Abstract
BackgroundSmall non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. Previously, only microRNAs (miRNAs) and piRNAs have been identified in the silkworm, Bombyx mori. Furthermore, only ncRNAs (50-500nt) of intermediate size have been systematically identified in the silkworm.ResultsHere, we performed a systematic identification and analysis of small RNAs (18-50nt) associated with the Bombyx mori argonaute2 (BmAgo2) protein. Using RIP-seq, we identified various types of small ncRNAs associated with BmAGO2. These ncRNAs showed a multimodal length distribution, with three peaks at ~20nt, ~27nt and ~33nt, which included tRNA-, transposable element (TE)-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. The tRNA-derived fragments (tRFs) were found at an extremely high abundance and accounted for 69.90% of the BmAgo2-associated small RNAs. Northern blotting confirmed that many tRFs were expressed or up-regulated only in the BmNPV-infected cells, implying that the tRFs play a prominent role by binding to BmAgo2 during BmNPV infection. Additional evidence suggested that there are potential cleavage sites on the D, anti-codon and TψC loops of the tRNAs. TE-derived small RNAs and piRNAs also accounted for a significant proportion of the BmAgo2-associated small RNAs, suggesting that BmAgo2 could be involved in the maintenance of genome stability by suppressing the activities of transposons guided by these small RNAs. Finally, Northern blotting was also used to confirm the Bombyx 5.8 s rRNA-derived small RNAs, demonstrating that various novel small RNAs exist in the silkworm.ConclusionsUsing an RIP-seq method in combination with Northern blotting, we identified various types of small RNAs associated with the BmAgo2 protein, including tRNA-, TE-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. Our findings provide new clues for future functional studies of the role of small RNAs in insect development and evolution.
Highlights
Small non-coding RNAs are important regulators of gene expression in eukaryotes
Mature miRNAs are a class of ~22nt small RNA molecules that bind to complementary sequences in target genes and regulate gene expression by modulating mRNA translation or stability [1]. miRNAs have been implicated in many cellular processes, including immune mechanisms of biological molecules, stem cell differentiation, cell signal transduction, tumorigenesis, neuronal development and apoptosis [1,2]. smallinterfering RNAs (siRNAs) are a class of double-stranded RNA molecules between 20 and 25nt in length
RNA immunoprecipitation (RIP) of Bombyx mori argonaute2 (BmAgo2) from BmN cells infected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) virus Small RNAs and their targets bind the Ago-containing RNA-induced silencing complex (RISC) complexes, in which the Ago proteins form stable Ago ribonucleoproteins that can be biochemically analyzed [53,57,58]
Summary
Small non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. TRNAs are not very stable and, under stress, can be sequence- cleaved into different sized fragments termed “tRNAderived fragments,” “tRFs” or “sitRNAs.” These fragments could be a novel class of small RNAs. Due to the recent development and application of high-throughput sequencing, many novel small RNAs have been identified, including tRNA-, rRNA-, snoRNAand snRNA-derived small RNAs [16,17,18,19,20,21]. TRNAs are not very stable and, under stress, can be sequence- cleaved into different sized fragments termed “tRNAderived fragments,” “tRFs” or “sitRNAs.” These fragments could be a novel class of small RNAs Because they associate with the Ago protein, it seems likely that they play a role similar to siRNAs in RNAi silencing [16,17]
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