Sphingomyelin regulates the transcriptional machinery in nuclear lipid microdomains
Nuclear lipid microdomains rich in sphingomyelin and cholesterol content regulate double-stranded exonuclease-resistant RNA. The study aimed to elucidate the importance of nuclear lipid microdomains in safeguarding nuclear RNA from digestion and to scrutinize all RNA present. Thus, we investigated the impact of sphingomyelinase on nuclear lipid microdomain RNA and conducted RNA extraction, library preparation, and sequencing. Sphingomyelinase treatment makes the RNA susceptible to RNase treatment. Nuclear lipid microdomains exhibit 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 is 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 for miRNAs. Moreover, we demonstrate the presence of ciRNA. 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. Overall design: 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.