Project description:Small non-coding RNAs (sncRNAs) are key molecules regulating gene expression. High-throughput RNA-seq greatly advanced sncRNA discovery; however, traditional cDNA library construction protocols generate biased sequencing results, in part due to RNA modifications that interfere with adapter ligation and reverse transcription processes, preventing the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (Panoramic RNA Display by Overcoming RNA modification Aborted Sequencing), employing a combination of enzymatic treatments to remove key RNA modifications that block adapter ligation and reverse transcription during cDNA library construction. PANDORA-Seq enables the discovery of thousands of modified sncRNAs previously undetected, mostly tRNA-derived small RNAs (tsRNAs) and rRNA-derived small RNAs (rsRNAs), which are tissue/cell-specifically detected across mouse brain, liver, spleen, sperm, mouse and human embryonic stem cells, and HeLa cells. Moreover, PANDORA-Seq reveals dynamic changes of tsRNAs and rsRNAs during reprogramming of induced pluripotent stem cells (iPSCs), pointing to future investigations on their potential regulatory functions.
Project description:Emerging small noncoding RNAs (sncRNAs), including tRNA-derived small RNAs (tsRNAs) and rRNA-derived small RNAs (rsRNAs), are critical in diverse biological processes, such as neurological diseases. Traditional sncRNA-seq protocols often miss these sncRNAs due to their modifications. We have recently developed PANDORA-seq, a method enabling more comprehensive detection of modified sncRNAs by overcoming the RNA modifications. Using PANDORA-seq, we have revealed an updated sncRNA profile enriched by tsRNAs/rsRNAs in the mouse cortex and found a particularly significant downregulation of mitochondrial tsRNAs and rsRNAs in an Alzheimer's disease (AD) mouse model, compared to genomic tsRNAs and rsRNAs. Moreover, our integrated analysis of cortex gene expression and sncRNA profiles reveals that those downregulated mitochondrial sncRNAs are negatively correlated with enhanced lysosomal activity, suggesting a crucial interplay between mitochondrial RNA dynamics and lysosomal function in AD. Given the versatile tsRNA/tsRNA molecular actions in cellular regulation, our data provides insights for future mechanistic study of AD with potential therapeutic strategies.
Project description:Purpose: To reveal the small RNA profiles in the sperm using traitional and PANDORA-seq methods Methods: 8 week male WT mice were exposed to DCHP by oral gavage for 4 weeks. Sperm RNA was extracted for further processing Results: PANDORA-seq revealed an abundant amount of tsRNA and rsRNA that were undetected by traditional-seq. Importantly, PANDORA-seq revealed that many differentially regulated sncRNAs also undetected with traditional-seq
Project description:Purpose: To reveal the small RNA profiles in the intima using traditional and PANDORA-seq methods. Methods: 3-week-old LDLR-/- male mice were fed a high cholesterol or low cholesterol diet for 9 weeks. Intimal RNA was extracted by injecting Trizol into the aorta and collecting the flow through for further processing. Results: PANDORA-seq revealed an abundant amount of tsRNA and rsRNA that were undetected by traditional-seq. Importantly, PANDORA-seq revealed that many differentially regulated sncRNAs also undetected with traditional-seq.