IC4R002-lncRNA-2014-25517485

Project Title

Genome-wide screening and functional analysis identify a large number of long noncoding RNAs involved in the sexual reproduction of rice

The Background of This Project

• Long noncoding RNAs (lncRNAs) play important roles in a wide range of biological processes in mammals and plants. However, the systematic examination of lncRNAs in plants lags behind that in mammals. Recently, lncRNAs have been identified in Arabidopsis and wheat; however, no systematic screening of potential lncRNAs has been reported for the rice genome.
• Non-protein-coding RNAs (ncRNAs) constitute a substantial portion of transcribed sequences with structural, regulatory or unknown functions. Because of these important biological roles, ncRNAs have been of great research interest in recent years. Attention was previously given to small regulatory RNAs (sRNAs), such as microRNAs (miRNAs), which are less than 200 nucleotides in length. ncRNAs longer than 200 nucleotides (long non-coding RNAs (lncRNAs)) were found to have functions associated with virtually every biological process in mammals, and these initial reports initiated a wave of research on lncRNAs that followed the path of sRNA research. Recently, lncRNAs have emerged as potent regulators, particularly in mammals. However, studies on lncRNAs in plants remain at the early stage; only a few lncRNAs have been shown to regulate plant development, especially during reproduction.
• Sexual reproduction is one of the most essential biological processes and occurs in a vast number of species. Numerous studies have been devoted to the identification of reproduction-related genes, making great progress in understanding the reproductive processes of both animals and plants. However, the complex regulatory networks involving these genes remain largely unknown. Intriguingly, many lncRNAs have recently been proven to play important roles in reproductive processes through the regulation of related genes in various species. In mammals, lncRNAs, such as Xist, H19, Kcnq1ot1, bxd and HOTAIR, have been found to be crucial for the precise control of embryogenesis. Notably, several plant lncRNAs have also been demonstrated to participate in reproductive regulation, including COLDAIR, COOLAIR, LDMAR, CsM10 and Zm401, indicating that one of the principal functions of plant lncRNAs might be to regulate plant reproduction. More interestingly, Komiya et al. found that a number of large intergenic non-coding RNAs (lincRNAs) could generate 21-nucleotide phasiRNAs, which associate with the germline-specific Argonaute (AGO) proteinMEL1 in rice, indicating that rice lncRNAs might play a role in the development of pre-meiotic germ cells. Genome-wide analysis is necessary to discover new lncRNAs and is important for the further functional analysis of these RNAs. More than 8,000 lncRNAs have been identified in humans using bioinformatic methods, and approximately 4,000 lncRNAs have been identified in mice. In plants, 6,480 transcripts have been classified as lncRNAs in Arabidopsis, and 125 putative stress-responsive lncRNAs have been identified in wheat. Although rice is a model species for plant development studies and represents a staple food for nearly half of the global population, rice lncRNAs remain poorly characterized, and no systematic screening of potential lncRNAs in the rice genome has been reported.

Plant Culture & Treatment

• Total RNA was obtained from rice anthers before flowering, pistils before flowering, spikelets 5 DAP and shoots 14 DAG; these samples were used for sequencing. The preparation of whole transcriptome libraries and deep sequencing were performed by the Annoroad Gene Technology Corporation (Beijing, PR China). Whole transcriptome libraries were constructed using TruSeq Stranded Total RNA with Ribo-Zero Gold (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system and qPCR (Kapa Biosystems, Woburn, MA, USA). The resulting libraries were sequenced initially on a HiSeq 2000 instrument that generated paired-end reads of 100 nucleotides. The sequencing data have been submitted to the NCBI Sequence Read Archive (SRA accession number SRP047482).

Figure 1 An integrative computational pipeline for the systematic identification of lncRNAs in rice.

Research Findings

• To systematically identify lncRNAs related to rice reproduction, we performed whole transcriptome ssRNA-seq of rice anthers, pistils, seeds that were harvested 5 DAP and shoots that were harvested 14 DAG (the sequencing results included 3.89×10 8 reads; Additional file 1; Sequence Read Archive (SRA) accession number SRP047482). We then developed a rice lncRNA computational identification pipeline based on RNA-seq data (Figure 1) using 4 whole transcriptome ssRNA-seq data sets and 40 available poly(A) RNA-seq data sets (1.23×10 9 reads). These datasets covered most of the organs and stages involved in rice reproduction (Additional file 1) and were suitable for the identification of reproduction-related lncRNAs. Our lncRNA identification strategy comprised three key procedures (Figure 1).First, the rice transcriptome was reconstructed from all of the RNA-seq datasets using Cufflink2.0. After filtering out infrequently expressed transcripts (those showing FPKM (fragments per kilobase of transcript per million mapped reads) scores <0.5 in all samples) and transcripts without strand information, we recovered 77.4% (30,219/39,045) of the non-transposable element (non-TE)-related mRNAs in the datasets (the mRNAs discussed in the following sections are non-TE related mRNAs unless otherwise specified). The efficient recovery of known protein-coding genes indicated that the dataset employed here was suitable for the recovery of novel transcribed regions of the rice genome.

Table 1 Effect of ozone stress on phenotypic values

Labs working on this Project

• Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, School of Life Science, Sun Yat-Sen University, Guangzhou 510275, PR China

Corresponding Author

• Yue-Qin Chen (lsscyq@mail.sysu.edu.cn)