Research On Rian Gene Expression Regulations And Its Relationships With DLK1-DIO3Imprinting Region

Currently, it has been dramatically demonstrated that pervasive mammalian genome is transcribed into long and short non-coding RNAs （ncRNAs） in a developmentally regulated pattern. Long ncRNA （lncRNA） was defined as more than200nt ncRNA, although the molecular functions of lncRNAs remained unknown, emerging evidence implicates the functional involvement of lncRNAs in the regulation of gene expression through the modification of chromatin, transport of specific mRNAs, control of pre-mRNA splicing and maintenance of subnuclear structures. LncRNA Rian gene （NR₀28261） was an identified maternal expressed imprinting gene located in Dlkl-Dio3region of mouse distal chromosome12, covered approximately60kb of mouse genome, which contained three of transcripts Meg8, Irm and AB063319, but their characteristics and roles were still lack of systematical recognition. Here, we focused on Rian gene, and mainly analyzed its transcripts expression patterns across mouse development, Rian expression regulation mechanism, then preliminary explored the potential functions at Dlkl-Dio3imprinting region.We first collected preimplanation embryos at oocyte,2-,4-,8-cell, morula and blastula stages to analyze the expression profiles of Rian and the other imprinting genes at Dlkl-Dio3. Quantitative real-time RT-PCR （QRT-PCR） results showed Meg8, Irm, AB063319, Gtl2, Mirg and Dio3all started to express at morula stage excepted for Dlk1, and their expression levels were up-regulated more than two times at blastocyst stage. The expression of Irm was obviously higher than the other two Rian transcripts, the fluorescence in situ hybridization （FISH） results showed Irm was expressed both in inner cell mass and trophoblast cells of blastula, suggesting Irm could be regarded as main transcript of Rian and functional at preimplanation stages. Alternatively, we analyzed Rian imprinted pattern at preimplanation stages, the results showed Dlkl-Dio3imprinting control region IG-DMR already maintained differentially methylated at8-cell stage, but Gt/2-DMR was still unmethylated, suggesting the imprinted pattern of the Dlkl-Dio3region was established by IG-DMR. Then, we characterized the spatiotemporal expression pattern of three mature spliced transcripts Meg8, Irm and AB0633I9of Rian gene at later-mid-gestation. The in situ hybridization results showed they were co-expressed in multi-tissues, especially in neural, muscle and endocrine tissues. However, Irm displayed more restricted expression in tongue and skeletal muscle than Meg8and AB063319. QRT-PCR results showed that Meg8, Irm and AB063319 were extremely similarly expressed in main organs including brain, tongue, heart, lung, liver and kidney during mouse development of E12.5, E15.5and E18.5. Furthermore, Rian transcripts were more restricted expressed in brain, muscle and reproductive organs in postnatal and adult mouse, which expression patterns were similar with maternal lncRNAs Gtl2and Mirg, but not overlapping with paternal coding protein genes Dlkl, suggesting these maternal lncRNAs might be coordinately regulated.Considering Rian were regulated by strictly temporal expression specificity, we further analyzed Rian epigenetic regulation mechanism. After demethylation treatment （5-Aza-2’-deoxycytidine,5-AZA） in N2A cells, Rian, Gtl2and Mirg expression up-regulated in large degree, and Rian and Mirg expression also up-regulated by histone deacetylases inhibitor treatment （4-phenylbutanonic acid,4-PBA）, but not Gtl2. Alternativly, the reason why Rian differently expressed in TM3and MLTC-1cells was mainly regulated by Gtl2-DMR methylation but not IG-DMR or Rian-MR. We also found Irm was obviously activated by histone acetylation roles at its transcription start region in MLTC-1cells by ChIP assay. Rian main transcript Irm（Rian/Irm） was mainly localized in several large speckles along with some weak staining of the nucleoplasm detected by FISH in vitro N2A and MLTC-1cells, which was similar in vivo tissues subcellular location. Of note, we also found Irm expression signals in nuclear periphery of N2A cells, suggesting Rian possibly possessed variant functions in different cell types.At last, we preliminarily explored Rian/Irm potential functions in Dlkl-Dio3. Transient transfection assays found overexpressed full-length and truncation of Irm both inhibiting Dlkl and Dio3expression in an extent in N2A cells, but only found full-length of Irm partly inhibiting Dio3in NIH3T3cells. In contrast, we detected the expression of maternal lncRNAs were up-regulated a little in overexpression N2A cells, especially Meg8nearly increased by50%.These results described above revealed Rian possessed complicated transcription regulated mechanism, and significant temporal expression specificity across embryonic development, implying that Rian was considered as potential functional RNA molecular in organisms, and participated in epigenetic regulations within Dlkl-Dio3imprinting region. It will supply the extent theory foundation for well understanding lncRNA function in embryonic development and genome imprinting.