A novel nucleocytoplasmic shuttling sequence of DAZAP1, a testis-abundant RNA-binding protein

Abstract

Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous RNA-binding protein highly expressed in the human and the mouse testes. It shows a dynamic subcellular localization during spermatogenesis, present predominantly in the nuclei of late-stage spermatocytes and round spermatids and translocated to the cytoplasm during spermatid elongation. To test the hypothesis that DAZAP1 shuttles between the nucleus and the cytoplasm, we studied the nuclear transport of DAZAP1 in somatic cells using immunostaining, heterokaryon formation, and mutagenesis. DAZAP1 is detected exclusively in the nucleus and has the ability to shuttle between the nucleus and the cytoplasm using a highly conserved 25 amino acid segment, designated ZNS, at its C terminus. ZNS shares no sequence homology with other known nuclear localization or export signals. Attachment of ZNS to a red fluorescent protein DsRed2 confers the nucleocytoplasmic shuttling ability to that protein. The nuclear localization of DAZAP1 depends on active transcription. In the presence of an RNA polymerase II inhibitor, DAZAP1 is retained in the cytoplasm. DAZAP1 colocalizes with hnRNP A1 and hnRNP C1 in the nucleus and is a component of the heterogeneous nuclear ribonucleoprotein particles. Our results suggest that DAZAP1 plays a key role in mRNA transport during spermatogenesis.

FIGURE 1.

Nuclear localization of DAZAP1. (A) Immunostaining of DAZAP1 and its derivatives. The two top left panels show the endogenous DAZAP1 in 293T and COS7 cells, respectively, detected with an anti-DAZAP1 antibody. The remaining panels show the expression of Xpress-tagged DAZAP1 and its derivatives in COS7 cells transfected with the respective expression vectors as indicated at the left side of each panel. The structures of DAZAP1 and its derivatives are shown in B. The expressed proteins were detected using an anti-Xpress antibody, and the locations of the signals are summarized in B. (B) Structures of DAZAP1 and its derivatives and their subcellular localization. DAZAP1 contains two RNA-binding domains (RBDs) at the N-terminal portion and a proline-rich C-terminal portion. The C25 segment that is required for the nuclear localization of DAZAP1 is boxed. (N) nuclear, (C) cytoplasmic.

FIGURE 2.

Mutagenesis analyses of the nuclear localization signal of DAZAP1. (A) Subcellular localization of the red fluorescent protein DsRed2 and its fusions with C25 and its derivatives in COS7 cells transfected with the respective expression vectors. (B) Evolutionary conservation of the C25 sequence. Sequences at the C termini of putative DAZAP1 orthologs in the various animals as well as that at the C terminus of the S. pombe mRNA cleavage factor protein are shown, with the positions in the human sequence indicated at the top. Chimpanzee, bovine, and rat DAZAP1 have the same C25 sequence as that of the humans and the mice. Different amino acids are shown in red. (C) Sequences of C25 and its derivatives in the DsRed2 fusion proteins. The first three residues shown in parentheses in front of C25 are encoded by the polylinker of the vector. These residues are removed from all the derivatives. The amino acid residues essential for nuclear localization are indicated by asterisks at the bottom. The subcellular localization of the fusion proteins are indicated at the right. The percentages of cells with nuclear restricted expression (N) and both cytoplasmic and nuclear expression (C + N) are indicated. For fusion proteins D2, D8, and D10, which have two different expression patterns, the patterns present in the majority of cells are shown in A.

FIGURE 3.

Nucleocytoplasmic shuttling of DAZAP1. Heterokaryons between mouse 3T3 cells and HeLa cells expressing Xpress-tagged DAZAP1 (A) or its derivatives (B), and either GFP-hnRNP A1 or GFP-hnRNP C1 are shown. Pictures within a frame show the same set of nuclei stained for the Xpress epitope (red), GFP (green), and DNA (blue). The nuclei of 3T3 cells are indicated. The presence of DAZAP1 and HnRNP A1 but not hnRNP C1 in the mouse nuclei indicates that the former two proteins can shuttle between the nucleus and the cytoplasm.

FIGURE 4.

Transcription-dependent nuclear localization of DAZAP1. COS7 cells transfected with pEGFP-hnRNP A1 (A,B) or pEGFP-hnRNP C1 (C,D) were treated with (B,D) or without (A,C) actinomycin D, and the expression of GFP fusion proteins and endogenous DAZAP1 was examined. Pictures in a frame show the same cells. E–H show the accumulation of DAZAP1 in the cytoplasm at various time points after actinomycin D treatment.

FIGURE 5.

Association of DAZAP1 with hnRNP particles. HnRNP particles were immunoprecipitated from the nucleoplasm of COS7 cells transfected with an expression vector of Xpress-tagged DAZAP1 and Western blotted with antibodies against the various proteins shown at the left. Preimmune serum was used as a control for the specificity of immunoprecipitation.