Silkworm A To I Rna Editing

Before maturing of the eukaryotic mRNA, Pre-mRNA undergoes several processes of post-transcriptional modification, including alternative splicing, editing and polyadenylation. One type of RNA editing is mediated by Adenosine deaminases that act on RNA (ADARs), which is called A-to-I editing. ADARs are RNA editing enzymes that change adenosines to inosines via a hydrolytic deamination reaction on double- stranded (ds) RNA. Like most dsRNA binding proteins, the enzymes will bind to any dsRNA without apparent sequence specificity. However, once bound, ADARs deaminate certain adenosines more efficiently than others. After editing, inosine(I) is translated as guanosine (G), and most enzymes recognize inosine as guanosine. Thus, ADARs change the primary sequence information in a RNA molecule. In addition, because inosine base-pairs with cytidine, ADARs can change the structure of RNA molecule by changing an AU base-pair to an IU mismatch.Here, as compared to the known editing sites in D. melanogaster, we decided to determine whether transcripts of these genes were targets for ADARs in Bombyx mori (silkworm). Using the BLAST algorithm with full coding region or the exon that the editing sites located, we identified their orthologues in silkworm genome, and specific primers were designed follow the sequences. Whole RNA of silkworm was isolated from unstaged embryo, larvae, pupae, and adult respectively. Genomic DNA was extracted from embryo of silkworm. Reverse transcription-polymerase chain reaction (RT-PCR) was performed and the PCR product was subjected to direct sequence analysis without coloning. Comparing the sequences of cDNA and genomic DNA, we found A to G discrepancies between cDNAs and the corresponding genomic sequence. Further analyse indicates that some of these sites were A-to-I editing sites. Ultimately, we found 9 A-to-I editing sites in 3 genes in silkworm.Among the tree genes that involve the phenomenon of A to I RNA editing, the coding region of synaptotagmin was obtained via PCR amplification. Amino acidsequences were gained follow the nucleic acid sequences, and the tertiary structure of synaptotagmin protein was predicted using RasMol. From the images synthesized by RasMol we found that protein contain editing sites has an addictive alpha helix comparing with the one without editing. In order to study on the function of A to I RNA editing in this gene, we cloned the coding region into the expression vector pGEX-4T-l. Recombinant protein was expressed successfully in E.coli BL21(DE3) induced with IPTG. We hope to get more information via comparing the activity of the two types of protein.Simultaneously, as we were searching for the editing sites of ADARs in silkworm, an interesting alternative splicing region was discovered serendipitously. This phenomenon exits in silkworm nicotinic acetylcholine receptor subunit Dcx6. We found that this gene has two special exons which have the same size of 45nt and share 70% identity with each other. PCR product of this gene was cloned into T vector. Sequence of the clones indicated that there were three types of splicing. The colones contain either of the exon or both. After analyzing the sequences of the clones, we found some interesting relationships between editing and alternative splicing.