Mechanism Of Nucleus LncRnas Binding To Specific Genomic Sites To Regulate The Expression Of Neighboring Genes

A large number of studies have shown the complexity of the mammalian transcriptomes.According to ENCODE project statistics,more than 90% of the nucleotides in the human genome can be transcribed.Although transcription seems to be quite common,high-throughput sequencing has found that there are a large number of non-coding RNAs(nc RNAs)in the genome,and protein-coding RNAs account for only about 1-2%.Because nc RNAs cannot encode proteins and lack conservation during biological development and evolution,they were once considered to be "dark matter" in the genome.However,large-scale whole-genome sequencing analysis revealed that nc RNAs show tissue-specific expression characteristics.Nc RNAs longer than 200 nucleotide sequences are termed as long non-coding RNAs(lnc RNAs).Although lnc RNA does not encode protein,it shares some common features with m RNA(both are transcribed by RNA polymerase II and further capped and spliced),and it plays an important role in multiple biological processes,such as X chromosome inactivation and regulation of imprinted gene expression.Lnc RNA also has a specific subcellular localization.The high expression in the nucleus is defined as nucleus lnc RNAs,and the high expression in the cytoplasm is defined as cytoplasm lnc RNAs.Generally,nucleus lnc RNAs can be folded into complex secondary structures,as molecular scaffolds to connect transcription factors or other chromatin modification complexes,and can also bind to genomic specific sites through cis or trans interactions to activate or inhibit gene expression,maybe by establishing epigenetic modifications.Epigenetic modifications include DNA methylation,histone modifications and chromatin remodeling.In previous studies,some activating histone modifications such as H3K4me3,H3K9 ac,H3K27ac,etc.,or inhibitory histone modifications such as H3K27me3,H3K9me3,etc.,have been identified.These apparent modifications have important regulatory effects on gene expression,and they are regulated by specific modifiers.For example,the chromatin modification complexes that catalyze the establishment of H3K4me3 mainly include SET1 and MLL.Current studies have proved that some nucleus lnc RNAs,such as Xist,Hotair,Dum,Swingn,etc.,can recruit specific epigenetic modifiers to establish related epigenetic modifications at specific genomic sites and regulate neighboring gene expression.However,the sequence characteristics and mechanism of the binding of lnc RNA to specific sites in the genome remain to be elucidated.By analyzing the studies of others and ours,we propose the hypothesis that the free sequence(FS)in lnc RNA may be combined with specific genomic sites through the principle of base complementary pairing,thereby establishing specific epigenetic modifications by related factors to regulate the expression of neighboring genes.In order to verify the hypothesis,this study firstly used the differential expression analysis of the transcriptome sequencing data(GSE58757)of the nucleus and cytoplasm of mouse embryonic stem cells to obtain specifically and highly expressed lnc RNAs in the nucleus(2,133)or in the cytoplasm(4,020;Log2Fold Change> 1 & P value <0.05).Then,RNAfold was used to predict the secondary structure of lnc RNAs based on the minimum free energy method,and sequences less than 20 nt of consecutive bases without complementary pairing were extracted and defined as free sequences(FSs).The FSs of each lnc RNAs were used to perform a genome-wide screening to predict the binding sites in the genome by Blastn(the number of mismatched bases < 2).The results showed that the average number of FSs and the number of predicted binding sites in the genome obtained by nucleus lnc RNAs were significantly higher than those of cytoplasm lnc RNAs(P value < 0.01),especially at the 5' and 3' ends.Next,we emplyed Chromatin Isolation by RNA Purification(Ch IRP)to detect the binding sites of two nucleus lnc RNAs(linc1393 and linc1281)and one cytoplasm lnc RNA(linc1450,as a negative control).The results shown that cytoplasm lnc RNAs have almost no specific binding to the genome.The binding sites of nucleus lnc RNAs linc1393 and linc1281 in the genome are 3,627 and 4,087,respectively(P value < 0.01).These binding sites are highly correlated with the binding sites predicted by FSs.(R = 0.75),especially related to the 5' and 3' ends FSs.In order to detect the regulatory effect of lnc RNAs on specific genes through binding sites,we used RNA sequencing data(GSE30245)from lnc RNAs knock-out mouse embryonic stem cells to analyze the distance between the differential expression gene and the closest binding site,and the results showed that the expression fold change of the differential expression gene close to the binding site was significantly higher than that of the differential expression gene far away from the binding site,suggesting that lnc RNAs have a regulatory effect on the expression of genes near the binding site.In order to further clarify the specific regulatory mechanism,we conducted in-depth research on specific nucleus lnc RNA linc1393.Linc1393 knockout resulted in the down-regulation of a large number of gene expressions,indicating that linc1393 has an activating effect on gene expression,and a large number of activated histone modifications within a certain range(±5.0Kb)upstream and downstream of the binding sites of linc1393 were observed,especially H3K4me3.Therefore,we believe that linc1393 mediates the establishment of activated histone modifications around the binding sites to activate the expression of neighboring genes.RNA pulldown combined with mass spectrometry results showed that linc1393 physically binds to the histone methyltransferase SET1 A related to the establishment of H3K4me3.The RIP-q PCR experiment also verified this result.The results showed that linc1393 could recruit SET1 A and other factors to establish H3K4me3 around its genomic binding sites to activate neighboring gene expression.In summary,through the above results,we concluded that:(1)The free sequences,especially at the ends of nucleus lnc RNAs,mediate the genomic binding specifically based on the principle of complementary base pairing;(2)Nucleus lncRNAs have a significant regulatory effect on the expression of genes adjacent to the binding site;(3)Nucleus lncRNAs can regulate the expression of neighboring genes by establishing related histone modifications around the binding site.