Elsevier

Fish & Shellfish Immunology

Volume 94, November 2019, Pages 634-642
Fish & Shellfish Immunology

Full length article
Immunity-associated long non-coding RNA and expression in response to bacterial infection in large yellow croaker (Larimichthys crocea)

https://doi.org/10.1016/j.fsi.2019.09.015Get rights and content

Highlights

  • Long non-coding RNA high-throughput sequencing of challenged spleen was conducted in large yellow croaker.

  • A set of 163 lncRNAs were identified as being specifically expressed in the spleen and may be involved in the immune response.

  • LncRNAs show tissue specific expression in different tissues.

Abstract

Long non-coding RNA refers to an RNA transcript of a non-coding protein with a sequence length greater than 200 bp. More and more reports indicated that lncRNA was involved in the regulation of gene expression as a signalling molecule, an inducing molecule, a leader molecule and a scaffold molecule. Previous studies have sequenced the draft genome and several transcriptome data sets for protein-coding genes of the large yellow croaker (Larimichthys crocea), but little is known about the expression and function of lncRNAs in this species. In order to obtain a catalogue of lncRNAs for this croaker, Vibrio parahaemolyticus infection challenge experiment was conducted and long non-coding RNA sequences were obtained. Using high-throughput sequencing of lncRNA, a total of 73,233 high-confidence transcripts were reconstructed in 32,726 loci, recovering most of the expressed reference transcripts, and 6473 novel expressed loci were identified. The tissue expression profile revealed that most lacunas were specifically enriched in distinct tissues. A set of 163 lncRNAs were identified as being specifically expressed in the spleen and may be involved in the immune response. It is the first time to identify specific lncRNAs in the L. crocea systematically in this croaker, aiming to benefit the future genomic study of this species.

Introduction

Large-scale transcriptomic studies have led to surprising discoveries, including the fact that less than 10% of the mammalian genome is dedicated to protein coding and that the genome contains a vast amount of non-protein coding transcripts, which has resulted in debate about the role of non-coding RNAs (ncRNAs) in cell biology [1,2]. In contrast to protein-coding genes, it is possible that the non-coding portion of the genome is related to organism complexity and crucial regulatory processes [3,4]. Traditionally, the regulatory functions of RNA were thought to be limited to ribosomal, messenger and transfer RNA roles, however, the FANTOM and ENCODE projects have annotated thousands of non-coding RNAs including tRNA, rRNA, microRNA and other non-coding RNA genes [5,6], and reported on the function of ncRNAs in various life activities.

In this study, we mainly explore long non-coding RNA (lncRNA). LncRNAs are an important class of ubiquitous nucleotides involved in a variety of biological functions [7]. LncRNAs regulate the expression of other genes by the identity of the signal molecule [8]. For example, the lncRNA-Xist gene and Xist gene function are known to cause the X chromosome to lose activity. An X chromosome in a mammalian female self-regulates silencing by the X inactivation centre (Xic) in order to maintain a dose-compensating effect. Binding of polycomb repressive complex 2 (PRC2) to the repeat-A element at the 5′ end of Xist RNA activates the gene. With Xist gene expression, its transcript is transcribed from the inactive X chromosome and encapsulated on the X chromosome, inhibiting gene expression at the overall level of the chromosome [9,10].

LncRNAs will indirectly regulate gene transcription by inducing target protein or miRNA to science the functional sequence, that is, lncRNAs bind to target proteins or miRNAs to dilute their levels within the cell, affecting their function [[11], [12], [13]]. LncRNAs serve as central platforms for the assembly of multiple related molecules [14]. Although the complex scaffold molecule is traditionally considered to be a protein [15], an increasing number of lncRNAs have been found to be involved in gene-regulation in the form of scaffolds. LncRNAs have multiple domains that can bind to different protein complexes, and some domains can bind to multiple elements, leading to time- and space-dependent transcriptional activation or transcriptional repression. For example, lncRNA-TERC (telomerase RNA) acts as a scaffold to activate telomerase catalytic activity. Telomerase catalytic activity requires the use of two universal telomerase subunits: a complete RNA subunit (TERC) that provides repeat template synthesis and support, and a catalytic protein subunit (TERT), as well as several species-specific accessory proteins. TERC has structural and catalytic activities that contribute to TERT-binding and provide stability [16].

