Project description:RNA internal modifications play critical role in development of multicellular organisms and their response to environmental cues. Using nanopore direct RNA sequencing (DRS), we constructed a large in vitro epitranscriptome (IVET) resource from plant cDNA library labeled with m6A, m1A and m5C respectively. Furthermore, after transfer learning, the pre-trained model was used to detect additional RNA internal modification such as m1A, hm5C, m7G and Ψ modification. Finally, we illustrated a global view of epitranscriptome with m6A, m1A, m5C, m7G and Ψ modification in rice seedlings under normal and high salinity environment. In summary, we provided a strategy for creating IVET resource from cDNA library and developed a computational method that use IVET-based transfer learning termed TandemMod for profiling epitranscriptome landscape with co-occupancy of multiple types of RNA modification in plants responsive to environmental signal.
Project description:tRNAs are important regulators of protein synthesis, and dysregulation of their abundance and modifications status is involved in many human diseases, including cancer. Despite the rapid development of novel tRNA sequencing approaches, due to tRNAs stable secondary structure and abundant modification sites, human tRNA landscape has remained mostly unexplored. Here, we evaluated the new nanopore RNA004 chemistry that is combined with an upgraded Dorado RNA base-caller model for tRNA quantification and site-specific modification determination in human cancer models. We demonstrate that this technology is sensitive to changes in tRNA expression between cancer cell types and response to external stress conditions, with highly reproducible results. We also show that analysis of base-calling error rate confirms the presence of known modifications, among them is the cancer-associated tRNAPhe-Wybutosine modification by TRMT12 (TYW2) . Furthermore, utilizing the new feature of RNA004 chemistry for the detection of common modifications, we show site-specific identification of m5C and pseudouridine at their annotated positions as well as in their known tRNA isoacceptors. We also reveal unique new modification sites and pinpoint a possible limitation of this method in differentiating between m1A and m6A chemical isomers. Overall, RNA004 tRNA-seq has the potential to improve human tRNAome characterization of tRNA abundance and site-specific modifications simultaneously.
Project description:To detect the modifed bases in SINEUP RNA, we compared chemically modified in vitro transcribed (IVT) SINEUP-GFP RNA and in-cell transcribed (ICT) SINEUP RNA from SINEUP-GFP and sense EGFP co-transfected HEK293T/17 cells. Comparative study of Nanopore direct RNA sequencing data from non-modified and modified IVT samples against the data from ICT SINEUP RNA sample revealed modified k-mers positions in SINEUP RNA in the cell.