J Vet Sci.  2018 Jan;19(1):3-12. 10.4142/jvs.2018.19.1.3.

Chicken RNA-binding protein T-cell internal antigen-1 contributes to stress granule formation in chicken cells and tissues

• Sun Y
• Zhang P
• Zheng H
• Dong L
• Tan L
• Song C
• Qiu X
• Liao Y
• Meng C
• Yu S
• Ding C

• KMID: 2402689
• DOI: http://doi.org/10.4142/jvs.2018.19.1.3

Abstract

T-cell internal antigen-1 (TIA-1) has roles in regulating alternative pre-mRNA splicing, mRNA translation, and stress granule (SG) formation in human cells. As an evolutionarily conserved response to environmental stress, SGs have been reported in various species. However, SG formation in chicken cells and the role of chicken TIA-1 (cTIA-1) in SG assembly has not been elucidated. In the present study, we cloned cTIA-1 and showed that it facilitates the assembly of canonical SGs in both human and chicken cells. Overexpression of the chicken prion-related domain (cPRD) of cTIA-1 that bore an N-terminal green fluorescent protein (GFP) tag (pntGFP-cPRD) or Flag tag (pFlag-cPRD) induced the production of typical SGs. However, C-terminal GFP-tagged cPRD induced notably large cytoplasmic granules that were devoid of endogenous G3BP1 and remained stable when exposed to cycloheximide, indicating that these were not typical SGs, and that the pntGFP tag influences cPRD localization. Finally, endogenous cTIA-1 was recruited to SGs in chicken cells and tissues under environmental stress. Taken together, our study provide evidence that cTIA-1 has a role in canonical SG formation in chicken cells and tissues. Our results also indicate that cPRD is necessary for SG aggregation.

Keyword

T-cell internal antigen-1; chickens; prion-related domain; stress granule; tissues

MeSH Terms

Chickens*
Clone Cells
Cycloheximide
Cytoplasmic Granules
Humans
Protein Biosynthesis
RNA Precursors
RNA-Binding Proteins*
T-Lymphocytes*
Cycloheximide
RNA Precursors
RNA-Binding Proteins

Figure

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  Fig. 1 Schematic representation of 7 expression plasmids, pctGFP-cTIA-1, pctGFP-cRRM, pctGFP-cPRD, pFlag-cTIA-1, pFlag-cRRM, pFlag-cPRD, and pntGFP-cPRD. GFP, green fluorescent protein; cTIA-1, chicken T-cell internal antigen-1; RRM, RNA recognition motif; PRD, prion-related domain; cRRM, chicken RRM; cPRD, chicken PRD.

• 

  Fig. 2 Cytoplasmic fluorescent dots were detected in pctGFP-cTIA-1-transfected HeLa and DF-1 cells that were exposed to environmental stressors. (A) HeLa and DF-1 cells were transfected with pEGFP-N1 and pctGFP-cTIA-1, respectively, followed by treatment at 24 h post-transfection with mock treatment, treatment with arsenite (ARS), heat shock, or infection by Newcastle disease virus (NDV) at an multiplicity of infection of 1 for 18 h. The cells were collected, fixed, and visualized by confocal microscopy. (B) The percentages of cells containing stress granule (SGs) per high-powered fields (HPF) were quantified visually by manual counting. A total of 10 pictures in 20 HPF were randomly captured in different fields. The data are expressed as the mean of three independent experiments. GFP, green fluorescent protein; cTIA-1, chicken T-cell internal antigen-1.

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  Fig. 3 Exogenously expressed pctGFP-cTIA-1 localized with endogenous stress granule markers in HeLa cells. HeLa cells transfected with pctGFP-cTIA-1 were mock treated or treated with arsenite (ARS) for 30 min. The cells were fixed and stained with an anti-G3BP1, PABP, or eIF3 antibody, respectively, and then visualized by confocal microscopy. GFP, green fluorescent protein; cTIA-1, chicken T-cell internal antigen-1; G3BP1, GTPase-activating protein-binding protein 1; DAPI, 4′, 6-diamidino-2-phenylindole; PABP, poly(A)-binding protein; eIF3, eukaryotic initiation factor 3.

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  Fig. 4 pctGFP-cPRD induced the formation of atypical large granules that were stable under cycloheximide (CHX) treatment. (A) HeLa cells transfected with pctGFP-cTIA-1, pctGFP-cPRD, or pctGFP-cRRM were mock treated or treated with arsenite (ARS) for 30 min. The cells were fixed and stained with an anti-G3BP1 antibody and then visualized by confocal microscopy. (B) HeLa cells transfected with pctGFP-cTIA-1, pctGFP-cPRD, or pctGFP-cRRM were treated with CHX. After 2 h, the cells were mock treated or treated with ARS for 30 min. The cells were fixed and stained with anti-G3BP1 antibody and then visualized by confocal microscopy. G3BP1, GTPase-activating protein-binding protein 1; DAPI, 4′, 6-diamidino-2-phenylindole; GFP, green fluorescent protein; cTIA-1, chicken T-cell internal antigen-1; cPRD, chicken prion-related domain; cRRM, chicken RNA recognition motif.

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  Fig. 5 The effect of the GFP tag on cPRD localization. HeLa cells transfected with pFlag-cTIA-1 (A) or pFlag-cRRM (B) were mock treated or treated with arsenite (ARS) for 30 min. Cells were fixed and stained with anti-Flag (only for pFlag-cTIA-1 transfection group) and anti-G3BP1 antibody and then visualized by confocal microscopy. (C) HeLa cells transfected with pctGFP-cPRD, pntGFP-cPRD, or pFlag-cPRD were mock treated or treated with ARS for 30 min. Cells were fixed and stained with anti-Flag (only for pFlag-cTIA-1 transfection group) and anti-G3BP1 antibody and then visualized by confocal microscopy. (D) HeLa cells transfected with pctGFP-cPRD, pntGFP-cPRD, or pFlag-cPRD were treated with cycloheximide (CHX). After 2 h, the cells were mock treated or treated with ARS for 30 min. The cells were fixed and stained with an anti-G3BP1 antibody and then visualized by confocal microscopy. cTIA-1, chicken T-cell internal antigen-1; G3BP1, GTPase-activating protein-binding protein 1; DAPI, 4′, 6-diamidino-2-phenylindole; cRRM, chicken RNA recognition motif; GFP, green fluorescent protein; cPRD, chicken prion-related domain.

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  Fig. 6 (A) HeLa cells were mock treated or treated with arsenite (ARS), heat shock, or infected with a virus. The cells were fixed and stained with an anti-TIA-1 antibody and then visualized by confocal microscopy. Arrows, stress granule (SG) dots. (B) The transcription levels of cTIA-1 were detected by performing real-time polymerase chain reaction. Fold-expressions were calculated based on the level of the cTIA-1 gene in the heart. (C) Two groups of three 7-day-old specific-pathogen-free chickens were mock treated or treated with heat shock. At the indicated time, the mock- and heat-shock-treated chicken hearts were dissected, sectioned, and stained with anti-TIA-1 antibody and then visualized by confocal microscopy. Arrows, SG dots. TIA-1, T-cell internal antigen-1; DAPI, 4′, 6-diamidino-2-phenylindole; NDV, Newcastle disease virus; cTIA-1, chicken TIA-1.

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