An increasing number of reports show that lncRNAs play an important role in the process of life [[17], [18], [19], [20]]. In addition, the continuous development of sequencing technology means more and more fish genomes have been published, and there are more and more accumulated transcriptome data. However, Studies on lncRNA in fishes immunity are still rare, research of lncRNA has been reported in zebrafish [21,22], salmon [23] and rainbow trout [24,25]. Pauli A et al. (2012) systematically identified long non-coding RNAs expressed during zebrafish embryogenesis. They performed time series of RNA-seq experiments in eight stages during early development in zebrafish, found that zebrafish lncRNA has many of mammalian counterparts: relatively short length, low exon number, low expression and a conservative level comparable to introns. And the temporal expression profile of lncRNAs reveals two novel properties: lncRNAs are expressed in a narrower window of time than protein-encoding genes and lncRNAs are particularly enriched in early embryos [22]. Heena et al. (2015) integrated and provided all relevant dataset resources for zebrafish lncRNA to provide analysis of spatiotemporal expression patterns of lncRNA in the context of various regulatory markers including histone modifications and transcription factors [21]. Sebastian Boltaña et al. (2016) reported the transcriptomic regulation of lncRNA in Atlantic salmon during ISA virus infection [23]. At the same time, the long non-coding RNA regulatory mechanism of Rainbow Trout was integrated and reported [24,25].

Larimichthys crocea is one of the most economically important marine fishes farmed in China. However, the population is suffering from various infectious diseases due to the increasing density and scale of the mariculture. Vibrio parahaemolyticus is a common pathogen causing Vibrio, a disease with high morbidity and mortality that can result in serious economic loss [26]. The whole-genome sequence of the large yellow croaker demonstrated the existence of a well-developed innate immune system and laid the foundation for genome-wide studies in this species [27]. However, few studies have focused on lncRNAs and their biological function in the croaker. In this study, we report on lncRNA related to the immune mechanisms of this croaker in order to better understand the complex immune system and provide basic information about the innate immunity of the species. Furthermore, deciphering the expression pattern of lncRNAs in the large yellow croaker would enable a better understanding of how genes are regulated in teleost.

Section snippets

Sample preparation

Healthy large yellow croakers (weight 150 ± 15g) were obtained from Zhejiang Dahaiyang Technology Co., Ltd. (Zhoushan, Zhejiang Province, China). Fish were maintained at 25 °C in an aerated seawater tank and fed a commercial diet for two weeks prior to the beginning of the experiment. Water in the tank was changed daily. After acclimation, two groups of 10 individuals were randomly chosen for challenge experiments. L. crocea were then intraperitoneally infected with V. parahaemolyticus (1 × 108

RNA-sequence data generation and processing

In the study, L. crocea was challenged with Vibrio parahaemolyticus and V. parahaemolyticus. The spleen tissue of L. crocea was extracted, then the spleen RNA was purified and converted into cDNA libraries that were subjected to paired-end 150 bp sequencing on Illumina's HiSeq platform. A high-throughput sequencing technique was used to carry out the long chain non-coding sequencing of the library, and about 10 G of the raw data were obtained in each of the six libraries. The Q30 (Phred Quality

Discussions

In the previous studies, most of them were focused on protein-coding gene function [[45], [46], [47], [48]], such as the study of innate immunity genes of teleost fish. Gao et al., 2016, reported the post-innate immune responses in various tissues of the Asian swamp eel after Aeromonas hydrophila, Acanthocephalan pallisentis, and poly I:C challenges [49]. Under stress by immunostimulants lipopolysaccharide (LPS), poly I:C, Edwards disease and streptococcus, the rainbow trout congenital gene

Conflicts of interest

The authors declare there is no conflict of interest regarding the publication of this paper.

Acknowledgements

This study was supported by the projects from Science and Technology Major Project of the Ministry of Science and Technology of Zhejiang Province (No. 2016C02055-7) and Science and Technology Program of Zhoushan (No 2018C31091).

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