Systematic Identification and Characterization of Long Non-Coding RNAs in the Silkworm, Bombyx mori.

Yuqian Wu,Duolian Liu,Quan Zhang,Ping Zhao,Tingcai Cheng,Chun Liu,Renwen Long,Qingyou Xia

doi:10.1371/journal.pone.0147147

Abstract

Long noncoding RNAs (lncRNAs) are emerging as important regulators in various biological processes. However, to date, no systematic characterization of lncRNAs has been reported in the silkworm Bombyx mori. In the present study, we generated eighteen RNA-seq datasets with relatively high depth. Using an in-house designed lncRNA identification pipeline, 11,810 lncRNAs were identified for 5,556 loci. Among these lncRNAs, 474 transcripts were intronic lncRNAs (ilncRNAs), 6,250 transcripts were intergenic lncRNAs (lincRNAs), and 5,086 were natural antisense lncRNAs (lncNATs). Compared with protein-coding mRNAs, silkworm lncRNAs are shorter in terms of full length but longer in terms of exon and intron length. In addition, lncRNAs exhibit a lower level of sequence conservation, more repeat sequences overlapped and higher tissue specificity than protein-coding mRNAs in the silkworm. We found that 69 lncRNA transcripts from 33 gene loci may function as miRNA precursors, and 104 lncRNA transcripts from 72 gene loci may act as competing endogenous RNAs (ceRNAs). In total, 49.47% of all gene loci (2,749/5,556) for which lncRNAs were identified showed sex-biased expression. Co-expression network analysis resulted in 19 modules, 12 of which revealed relatively high tissue specificity. The highlighted darkgoldenrod module was specifically associated with middle and posterior silk glands, and the hub lncRNAs within this module were co-expressed with proteins involved in translation, translocation, and secretory processes, suggesting that these hub lncRNAs may function as regulators of the biosynthesis, translocation, and secretion of silk proteins. This study presents the first comprehensive genome-wide analysis of silkworm lncRNAs and provides an invaluable resource for genetic, evolutionary, and genomic studies of B. mori.

Highlights

Long non-coding RNAs have been arbitrarily defined as non-coding RNAs greater than 200 nucleotides in length
Our results indicate that a large number of silkworm Long non-coding RNAs (lncRNAs) show relatively low expression levels, high spatial specificity, and low levels of sequence conservation compared with silkworm protein-coding mRNAs
Sexed tissues including the anterior silk gland (ASG), the anterior section of middle silk gland (AMSG), the middle section of middle silk gland (MMSG), the posterior section of middle silk gland (PMSG), the posterior silk gland (PSG), gonad, fat body, Malpighian tubule (MpT), and brain were dissected from day 3 fifth instar male and female larvae, respectively

Summary

Introduction

Long non-coding RNAs (lncRNAs) have been arbitrarily defined as non-coding RNAs greater than 200 nucleotides in length. Several lncRNAs have been experimentally validated as important regulators of gene regulation, dosage compensation, development, and behavior in the fruit fly. The neural-specific Drosophila lncRNA CRG (CASK regulatory gene) participates in locomotion and climbing by enhancing its neighboring CASK expression via the recruitment of RNA polymerase II to the CASK promoter regions [23]. Another example of a behavior-related Drosophila lncRNA is Sphinx, whose 5'-flanking 300-bp sequence is conserved across Drosophila species. Additional public silkworm RNA-seq datasets (which represented 3 tissue samples), we systematically identified lncRNAs at the whole-genome level. Our results reveal that a proportion of lncRNAs in the silk gland gene co-expression network core may participate in the biosynthesis, translocation, and secretion of silk proteins

Materials and Methods

Results and Discussion

Conclusion