Screening And Studies On Long Non-coding RNA In Preimplantation Mouse Embryos

Preimplantation development is an amazing and precisely regulated process, and it is no doubt that lucubrating preimplantation development is important for both reproductive biology and regenerative medicine. A series of important developmental events happened only in preimplantation embryos, such as zygote genome activation(ZGA). Long non-coding RNAs(lnc RNAs) have emerged as a new aspect of biology and many lnc RNAs were identified in preimplantation embryos by sequencing, however, no one can answer the questions whether lnc RNAs are involved in regulation of preimplantation development, and if any, what role do these lnc RNAs play. Besides, mouse endogenous retrovirus(mERV) are mostly active in preimplantation embryos, but what role they play are unknown. Then we asked whether there are some mERV cognate lnc RNAs invoving in preimplantation development.Here, we directional screen m ERV cognae lnc RNAs using critically designed primers targeting m ERVs, and sudy the function of one of them by interference in preimplantation embryos. The main results are as follows. Thirty six novel transcripts were identified and the sequencing results were analyzed through UCSC blat tool(http://genome.ucsc.edu/index.html). Most of these 36 novel transcripts(30/38) are ERV cognate in consistent with our expectation. In sum, 23 are GLN associated and 5 are Mu ERVL associated; Molecular cloning by SSRT and RACE confirmed that lnc-GET are spliced, and polyadenylated transcripts, containing 2 variants lnc-GET1(6285 nt) and lnc-GET2(6107 nt), resulting from different splice donor of its only intron, while Dyei is transcribed from the GLN LTR, 665 nt, and has 3 exons. Subcellular localization analysis showed both lnc-GET and Dyei are specifically localized in the nucleus and mainly binding to the chromatin. Secondary structure computational analysis and mi RNA reverse northern blot analysis excluded the possibility that they are pri-mi RNAs, so they are lnc RANs. Using TM-q PCR and RNA-FISH, we found lnc-GET is 2- to 4-cell embryo-specific and Dyei is 4-cell embryo-specific; Depletion of lnc-GET caused the embryonic developmental arrest at 2-cell stage, however, Dyei depletion did not affect the embryonic development. These lnc-GET-depleted 2-cell embryos(lnc-GET-depleted 2C) were stained strong Brd U, but no CAF1, and evident H3S10 ph, indicating they are arrested at late G2 phase. γH2A.X is undetectable in lnc-GET-depleted 2C, indicating DNA damage is not related to the developmental arrest. Moreover, the expression of CDK1, maternal proteins OCT4 and SOX2 and the initiation of major ZGA in lnc-GET-depleted 2C were not been affected. We also didn’t find significant changes in the expression level of forward and reverse transcripts of pericentric satellites, and the DNA-FISH demonstrated pericentric rings has been reorganized into chromocenters; We performed low initial amount RNA-seq to compare the gene expression during najor ZGA between late 2-cell embryos injected with Control-LNA(Control-LNA L2C)(n=2225) and lnc-GET-depleted 2C(n=2042). Consequently, in lnc-GET-depleted 2C, 723 genes were up-regulated, 521 genes were down-regulated(FDR≤0.0001, RPKM ≥1, and fold change > 2), and unexpectedly up to 11060 genes happened abnormal alternative splicing events. These results indicate the lnc-GET-depletion induces dramatic major ZGA disorder. KEGG pathway analysis demonstrated that lnc-GET depletion mainly inhibits the MAPK signaling pathway, showing the key factors in ERK1/2-MAPK or LNK/p38-MAPK signaling pathway repressed dramatically. The inhibition of MAPK signaling pathway might be one of the reasons for the late 2-cell arrest by lnc-GET depletion. GO-BP analysis showed most abnormally spliced genes in lnc-GET-depleted 2C are also associated with cell cycle(especially the M phase) regulation. Lnc-GET might regulate cell cycle through affecting not only transcription, but also the alternative splicing of cell cycle-associated genes; We performed pd-MS to reveal the proteins interacting with lnc-GET, and 4 independent proteins were identified: hn RNP U, FUBP1, DAZAP1, and ILF2. These proteins are present in nucleus of normal late 2-cell and early 4-cell embryos, co-localizing with lnc-GET. Among them, ILF2, FUBP1, and hn RNP U are transcription factors, and ILF2, hnRNP U, FUBP1, and DAZAP1 are spliceosome components. These results fit well with the RNA-seq results, and strongly implicate the important roles of lnc-GET in transcription regulation and splicing contro; Based on Wilcoxon rank sum test corrected by Benjamin multiple test and Wilcoxon rank single test(p = 2.2×10-16), we found that genomic loci of DEGs distribute proximally to the GLK-LTRs. Therefore, lnc-GET may participate transcription regulation through mediating the cis-regulatory activity of GLK-LTRs.