Diverse interleukin-7 mRNA transcripts in Chinese tree shrew (Tupaia belangeri chinensis)

Abstract

Interleukin-7 (IL7) is a pleiotropic cytokine that is actively involved in the immune system. The Chinese tree shrew (Tupaia belangeri chinensis) has been proposed as an alternative experimental animal to primates in biomedical research. However, there is a lack of biological knowledge about the immune system of the tree shrew. In this study, we cloned the IL7 gene (tIL7) in the Chinese tree shrew and quantified the expression of mRNA transcripts in eight tissues (heart, liver, spleen, lung, kidney, intestine, skeletal muscle and brain) from 20 individuals. Eleven tIL7 mRNA transcripts were identified in different tissues. The canonical form (tIL7c) had a length of 1817 bp and encoded a predicted gene product with 177 amino acids. Phylogenetic analyses based on the amino acid sequences revealed a considerably large genetic difference between tree shrew and human. Quantification of mRNA expression of transcripts tIL7c, tIL7-sv1, tIL7-sv2 and tIL7-sv3 showed that these transcripts were expressed in all tissues, albeit the expression levels varied in different tissues. Transcripts tIL7c, tIL7-sv1, and tIL7-sv2 had the lowest expression in brain, and tIL7-sv3 had a dramatically high mRNA expression in skeletal muscle and heart. The mRNA expression levels of tIL7c and tIL7-sv1 were significantly increased upon ploy(I:C) stimulation in tree shrew primary renal cells. As with human full-length IL7, tIL7c, tIL7-sv1, tIL7-sv2 and tIL7-sv3 showed similar a subcellular localization pattern. Our results identified diverse tIL7 transcripts in the Chinese tree shrew, which may play a potential role in modulating IL7-regulated biological effects.

Figure 4. Schematic structure of IL7 mRNA and its transcripts in Chinese tree shrew.

(A) Eleven mRNA transcripts of tIL7 gene. All transcripts were amplified by using primer pair tIL7F and tIL7R. Exons were indicated as boxes. Broken lines indicated alternative splicing of exons in tIL7 transcripts. (B) A 21-bp insertion between exons 3 and 4 in transcripts tIL7-sv9 and tIL7-sv10 would result in a truncated peptide in the C-terminal of predicted protein. Transcripts tIL7-sv6, tIL7-sv9 and tIL7-sv10 complied with the splicing rule and were GT-AG introns.

Figure 1. Nucleotide and deduced amino acid sequence of the IL7 gene in Chinese tree shrew.

The six exons were marked by arrows and alternative splicing fragment of transcript tIL7-sv6 in the 5′-UTR was shaded. Potential polyadenylation signal AATAAA was marked with a box. Three predicted N-glycosylation sites were marked with dots below the respective amino acid. Three single nucleotide polymorphisms were underlined in this gene and were marked by “ = ”.

Figure 2. Alignment of IL7 amino acid sequences in 17 vertebrate species.

The six conserved cysteine residues were marked by dark gray. “*” indicated that all residues in that column were identical in all sequences. Conserved substitutions were marked by “:”. Semi-conserved substitutions were marked by “.”. Signal peptide region was marked in box. Sequence ID information was presented in Table S1.

Figure 3. Phylogenetic trees of the IL7 gene based on nucleotide sequences (A) and deduced protein sequences (B).

The trees were reconstructed using the neighbor-joining method under the complete deletion option, with 1000 bootstrap replications. Sequence ID information was presented in Table S1. The IL7 sequence of tree shrew retrieved from the Ensembl database was marked by “*”.

Figure 5. Expression patterns of tIL7 and its transcripts in eight different tissues from 20 adult Chinese tree shrews.

Relative mRNA levels of tIL7c (A), tIL7-sv1 (B), tIL7-sv2 (C), tIL7-sv3 (D) were normalized to the amount of β-actin mRNA. (E) Overall expression profile of the four transcripts of tIL7. (F) mRNA expression of the tIL7c and its transcripts in primary renal cells transfected with 1 µg/mL short and long poly(I:C) at 6, 12 and 24h. NC – non-transfected cells, poly(I:C) L – long poly(I:C), poly (I:C) S – short poly(I:C). The graph shows the mean ± SD of three independent tests.

Figure 6. Subcellular localization of EGFP-tagged tIL7c and tIL7-sv isoforms in HeLa cells.

HeLa cells were transfected with pEGFP-N2 empty vector and pEGFP-N2 vector with insert of tIL7c or each of the three tIL7c transcripts (tIL7-sv1, tIL7-sv2 and tIL7-sv3) with (SP+) and without (SP–) the signal peptide. Immunofluorescence images were taken at 48 h after transfection. The scale marked in each section of the figure referred to 20 µm.