Characterization And Functional Analysis Of Tudor-SN Gene In Silkworm, Bombyx Mori

During a decade after the discovery of RNAi phenomenon in C.elegans 1998, the technology of RNAi has been widely performed in various organisms. However, proteins participating to the well-known mechanism are identified to be somehow different in distinct species, due to the progressive dissection of RNAi mechanism. Meanwhile there are many proteins involving into RNAi pathway remaining to be elusive. Tudor-SN(TSN)is one of them, which is the first one isolated from RNA induced silencing complex(RISC), but the specific action of Tudor-SN in the RISC complex is still unknown. Full-length Tudor-SN consists of five staphylococcal nuclease domains (four complete SN domains and a partial SN domain) and a Tudor domain and is extensively conserved in many species. To be surprised, TSN protein plays different roles in different organisms, and is related to multifarious important pathways, such as RNA transport, transcriptional co-activation, RNA splicing, digesting edited microRNA and so on. The versatile protein is known to participate in so many pathways owing to its complicated protein structure. As concerned as silkworm, the model animal of Lepidoptera, it has been sequenced gnomonically. And RNAi has been adopted to study functions of its genes for several years. However, reports about the RNAi mechanism of silkworm are very scarce. Considering all above, we investigated TSN of silkworm by cloning its CDS, comparing its putative amino acid sequence, sub-cellular localizing, making interacting between TSN with AGO1 and AG02 in silkworm cells by Bimolecular Fluorescence complementation (BiFC) constructing transgenic cell line expressing cyclic luciferase for real-Time sensing of caspase-3 activities to test cell apoptosis after treatment of dsRNA, by the way analyzing the function of tsn in RISC with the method of transient transfection of dsRNA at different dosages and constructing a transgenic cell line over expressing tsn gene to test the efficiency of RNAi. So all results are as follows:1. Analysis of sequence and expression profile of tsn gene in silkwormThe full-length cDNA sequences are 3362bp in length and include a complete open reading frame (ORF) of 2267bp that encodes a deduced protein of 888 amino acids, 186bp of 5'-UTR and 509bp of 3'-UTR.Molecular mass is 98.88kD and PI is 8.55. TSN protein contains four complete SN domains, a partial SN domain and Tudor domain with five conserved sites for formation of aromatic cage, which determine the interaction of TSN with other proteins. TSN in silkworm has 52%,51%,43% identity and 70%,67%,63% similarity with fly, nematode and human respectively. Using RT-PCR, tsn gene was found to express in all the tissues tested, mainly in testis, ovary and silk gland, but a little in blood and malpighian tubles. Besides, tsn gene was mostly expressed in cytoplasm of cell confirmed by immunostaining.2.Interactions between TSN with AGO 1 and AG02 in silkworm cellsBy construction of bimolecular fluorescence complementation assay in BmN4 cells, we confirmed the interactions between TSN with AGO1 and AG02. We found that there was a complex formed in cytoplasm based on the combinations of TSN and AGO proteins. And the complexes look like to be localized in some organelle. Because of the resent report, that AGO1 and AG02 involved in repression of translation in P-boby, we speculated that TSN may be associated with repression of translation induced by AGO1 and AG02 respectively.3. Analysis of antagonism between RNA editing and RNAi pathway in silkworm BmN4 cellsThere is actually an antagonism between A-I RNA editing and RNAi pathway reported in a few species. The edited dsRNA by deaminase(ADAR) will be bound and digested by TSN. In genome of silkworm, there is a adar gene. And we found adar gene was mainly expressed in head and integument. In silkworm cells, we preformed RNAi against adar and tsn genes to test RNAi efficiency compared with only dsRNA against firefly luciferase gene. We found 72 hours after co-transfections knocking down the expression of adar and tsn genes did not affect the efficiency of RNAi. So, our data indicated that there was not an obvious antagonism between RNA editing and RNAi pathway in BmN4 cells, in our conditions.4. Apoptosis test in BmN4 cells after RNAi against adar and tsn genesBecause of significance of RNA editing induced by ADAR for cell and body survival, and that tsn knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility, so we presumed from all above maintained apoptosis would be turned up after knocking down those two genes. Subsequently, we constructed a transgenic cell line over-expressing cyclic luciferase for real-Time sensing of caspase-3 activities. But with this kind of transgenic cells we did not find any phenotype caused by dsRNA treatment morphologically and molecularly, compared with positive control, which was transfected with plasmid over-expressing human p53 gene.5. Analysis of RNA efficiency after RNAi against tsn gene and over-expressing of tsn gene in BmN4 cellsBy the method of transient detection after RNAi, that is instantaneous detection after transfection 24h to 48h, we found the expression level of report gene gradually increased during the dsRNA dosages from 2.5ng to 40ng,36 hours after transfection, indicating the increasing of RNAi efficiency. On the contrary,80ng dsRNA treatment leaded to decrease the RNAi efficiency. The former was due to knocking down of tsn gene, but the latter was caused by mechanism of non-specific of RNAi. On the other hand, we constructed a transgenic cell line to over-express tsn gene in BmN4 cells. Subsequently, we performed RNAi against report gene in this kind of cells. However, we did not find any difference in RNAi efficiency, comparing with the normal cells.In conclusion, we has sub-cellularly localized tsn gene in cytoplasm of silkworm cells. By construction of bimolecular fluorescence complementation assay in BmN4 cells, we confirmed the interactions between TSN with AGO1 and AG02. We has constructed a transgenic cell line over-expressing cyclic luciferase for real-Time sensing of caspase-3 activities. But with this kind of transgenic cells we did not find any phenotypes caused by dsRNA treatment morphologically and molecularly. Our data indicated that there was not an obvious antagonism between RNA editing and RNAi pathway in BmN4 cells, in our conditions. By the method of transient detection after RNAi, we found TSN protein played some roles in RNAi of silkworm. But over-expression of tsn gene could not directly increase RNAi efficiency in BmN4 cells.