Project description:We report the identification and quantification of Watson-Crick modifications in tRNA and rRNA through the use of high throughput sequencing. We apply the recently published DM-tRNA-Seq method to generate demethylase treated and untreated 293T samples, and using computational methods we are able to flag sites using a modification index. This index allows us to generate site-resolved information about modification that we can use to identify and quantify Watson-Crick face modifications in tRNA and rRNA. With the demethylase treated samples, we are able to validate numerous nucleotide modifications from demethylase substrates, and the absence of demethylase action also serves to aid in identification. We find numerous novel modification sites in tRNA as well as striking comparisons between tissues cultures lines. Our study reports a comprehensive analysis of the tRNA modification landscape by identifying sites of modification as well as quantifying modification levels.
Project description:Purpose: High-throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, such those derived from tRNAs. Also recent advances in high-throughput RNA sequencing has revealed the complex RNA modification landscape and the complex role these nucleosides modifactions have in cell signalling, stem cell biology, development and cancer. The goal of this study is to establish how m5C-tRNA methylation and tRNA-derived small RNAs can affect stem cell fucntion in cancer. Methods: four replicates of tRNAs and RNA buisulphite sequencing of wild-type (WT) and NSun2 -/- mouse skin squamous tumours were generated by deep sequencing, using Illumina HiSeq platform. Results: Our analyses reveal that inhibition of post-transcriptional cytosine-5 methylation locks stem cells in this distinct translational inhibition programme that results in tumour progression but that also sentizes cancer cells to genotoxic stress. Transfer RNA (tRNA) sequencing and RNA Bisulphite sequencing of wild-type (WT) and NSun2 -/- mouse skin squamous tumours
Project description:Purpose: High-throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, such those derived from tRNAs. Also recent advances in high-throughput RNA sequencing has revealed the complex RNA modification landscape and the complex role these nucleosides modifactions have in cell signalling, stem cell biology and development. The goal of this study is to establish how tRNA-derived small RNAs can affect stem cell function. Methods: four replicates of tRNAs sequencing of wild-type (WT) and NSun2 -/- mouse embryonic frontal lobes at E18.5 were generated by deep sequencing, using Illumina HiSeq platform. Results: Our analyses reveal that deletion of the cytosine-5 tRNA methylase NSUN2 increases tRNA cleaveage of NSUN2 tRNA substrates.
Project description:Methylation of guanosine on position N7 (m7G) on internal RNA positions have been found in all domains of life and have been implicated in human disease, but m7G modifications has so far only been mapped in a limited number of RNA molecules. Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection of m7G modifications. In our method, m7G modified positions are converted to abasic sites by mild reduction, recorded as cDNA mutations through reverse transcription, sequenced and subsequently detected by identification of positions with increased mutation rates in the reduced sample compared to the control. We show that m7G-MaP-seq efficiently detects m7G modifications in rRNA, including a previously uncharacterised rRNA modification in Arabidopsis thaliana. Furthermore, we identify m7G tRNA modifications in budding yeast, human and arabidopsis tRNA and show that m7G modification occurs before tRNA splicing. We do not find any evidence for internal m7G modifications being present in other small RNA, such as miRNA, snoRNA and sRNA. Likewise, high coverage m7G-MaP-seq analysis of mRNA from E. coli or yeast cells failed to identify any internal m7G modifications.
Project description:We report a m3C-specific high-throughput sequencing techinque, Hydrazine-Aniline Cleavage sequencing (HAC-seq) to profile the m3C methylome at single-nucleotide resolution. We apply HAC-seq to analyze ribosomal RNA-depleted total RNA from MCF7 cells. We find that tRNA are the predominant m3C-modified RNA species. We find no evidence of m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNA in MCF7 cells.