The Drosha Protein Interaction With Thyroid Hormone Receptor And Its Regulation On MicroRNA During Japanese Flounde Metamorphosis

Japanese flounder(Paralichthys olivaceus) belongs to the Bothidae families of Pleuronectiformes, which is one of the most important economic marine fishes in China.Its typical postembryonic metamorphosis is a good model to study the mechanism of asymmetric development. As Japanese flounder mature, bilaterally symmetric larvae become markedly asymmetrical as one eye translocates across the dorsal midline to the opposite side of the head, resulting in an ocular side that will face the water column, and a blind side that will face the substrate. During the transitional metamorphic period, fish swim at an increasing angle to ultimately settle onto the substrate. Particularly rapid and dramatic remodeling occurs during metamorphosis. Their structures and functions also change, which were thought to be caused by the tissue-specific expression of gene.Thyroid hormone directly or indirectly induced tissue-specific expression of gene.Exogenous thyroid hormone can lead to Japanese flounder metamorphosis in advance,while thiourea can make Japanese flounder metamorphosis stagnate.Micro RNA(mi RNAs) plays an important role during Japanese flounder metamorphosis. Micro RNAs are a class of small(~22 nucleotides) non-coding RNAs that control gene expression by targeting m RNAs and triggering either translational repression or RNA degradation. Two enzymes; Drosha and Dicer, are pivotal in the processing of pri-mi RNA into mature double stranded mi RNA fragments. Drosha is a nuclear enzyme that cleaves primary mi RNA transcripts(pri-mi RNA) into short(~70nucleotides) double-stranded RNA precursors that contain a 3’overhang, known as pre-mi RNA. The pre-mi RNAs are then exported to the cytoplasm where they are cleaved by Dicer into mature double-stranded mi RNA fragments of approximately 22 nucleotides in length.In this study, q RT- PCR and co-immune technique were used to clarify the relationship between the thyroid hormone receptor and RNase Ⅲ enzyme – Drosha. We further revealed target genes of mi RNAs using bioinformatics and dual luciferase report gene system and verified their functions during Japanese flounder metamorphosis,which is helpful because it provides a good foundation for research of metamorphosis of Japanese flounder induced by thyroid hormone. In this study, we focused on four sections:1. The effects of thyroid hormones on expression of Drosha gene in Japanese flounderFirst, we cloned and characterized c DNA sequences of Drosha from Paralichthys olivaceus. The deduced Drosha protein contained two RNA-binding sites. The amino acids sequence comparison showed that the Drosha protein of Japanese flounder had a71-91% similarity with other animals, indicating the high level of gene conservation.Second, real-time quantitative PCR results revealed that Drosha m RNA were rifely expressed in all detected tissues, but the expression pattern were different among each other. Tissue distribution analysis indicated that Drosha were abundantly expressed in brain and musle, and less expressed in gill and kidney. The expression of Drosha highly increases in almost all stages except in unfertilized eggs where it doesn’t express. The expression of Drosha was downregulated by exogenous thyroid hormones and thiourea,which showed that thyroid hormones may influence the transcriptional regulation of Drosha gene. And Drosha gene controlled the mature of micro RNAs, then regulated Japanese flounder metamorphosis.2. Identification of interaction between THR and Drosha protein by Co-Immunoprecipitation(Co-IP)The tissues and cells used in this study were treated with TH and the controlgroups were normal tissues and cells. Through Co-IP, the total protein was obtained by using IP Lysis/Wash Buffer and the THR(or Drosha) immunoprecipitate complex was purified via Co-IP with specific THR or(Drosha) antibody. The complex was separated by SDS-PAGE and detected with anti-Drosha(or THR)by Western Blotting. The results showed Drosha was detected by anti-Drosha in the immunoprecipitates precipitated with anti- THR and THR was detected by anti-THR in the immunoprecipitates precipitated with anti-Drosha in TH group,but they were not detected in control groups.The results confirmed the interaction between THR and Drosha.3.The expression changes of target gene and micro RNA induced by Drosha-si RNAWe took the Drosha gene as a target and designed and synthesized three si RNA sequences. We then transfected flounder embryonic cells by lipofecamine TM RNAi MAX, and established the best silence sequence,dose and time of the transfection by detecting the expression of Drosha m RNA and protein using Realtime-PCR and Western blot technology. The results showed(1) The expression of Drosha m RNA and protein were significantly decreased in Drosha-si RNA group than those in the control group(P<0.05).