Study On Bombyx Mori Protein BmAGO2-binding RNA

Argonaute (AGO) protein immunoprecipitation have been widely used in isolation andidentification of miRNA target genes. AGO protein was the core catalytic element ofRNA-induced silencing complex (RISC), which associated with small RNA and its targetedgenes and splice the targeted genes by guiding small RNA. In our previous studies, byco-immunoprecipitation method for isolating the BmAGO2-associated small RNAs (less than50nt), a lot of different types of small RNAs by high-throughput sequencing have been found,including miRNAs, piRNAs, TE-siRNAs and unannotated small RNAs and so on. These smallRNAs may be formed analogous miRISC by combining BmAGO2protein, and then combinedwith targeted mRNAs. This study by co-immunoprecipitation and high throughput sequencingmethod can also analyze and identify the potential targeted mRNA of BmAGO2-associated smallRNAs and build a foundation for the function of BmAGO2-associated small RNAs.The recombinant virus, ie1-vbacmid-BmAGO2, was used to infect BmN cells. AfterHIS-BmAGO2protein expression in BmN cells, we isolated HIS-BmAGO2protein andBmAGO2-associated RNAs by immunoprecipition. The RNAs (greater than200nt) wereisolated and sequenced by using high-throughput sequencing technology. The high-throughputsequencing yielded a total of11,528,900reads. Further analysis obtained851BmAGO2-associated mRNA,1,694transposable elements (TE). GO enriched analysis ofBmAGO2-associated mRNA showed that they are involved in cell structure, cell developmentand cells differentiation. Based on BmAGO2-associated miRNAs and mRNAs, using miRandaRNAhybrid software analysis, we predicted a large number of binding sites of miRNA targetgenes, and confirmed that a miRNA had plurality of target mRNAs, a mRNA may also beaffected a plurality of miRNAs regulation at the same time. The analysis of the unannotatedBmAGO2-associated siRNA and its potential targeted mRNAs found that a total of115BmAGO2-associated mRNAs could match with siRNAs, showing silkworm gene expressioncould be regulated by siRNAs after infectiyng by baculovirus, might be affected by siRNApathway.The majority of BmAGO2-associated small RNAs was TE-derived small RNAs.BmAGO2-associated TE-siRNAs and BmAGO2-associated TEs, which adundance was greater than or equal to50, matching comparison found that111TEs could generate small RNAs, andthese small RNA reverse complementary matching TE. By siRNAs homologous to TEsassociated with AGO2proteins and mediated RNAi, TEs activities are suppressed. Theabundance of bm1645and bm1645-derived siRNA was higher than the other BmAGO2associated TEs and the other TE-derived siRNA. bm1645was a pool of small RNAs. Thefragments of bm1645in generating TE-siRNA was not random, and they were distributed in5major areas. The specific function of the mechanism remains to be further studied.We randomly selected five high expression abundance TEs: Aquila, BmpiggyBac-MER85,bm1456, bm1645and bm1866. Using recombinant plasmids pIEx-1-BmAGO2, we obtainedover-expression BmAGO2in BmN cells. The result of qRT-PCR analysis demonstrated thatup-regulation for the transcriptional level of Aquila and bm1456but down-regulation forBmpiggyBac-MER85, bm1645and bm1866. Meanwhile, by the method of RNAi down-regulatethe transcriptional level of BmAGO2, BmAGO3and BmSIWI in BmN cells, we confirmed thetranscriptional level of5TEs was up-regulation in varying degrees by detection of qRT-PCR. Aspivotal constituent of RISC, lower expression quantity of AGO protein would influence theactivity of RISC. In conclusion, we conjectured that TEs activities are suppressed by TE-derivedsiRNA associated with BmAGO2proteins, TE-derived piRNA associated with BmAGO3andBmSIWI, and induce the activity of RISC. The specific mechanism remain needs more studies.