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. 2009 Aug;81(2):302-9.
doi: 10.1095/biolreprod.108.073221. Epub 2009 Feb 25.

Cloning, characterization, and expression analysis of the novel acetyltransferase retrogene Ard1b in the mouse

Alan Lap-Yin Pang  1 , Stephanie Peacock, Warren Johnson, Deborah H Bear, Owen M Rennert, Wai-Yee Chan
Affiliations

Cloning, characterization, and expression analysis of the novel acetyltransferase retrogene Ard1b in the mouse

Alan Lap-Yin Pang et al. Biol Reprod. 2009 Aug.

Abstract

N-alpha-terminal acetylation is a modification process that occurs cotranslationally on most eukaryotic proteins. The major enzyme responsible for this process, N-alpha-terminal acetyltransferase, is composed of the catalytic subunit ARD1A and the auxiliary subunit NAT1. We cloned, characterized, and studied the expression pattern of Ard1b (also known as Ard2), a novel homolog of the mouse Ard1a. Comparison of the genomic structures suggests that the autosomal Ard1b is a retroposed copy of the X-linked Ard1a. Expression analyses demonstrated a testis predominance of Ard1b. A reciprocal expression pattern between Ard1a and Ard1b is also observed during spermatogenesis, suggesting that Ard1b is expressed to compensate for the loss of Ard1a starting from meiosis. Both ARD1A and ARD1B can interact with NAT1 to constitute a functional N-alpha-terminal acetyltransferase in vitro. The expression of ARD1B protein can be detected in mouse testes but is delayed until the first appearance of round spermatids. In a cell culture model, the inclusion of the long 3' untranslated region of Ard1b leads to reduction of luciferase reporter activity, which implicates its role in translational repression of Ard1b during spermatogenesis. Our results suggest that ARD1B may have an important role in the later course of the spermatogenic process.

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Figures

FIG. 1.
FIG. 1.
Sequence alignment of mouse Ard1a and Ard1b gene products. The alignment of the ORFs (A) of Ard1a and Ard1b transcripts and polypeptide sequences (B) of ARD1A and ARD1B is shown. The region of ARD1A and ARD1B that shows similarity to GCN5-related N-acetyltransferases, a superfamily of enzymes that catalyze the transfer of an acetyl group from acetyl-CoA to primary amine of substrate proteins, is highlighted in gray. Sequence alignment was performed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/). The asterisk refers to identical nucleotide or amino acid residue, whereas the colon and period refer to conserved and semiconserved substitutions in amino acid residue, respectively, in all sequences in the alignment.
FIG. 2.
FIG. 2.
Genomic structure and expression pattern of Ard1a and Ard1b. A) Genomic organization of Ard1a and Ard1b. The ORFs are shown in solid black. Vertical arrows indicate the relative positions of canonical polyadenylation signal (AATAAA) in the genes, whereas horizontal arrows indicate the primers used for expression analysis of Ard1a and Ard1b. The relative positions of the original ESTs (BG082949 and BG069933) representing Ard1b are also shown. A(n) represents poly(A) tail. B) Analysis of tissue expression pattern of Ard1a and Ard1b by RT-PCR. Sizes of the expected PCR products are shown. The contrast of the whole panel for Ard1b was increased (LabWorks 4.0; UVP BioImaging Systems, Upland, CA) to reveal the presence of PCR product from the mouse ovary (lane 7). The nonenhanced version of the panel (framed) is also included. As determined in our previous study [20], the expression level of Ard1b in pachytene spermatocytes and round spermatids is 80-fold and 9-fold, respectively, higher than that in type A spermatogonia. Amplification of β-actin (Actb) was performed as an internal control. M, DNA ladder; 1, liver; 2, brain; 3, heart; 4, lung; 5, spleen; 6, testis; 7, ovary; 8, kidney; 9, Embryonic Day 10–12 embryo; 10, Sertoli cells; 11, type A spermatogonia; 12, pachytene spermatocytes; 13, round spermatids; −, no template control.
FIG. 3.
FIG. 3.
ARD1B interacts with NAT1 to display NAT activity. A) Protein-protein interaction between ARD1B and NAT1. CHO-K1 cells were transiently transfected with HA-tagged ARD1B (ARD1B-HA) and MYC-tagged NAT1 (MYC-NAT1) expression vectors (see Materials and Methods). Immunoprecipitation (IP) was performed using rabbit anti-HA antibody and normal rabbit IgG, followed by Western blotting analysis (WB). The presence of ARD1B-HA and MYC-NAT1 in the same immunoprecipitate indicates a physical interaction between the two proteins in mammalian cells. The same study was performed with ARD1A-HA replacing ARD1B-HA. Duplicate experiments were performed, and similar results were obtained. The immunoreactive band denoted by the asterisk represents nonspecific signal or ARD1A-HA proteins that had a partially truncated N-terminal. B) NAT assay. In vitro-translated proteins were allowed to interact before immunoprecipitation with rabbit normal IgG or rabbit anti-HA antibody. NAT assay was performed in the presence of [3H]acetyl-CoA and human ACTH 1–24 peptide. Annotation on the x-axis: Normal IgG, ARD1A-HA or ARD1B-HA alone immunoprecipitated by normal IgG; ARD1A or ARD1B, ARD1A-HA or ARD1B-HA alone immunoprecipitated by anti-HA antibody; NAT1:ARD1A or NAT1:ARD1B, the said protein complex immunoprecipitated by anti-HA antibody. Results were compared using paired two-tailed Student t-test. Data shown represent the averages of four independent experiments. The error bars represent the SDs of the measurement of radioactivity. cpm indicates the extent of incorporation of the [3H]acetyl group to human ACTH 1–24 peptide in the presence of the different immunoprecipitates.
FIG. 4.
FIG. 4.
Developmental expression pattern of ARD1A and ARD1B during spermatogenesis. A) Specificity test of antibodies. HeLa cells were first transiently transfected with empty phCMV3 vector (Vector) and expression vectors carrying ARD1A-HA (CS2+mARD1/HA), MYC-NAT1 (CS2+MT-mNAT1), and ARD1B-HA (phCMV3-Ard1b/HA) for 2 days. Western blotting was performed to test the specificities of anti-ARD1A and anti-ARD1B antibodies on immunoprecipitates pulled down with rabbit anti-HA antibody from transfected cell lysates. B) Expression pattern of ARD1A and ARD1B in testes isolated from mice of specified ages. The expression of β-actin was monitored as an internal control. The experiment was repeated, and similar results were obtained.
FIG. 5.
FIG. 5.
Repression of luciferase reporter activity in the presence of the Ard1b 3′ UTR. A) The relative luciferase reporter activity in GC-2spd(ts) cells transfected with reporter constructs in the presence or absence of the Ard1b 3′ UTR. Triplicate measurement was performed, and the data shown were the averaged results from three independent experiments. B) Determination of transcript levels by RT-PCR of both firefly (luc2) and Renilla (hRluc) luciferase genes in GC-2spd(ts) cells transfected under the same experimental conditions as in A. Numerical values represent the relative signal intensities of the PCR products, which were determined using the “Area Density” tool of LabWorks 4.0 (UVP BioImaging Systems). Data presented were obtained from two separate experiments (1 and 2). Amplification of Gapdh was performed as an internal control, and its relative signal intensities across all samples are shown. M, DNA ladder; −, no template control. The error bars represent the SDs of the measurement of reporter activity.
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