, respectively. As controls, the same experiments were performed in the absence of either tubulin or antitubulin antibodies. Purified actin was also loaded on the gel as a control (lane C). Sizes are indicated in kilodaltons.

FIG. 6

FIG. 6

Molecular mapping of the dsRBD and TBD. Bacterially expressed MBP-mStau (lanes 1), MBP-mStau deletion mutants (lanes 2 to 7), or MBP-aminopeptidase (lanes C) fusion proteins were electrophoresed on a polyacrylamide gel, transferred to nitrocellulose, incubated with either 32P-labeled 3′-UTR bicoid RNA (A) or tubulin and antitubulin antibodies (B), and revealed as described above. (C) Schematic representation of the mutant proteins. Their RNA- and tubulin-binding responses are indicated.

FIG. 7

FIG. 7

Subcellular localization of the hStau-GFP fusion proteins. COS7 cells were transfected with cDNAs coding for either the hStau-GFP (A and B) or TBD-GFP (C) fusion protein, or GFP alone (D). Untreated (A, C, and D) or Triton X-100-treated (B) cells were fixed and visualized by autofluorescence. Bar = 20 μm.

FIG. 8

FIG. 8

Colocalization of hStau with markers of the RER by confocal microscopy. A cDNA coding for an hStau-HA fusion protein was transfected into COS7 cells. Triton X-100-treated cells were fixed and double labeled with anti-HA (B) and anticalreticulin (A) or with anti-HA (E) and anticalnexin (D). Anti-HA was detected with Texas red-coupled anti-mouse IgG antibodies, using the Texas Red channel; anticalreticulin and anticalnexin were detected with fluorescein-conjugated anti-rabbit IgG antibodies, using the fluorescein channel. Panels C and F are superpositions of panels A plus B and D plus E, respectively. No overlap was observed between the fluorescein and Texas red channels. Bar = 10 μm.

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