HNS, a nuclear-cytoplasmic shuttling sequence in HuR - PubMed (original) (raw)

HNS, a nuclear-cytoplasmic shuttling sequence in HuR

X C Fan et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Proteins are transported into and out of the cell nucleus via specific signals. The two best-studied nuclear transport processes are mediated either by classical nuclear localization signals or nuclear export signals. There also are shuttling sequences that direct the bidirectional transport of RNA-binding proteins. Two examples are the M9 sequence in heterogeneous nuclear ribonucleoprotein A1 and the heterogeneous nuclear ribonucleoprotein K shuttling domain (KNS) sequence in heterogeneous nuclear ribonucleoprotein K, both of which appear to contribute importantly to the export of mRNA to the cytoplasm. HuR is an RNA-binding protein that can stabilize labile mRNAs containing AU-rich elements in their 3' untranslated regions and has been shown to shuttle between the nucleus and cytoplasm (18, 19). We have identified in HuR a shuttling sequence that also possess transcription-dependent nuclear localization signal activity. We propose that HuR first may bind AU-rich element-containing mRNAs in the nucleus and then escort them through the nuclear pore, providing protection during and after export to the cytoplasmic compartment.

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Figures

Figure 1

The HuR hinge region is essential for nuclear localization. (A) Schematic representation of wild-type and mutant HuR proteins assayed in B. (B) Subcellular localization of wild-type and truncated HuRs. NIH 3T3 cells were transiently transfected with expression vectors containing HuR-C-Flag, HuR-ΔhRRM3-C-Flag, or HuR-ΔRRM3-C-Flag, fixed with 3% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100, and stained with monoclonal anti-Flag antibody (Sigma).

Figure 2

HuR-ΔRRM3 shuttles between the nucleus and the cytoplasm. HeLa cells were transiently transfected with the HuR-ΔRRM3-C-Flag expression vector, fused with mouse NIH 3T3 cells to form heterokaryons by using 50% polyethylene glycol, and incubated for another 3 hr in the presence of the protein synthesis inhibitor cycloheximide. The coculture was then fixed and stained by using the anti-Flag mAb with costaining by Hoechst dye 33258, which distinguishes the human from the mouse nuclei (indicated by arrows).

Figure 3

Delineation of the HuR nuclear localization signal. (A) Schematic diagram and summary of the intracellular localization of PK fusion proteins containing HuR fragments M1 to M12. (B) NIH 3T3 cells were transiently transfected with myc-tagged PK expression vectors containing HuR fragments and processed as described in Fig. 1_B_, except for use of anti-myc antibody 9E10. myc-tagged PK was transfected as a control.

Figure 4

The HuR NLS confers shuttling activity. (A). Schematic representation of the NPc fusion proteins assayed in Fig. 4_B_. (B) Heterokaryon fusion assays of NPc-M1, -M7, and -M10. HeLa cells were transiently transfected with NPc fusion proteins containing HuR sequences M1, M7, or M10. Heterokaryon fusion and staining assays were performed as described in Fig. 2, except for use of anti-myc mAb 9E10. Mouse nuclei in the heterokaryons are indicated with arrows. The NPc-NLS was analyzed as a negative control.

Figure 5

The NLS activity of HuR depends on pol II transcription. The HuR-nucleoplasmin fusion proteins NPc-M1, -M7, and -M10, as well as the negative control NPc-NLS, were transiently transfected into mouse NIH 3T3 cells, incubated with actinomycin D at 5 μg/ml for 3 hr, and fixed and subjected to immunofluorescence staining by using the anti-myc 9E10 antibody.

Figure 6

Sequence comparisons of HNS. (A) Amino acid alignment of the hinge region of Hu family proteins. The hinge-region amino acid sequences of human Hu proteins Hel-N1 (HuB), HuC, and HuD and the Xenopus HuR homolog ElrA are compared with human HuR (28, 29). Residues identical to the HuR consensus are shown in dark gray, whereas conservative substitutions are shaded light gray. (B) Amino acid alignment of human HuR HNS and human hnRNP A1 M9 (36). Identical residues are shown in dark gray. The Gly-274 residue that is essential for shuttling in M9 has been indicated by an arrow.

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HNS, a nuclear-cytoplasmic shuttling sequence in HuR - PubMed (original) (raw)