Sphingomyelin and RNase-resistant RNA in Microdomains of the Inner Nuclear Membrane

Previous evidence has suggested the potential involvement of sphingomyelin and cholesterol located in specific microdomains associated with the inner nuclear membrane in regulating a double-stranded exonuclease-resistant RNA. The objectives of this study were to elucidate the importance of sphingomyelin and cholesterol in safeguarding nuclear RNA from digestion and to scrutinize all RNAs associated with nuclear microdomains. To address these objectives, we investigated the impact of sphingomyelinase or phosphatidylcholine-dependent phospholipase C on nuclear lipid microdomain RNA and subsequently conducted RNA extraction, library preparation, and sequencing of all RNAs present in nuclear microdomains. Only sphingomyelinase treatment rendered the RNA susceptible to RNase treatment. Nuclear lipid microdomains exhibited a higher abundance of retained introns, small nuclear RNA, and long intergenic non-coding RNA compared to whole nuclei, with a notable enrichment in miRNA. The high concentration (20%) of miRNAs in nuclear lipid microdomains was justified by the presence of specific nuclear circular RNA as exons circularized with 'retained' introns, referred to as exon-intron circular RNA (EIciRNA) that act as a sponge of miRNAs. Moreover, it was demonstrated the presence also of ciRNA, circular RNA composed exclusively by introns. The functional analysis indicates that all types of RNase-resistant RNA associated with nuclear lipid microdomains are involved in chromatin organization and brain pathophysiology. In conclusion, nuclear lipid microdomains represent a site of transcription regulation in which circular RNAs, miRNA and double-stranded mRNA, all resistant to RNase, are stabilized by nuclear sphingomyelin. The experimental plan includes: a) purification of nuclear lipid microdomains from HN9.10e cells in the absence or presence of liposomes with equimolecular concentrations of cholesterol and sphingomyelin; 2) localization in the cells of sphingomyelin and cholesterol/sphingomyelin complex with specific fluorescent probes; 3) verification of the impossibility of purifying nuclear lipid microdomains after treatment with sphingomyelinase which degrades sphingomyelin, an essential lipid for their structure; 4) lipid extraction and lipidomics analysis of whole nuclei and nuclear lipid microdomains; 5) extraction and sequencing of RNA in nuclei and nuclear lipid microdomains Please note that the "Pool-ID-1_S1_*_expression_values.xlsx' data was generated from all Pool-ID-1_S1_L00* (multiplexed) raw data pooled together and is linked to the Pool rep1 sample records. Briefly all samples were pooled together and sequenced in different lanes, then fastq files were demultiplexed, according to specific barcodes, and used for subsequent gene expression quantification.

既往研究证据表明，定位于与内核膜（inner nuclear membrane）相关的特定微结构域中的鞘磷脂（sphingomyelin）与胆固醇（cholesterol），可能参与调控双链核酸外切酶抗性RNA（exonuclease-resistant RNA）。本研究旨在阐明鞘磷脂与胆固醇在保护核RNA免受降解过程中的重要作用，并全面分析与核微结构域相关的所有RNA。
为达成上述研究目标，我们分别考察了鞘磷脂酶或磷脂酰胆碱依赖性磷脂酶C对核脂类微结构域RNA的影响，随后对核脂类微结构域中的全部RNA进行了提取、文库制备及测序。仅经鞘磷脂酶处理后的RNA，才对核糖核酸酶（RNase）处理表现出敏感性。
相较于全细胞核，核脂类微结构域中富集了更多的内含子滞留RNA、小核RNA（small nuclear RNA）与长链基因间非编码RNA（long intergenic non-coding RNA），且显著富集微小RNA（miRNA）。核脂类微结构域中微小RNA占比高达20%，这一现象可通过一类特定的核环状RNA（circular RNA）得以解释：这类RNA为带有“滞留”内含子的外显子环化产物，即外显子-内含子环状RNA（exon-intron circular RNA, EIciRNA），可作为微小RNA的海绵分子。此外，研究还证实了仅由内含子构成的环状RNA（ciRNA）的存在。
功能分析显示，所有与核脂类微结构域相关的核糖核酸酶抗性RNA，均参与染色质组织与脑病理生理学过程。
综上，核脂类微结构域是转录调控的重要位点，其中的环状RNA、微小RNA及双链信使RNA（mRNA）均对核糖核酸酶具有抗性，并可通过核鞘磷脂得以稳定。
本研究的实验方案包括：1）从HN9.10e细胞中分离纯化核脂类微结构域，分别设置添加等摩尔浓度胆固醇与鞘磷脂的脂质体（liposomes）组与空白对照组；2）利用特异性荧光探针，观察鞘磷脂及胆固醇-鞘磷脂复合物在细胞内的定位；3）验证经降解鞘磷脂的鞘磷脂酶处理后，无法再纯化得到核脂类微结构域（鞘磷脂是维持其结构的关键脂质）；4）对全细胞核与核脂类微结构域进行脂质提取及脂质组学（lipidomics）分析；5）对细胞核及核脂类微结构域中的RNA进行提取与测序。
请注意，"Pool-ID-1_S1_*_expression_values.xlsx"数据集由所有Pool-ID-1_S1_L00*（多重测序）原始数据合并得到，并与Pool rep1样本记录相关联。简言之，所有样本经合并后在不同测序泳道完成测序，随后根据特定条形码（barcodes）对FASTQ文件进行解复用处理，并用于后续的基因表达定量分析。