Investigation On Regulation Of Fibroin L Chain By MicroRNAs And Mutually Exclusive Splicing Of Bombyx Mori 14-3-3ζ Gene

MicroRNAs are natural, single-stranded, small noncoding RNA molecules that play important roles in posttranscriptional gene regulation by binding to target mRNAs 3'UTR and suppress its translation or initiate its degradation. So identifying the target gene is very important to study the function of microRNAs.Fibroin is a type of protein created by Bombyx mori (silkworms) in the production of silk, and it is a very important fabric of international trade in the world. Before that, some researchers had done some works about regulation of fibroin L chain by microRNAs in heterologous system. But the way microRNAs take effects are different between in animals and plants. So we use BmN cell as our research system, the EGFP as the report gene to carry out our experiments. We found that miR190 may regulate the expression of the Fib L chain. When refer to the expression analysis of microRNAs in tissues of B.mori, miR-190 expressed more in the silk gland than other tissues, also suggests the expression of Fib L chain may be regulated by the miR-190.In order to deeply study the regulation of fibroin L chain by microRNAs from protein level, through the prokaryotic protein expression system, we get the fibroin L chain protein, and use it to prepare the polyclonal antibody. These all help us make a solid foundation to further study the regulation of fibroin L chain by microRNAs.Mutually exclusive splicing is a strictly regulated type of Alternative splicing which plays important roles in posttranscriptional gene regulation. In this form, only one out of two or more mutually exclusive exons is included in the mature mRNA isoform, thus increased protein diversity. Many works have done to study the mechanism underlies the mutually exclusive splicing, and some models are also given to clarify the behavior.14-3-3ζgene is a member of 14-3-3 gene family which are conserved regulatory molecules expressed in all eukaryotic cells. They have the ability to bind a multitude of functionally diverse signaling proteins. Based on the research work of 14-3-3ζgene of Drosophila melanogaster, we find that silkworm's 14-3-3ζgene also has a couple of mutually exclusive exons 5a and 5b. Through comparative genome analysis of other seven Lepidoptera species, we find that two sequences, selector sequence (SS) and Anchor sequence (AS), in this gene are highly conserved and can be folded into well RNA pairings. Disrupting, compensatory, deletion and insertion mutagenesis show that these inter-intronic RNA pairing ensures mutually exclusive splicing. When RNA pairing can be formed, exon 5a was activated and simultaneous repression of the exon 5b within the loop. Otherwise, exon 5a was repressed and exon 5b was presented in the mature mRNA. Thus we raised the model that controlling the mutually exclusive splicing of silkworm's 14-3-3ζgene, and this work also gave an additional lay for regulating mutually exclusive splicing.