Molecularcloning,characterizationandexpressionanalysisofanInterleukin-2enhancerbindingfactor2homologuefromTetraodonnigroviridis资源-CSDN文库

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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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were also identified as component of spliceosome and participated in the regulation of RNA

splicing (Zhou et al., 2002). In addition, ILF2 may also function on regulating translation. As

reported, ILF2 and ILF3 can interact with the dsRNA-dependent protein kinase (Langland et al.,

1999; Parker et al., 2001), a putative regulator of translation initiation. ILF2 also can interact

with translational elongation initiation factor 2 alpha, beta, and gamma subunits (Ting et al.,

1998). Besides, ILF2 can associate with RNAs in ribonucleoprotein complexes and participate in

regulating of delayed translation of mRNAs (Curtis et al., 1995).

ILF2 has been well characterized in mammals, however, little is known about its existence

and biological function in bony fish. Fish, as a low vertebrate, is considered as an important

evolutionary link between invertebrate and high vertebrate. The investigation of immune-related

genes in fish can provide inspiration for elucidating the genesis and evolutionary progression of

those genes. Tetraodon nigroviridis, as a genome sequenced model organism, provided a good

model system for the study of cloning, identification and functional analysis of novel

immune-related genes in fish. In this paper, we successfully cloned and characterized an

immune-related gene ILF2 homologue from Tetraodon nigroviridis. The result of our research

will pave the way for the further investigation on immunological function of ILF2 in low

vertebrate and elucidating the composing and regulation mechanism of the IL-2 system. Besides,

it will also provide some proof for investigation genesis and evolution of the IL-2 system.

2. Materials and methods

2.1 Experimental fish

Green spotted pufferfish, Tetraodon nigroviridis, one year old of both sexes, weighing

approximately 4~6g, body length 4~5cm, was obtained from the Institute of Fisheries of

Zhejiang, China. The fish were kept in a recirculating water at 26 , and fed with commercial ℃

pellets at a daily ration of 0.7% of their body weight. All fish were held in laboratory for at least

two weeks prior to use in experiments to allow for acclimatization and evaluation of overall fish

health. Only healthy fish, as determined by general appearance and level of activity, were used

for studies.

2.2 Sequences retrieval

The Tetraodon nigroviridis genome database was searched by basic local alignment search

tool analysis using human ILF2 amino acid sequence (Genbank accession no. NP_004506.2),

and two stretches of sequences (chrUn_random:109879005,109882236;

chrUn_random:109889297,109892638) sharing high homology were found. Subsequently, both

sequences were retrieved and further analyzed using Genescan (Burge and Karlin, 1998),

BLAST (Altschul et al., 1990) and FASTA (Pearson and Lipman, 1988) programs. From this

analysis, a possible coding sequence was found and was exploited to design primers for

obtaining the full-length Tetraodon ILF2 cDNA.

2.3 RNA isolation and first strand cDNA synthesis

Healthy fish were sacrificed and total RNA was isolated from two main immune tissues,

spleen and head kidney using TaKaRa RNAiso Reagent (TaKaRa), according to the

manufacturer’s instructions. The concentration of total RNA was measured by spectrophotometry,

and cDNA was synthesized from 1μg total RNA using TaKaRa 3’ Full RACE Core Set (TaKaRa),

according to the manufacturer’s instructions and used as a template for gene cloning by PCR.

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