J Vet Sci.  2017 Dec;18(4):531-540. 10.4142/jvs.2017.18.4.531.

Molecular cloning and characterization of porcine ribosomal protein L21

• Sun WS
• Chun JL
• Kim DH
• Ahn JS
• Kim MK
• Hwang IS
• Kwon DJ
• Hwang S
• Lee JW

• KMID: 2398513
• DOI: http://doi.org/10.4142/jvs.2017.18.4.531

Abstract

Ribosomal protein L21 (RPL21) is a structural component of the 60S subunit of the eukaryotic ribosome. This protein has an important role in protein synthesis and the occurrence of hereditary diseases. Pig is a common laboratory model, however, to the best of our knowledge, its RPL21 gene has not been cloned to date. In this study, we cloned and identified the full-length sequence of the pig RPL21 gene for the first time. In addition, we examined its expression pattern and function by using overexpression or knockdown approaches. As a result, we obtained a 604 bp segment that contains a 483 bp open reading frame encoding 160 amino acids. The pig RPL21 gene is located in the "+" strand of chromosome 11, which spans 2167 bp from 4199792 to 4201958. Pig RPL21 protein has nine strands and two helices in its secondary structure. Pig RPL21 is predominantly expressed in ovary and lung, at lower levels in kidney, small intestine, and skin, and at the lowest levels in heart and liver. Furthermore, RPL21 expression is closely connected with cell proliferation and cell cycle arrest. The results are intended to provide useful information for the further study of pig RPL21.

Keyword

Sus scrofa; conservation; gene expression; molecular cloning; ribosomal protein L21

MeSH Terms

Amino Acids
Cell Cycle Checkpoints
Cell Proliferation
Chromosomes, Human, Pair 11
Clone Cells
Cloning, Molecular*
Female
Gene Expression
Genetic Diseases, Inborn
Heart
Intestine, Small
Kidney
Liver
Lung
Open Reading Frames
Ovary
Ribosomal Proteins*
Ribosomes
Skin
Sus scrofa
Amino Acids
Ribosomal Proteins

Figure

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  Fig. 1 Sequence analysis of the porcine ribosomal protein L21 (RPL21) gene and its inferred amino acid sequence. (A) The schematic representation of the porcine RPL21 gene mapped to chromosome 11. Blue boxes represent the gene's five exons; there are four intron regions between the exons. (B) Nucleotides and the inferred amino acid sequence of the pig RPL21 gene. Pink cylinders are helices, and yellow rectangles with an arrow represent strands in the deduced secondary structure. Blue lines represent coils. The start codon and stop codon are indicated by red boxes. A possible N-glycosylation site is shown in red letters. Sequences before the ATG refer to the noncoding region. The “A” in the initiation codon “ATG” is taken as +1. Numbers on the right side of panel B represent the deduced amino acid sequence. (C) The predicted three-dimensional structure of the pig RPL21 protein. Positions of the N and C termini are shown in capital letters.

• 

  Fig. 2 Conservation and phylogenetic analysis of pig ribosomal protein L21 (RPL21) protein. (A) Multiple sequence alignment of the RPL21 amino acid sequences across various species. In total, twenty conserved regions were identified and are indicated by yellow shading. The mutation site reported to cause hypotrichosis simplex is indicated with a red box. (B) Phylogenetic analysis of porcine RPL21 protein with its 12 homologous counterparts has been published previously (accession Nos. in National Center for Biotechnology Information are presented in brackets). Pig, dog, human, mouse, cattle, and rat share more than 95% identity at the amino acid level. The 13 species are Sus scrofa (S.scrofa); Mus musculus (M.musculus); Bos taurus (B.taurus); Canis lupus familiaris (C.lupus); Rattus norvegicus (R.norvegicus); Danio rerio (D.rerio); Homo sapiens (H.sapiens); Xenopus tropicalis (X.tropicalis); Drosophila melanogaster (D.melanogaster); Caenorhabditis elegans (C.elegans); Saccharomyces cerevisiae S288c (S.cerevisiae), and Schizosaccharomyces pombe 972h (S.pombe).

• 

  Fig. 3 The expression pattern of the ribosomal protein L21 (RPL21) gene in different tissues. (A) A standard curve was constructed with serial ten-fold dilutions of the pCX-RPL21 plasmid DNA by quantitative polymerase chain reaction. (B) The transcript abundance level of RPL21 mRNA in 7 pig tissues. RPL21 is relatively highly expressed in ovary and lung, less so in kidney, small intestine, and skin, and expressed at the lowest level in heart and liver. With the exception of kidney, this expression pattern is similar to that in human tissues (C). Data are presented as mean ± SEM values. Similar results were observed in more than two independent experiments.

• 

  Fig. 4 Overexpression of pig ribosomal protein L21 (RPL21) in a pig fibroblast cell line. (A) Schematic diagram of the vector used for RPL21 overexpression. (B) There was more than an 8-fold change in the expression level of RPL21 mRNA after transfection with pCX-RPL21 vector. (C) The deduced protein with a molecular weight of approximately 18.5 kDa was confirmed by western blotting. (D) Pig RPL21 localized to both cytoplasm and nuclei as detected by double immunofluorescence staining. Beta-actin was used as an internal control for the western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control for the real-time polymerase chain reaction. Data are presented as mean ± SEM values. *p < 0.05 vs. normal. Similar results were observed in more than two independent experiments. Scale bars = 10 µm.

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  Fig. 5 Knockdown of ribosomal protein L21 (RPL21) may inhibit the proliferation of porcine ear skin fibroblast (PEF) cells and induce cell cycle arrest. (A) Real-time polymerase chain reaction (PCR) results show a significant decrease in the level of RPL21 expression following RNA interference. (B) Knockdown of RPL21 significantly inhibited the proliferation of PEF cells. (C) The expression levels of p53 and p21 protein were investigated by western blotting. (D) Knockdown of RPL21 led to G2 arrest. Beta-actin was used as an internal control in the western blotting. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control for real-time PCR. Data are presented as mean ± SEM values. *p < 0.05, **p < 0.01 vs. normal. Similar results were observed in more than two independent experiments.

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