http://www.paper.edu.cn
Molecular cloning, characterization and expression analysis
of an Interleukin-2 enhancer binding factor 2 homologue
from Tetraodon nigroviridis
Xiang Lixin，Shao Jianzhong*
College of Life Sciences, Zhejiang University, Hangzhou, People’s Republic of China (310012)
E-mail：shaojz@zju.edu.cn
Abstract
Interleukin-2 enhancer binding factor 2 (ILF2) was reported to regulate transcription of interleukin-2
(IL-2), a central cytokine in the regulation of T-cell responses. This property of ILF2 was well
characterized in human and mammals, but little is known in bony fish. In this paper, an ILF2
homologue was cloned and well characterized from Tetraodon nigroviridi for the further investigation
of the function of ILF2 in bony fish. The full-length Tetraodon ILF2 cDNA was 1380 bp in size and
contained an open reading frame (ORF) of 1164 bp that translates into a 387 amino-acid peptide with a
molecular weight of 42.9 kDa, a 5’ untranslated region (UTR) of 57 bp, and a 3’ UTR of 159 bp
containing a poly A tail. The deduced peptide of Tetraodon ILF2 shared an overall identity of
58%~93% with other known ILF2 sequences, and contained two N-glycosylation sites, two
N-myristoylation sites, one RGD cell attachment sequence, six protein kinase C phosphorylation sites,
one amino-terminal RGG-rich single-stranded RNA-binding domain, and a DZF zinc-finger nucleic
acid binding domain, most of which were highly conserved through species compared. Constitutive
expression of Tetraodon ILF2 was observed in all tissues examined, including gill, gut, head kidney,
spleen, liver, brain and heart. The highest expression was detected in heart, followed by liver, head
kidney and brain. Stimulation with LPS did not significantly alter the expression of Tetraodon ILF2.
Gene organization analysis showed that the Tetraodon ILF2 gene have fifteen exons, one more than
other known ILF2 genes in human and mouse. Genes up- and down-stream from the Tetraodon ILF2
were Rpa12, Peroxin-11b, Smad4, Snapap and Txnip homologue, which were different from that in
human and mouse.
Keywords: Tetraodon nigroviridis; interleukin-2 enhancer binding factor 2; cloning; gene
organization; chromosome synteny; in vivo expression study.
1. Introduction
Nuclear factor of activated T cells (NF-AT) is a lymphoid-specific transcription factor that is
thought to be largely responsible for determining the cell type-specific expression of the
interleukin-2 (IL-2) gene. ILF2, also referred as NF45, is a component of the NFAT complex, as
well as ILF3. ILF2 was originally copurified with ILF3 as a heterodimer, by virtue of its ability to
bind to the NFAT binding site presenting on the interleukin-2 (IL-2) promoter, known as the
antigen receptor response element 2 (ARRE-2), from the nuclear extract of stimulated Jurkat
T-cell (Corthesy and Kao, 1994). Besides, ILF2 also has a potentiality to interact with RNA. It
contains an amino-terminal RGG-rich single-stranded RNA binding domain and a DZF
zinc-finger nucleic acid binding domain which is shared with ILF3 and other double-stranded
RNA-binding proteins involved in gene regulation (Zhao et al., 2005). ILF2 represents host
cellular factors that can interact specifically with viral RNAs, including hepatitis B virus RNA
(Shin et al., 2002), bovine diarrheal virus RNA (Isken et al., 2003) and adenovirus
virus-associated RNAII (Liao et al., 1998), as well as ILF3.
ILF2 contributes to gene regulation at levels of transcription, splicing and translation. It was
reported that ILF3 functioned as both positive and negative regulator in gene expression,
depending on the promoter context, while ILF2 acted as a regulator of ILF3 to stimulate its
ability to activate gene expression (Reichman et al., 2002). The function of ILF2 in up-regulating
IL-2 expression has been recently demonstrated in mammals (Zhao et al., 2005). ILF2 and ILF3
-1-
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