(2) The expression of Drosha m RNA and protein decreased significantly(sequence/time/concentration) in Drosha-si RNA2 group, transfection concentration at 2μg/ml group, transfection time at 48 h group compared with other groups(P<0.05).(3) The expression of pri-micro RNA did not change, while the expression of mature micro RNA was dramatically decreased in Drosha-si RNA group when compared with the control group.(4) The expression of CDKN2 m RNA was significantly increased while the expression of Cdc42 m RNA was dramatically decreased in Drosha-si RNA group when compared with the control group(P<0.05).4. In silico prediction of micro RNA targets and the functions of target genes during Japanese flounder developmentBased on the Japanese flounder mi RNAs sequence generated using high throughout Solexa sequencing method, the targets of pol-mi R-1,pol-mi R-133 a and pol-mi R-17 were predicted by employing RNAhybrid program(http://bibiserv2.cebitec.uni-bielefeld.de/rnahybrid?id=rnahybrid_view_submission)considering various parameters, including seed region pairing, minimum free energy and 3’end pairing. The targets effects during Japanese flounder development wereanalysed showing that HDAC4 was the direct target of mi R-1and mi R-133 a,α-TM was the direct target of mi R-133 a and Cdc42 was the direct target of mi R-17. We cloned and characterized c DNA sequences of HDAC4,α-TM and Cdc42 from Japanese flounder,Paralichthys olivaceus.Real-time quantitative PCR results revealed that HDAC4 andα-TM m RNA were widely expressed in all detected tissues, and abundantly expressed in muscle. The expression of HDAC4 m RNA was consistent with that of α-TM m RNA in almost all stages. HDAC4 m RNA levels at 3dph(day post hatching) and 36 dph were higher than that at other stages, while the first peak of α-TM m RNA levels at 3dph(day post hatching) and the second peak at 29 dph. Exogenous thyroid hormones either directly or indirectly promoted the expression of HDAC4 and α-TM m RNA during metamorphosis, indicating that HDAC4 and α-TM might directly or indirectly be regulated by thyroid hormone during muscle development in metamorphosis.Cdc42 m RNA and mi R-17 were universally expressed in all detected tissues and stages, and Cdc42 m RNA was abundantly expressed in ovary and brain, while mi R-17 was abundantly expressed in brain, gill, stomach,intestines,and significantly low in the ovary. Cdc42 m RNA levels was increasing and mi R-17 level was decreasing at stage of embryo development. Cdc42 m RNA levels at 17dph(day post hatching) and 36 dph were higher than that at other stages. The mi R-17 expression was found to be inversely correlated with Cdc42 m RNA expression in spatio-temporal of Japanese flounder. In addition, Cdc42 was downregulated, however, mi R-17 was upregulated by exogenous thyroid hormones, indicating mi R-17 was up-regulated in Japanese flounder and was a negative regulator of Cdc42.We previously investigated how TH regulated Japanese flounder metamorphosis from the perspective of micro RNAs. First, we cloned Drosha c DNA and analysed the temporal and spatial pattern of Drosha m RNA,then the effects of exogenous thyroid hormones during Japanese flounder metamorphosis.Second, we identified the interaction between THR and Drosha protein by Co-IP and Drosha control the target gene and micro RNA. Last, we predicted and verified the micro RNA targets and functions of the target genes during Japanese flounder development. In this study, we found a TH-mediated signaling pathway, that was TH-THR-Drosa-micro RNA-target gene-Japanese flounder metamorphosis.This study will help facilitate the understanding of Japanese flounder development and provide a novel theoretical basis on TH inducing metamorphosis.