Project description:Chemical modifications on mRNA are increasingly recognized as a critical regulatory layer of the flow of genetic information, but quantitative tools to monitor RNA modifications in a whole-transcriptome and site-specific manner are lacking. Here we describe a versatile directed evolution platform that rapidly selects for reverse transcriptases that install mutations during reverse transcription at sites of a given type of RNA modification, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N1-methyladenosine (m1A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m1A sites. We apply the evolved reverse transcriptase to identify thousands of statistically confident m1A sites in human mRNA, some of which can be detected in antibody-free RNA-seq libraries. Together, this work develops and validates the reverse transcriptase evolution platform and provides new tools, analysis methods, and datasets to study m1A biology.
Project description:The 293T cells overexpressing human telomerase reverse transcriptase (hTERT) were lysed and co-immunoprecipitation was performed using hTERT antibody. Then protein mass spectrum was conducted in order to identify the hTERT-interacting proteins.
Project description:To investigate the function of N1-methyladenosine methylome (m1A) in ocular melanoma, we analyzed m1A enrichment level in ocular melanoma and melanocyte cell lines and established ALKBH3 knock down cell lines in 92.1.
Project description:N1-Methyladenosine (m1A) is a prevalent post-transcriptional RNA modification, yet little is known about its abundance, topol- ogy and dynamics in mRNA. Here, we show that m1A is prevalent in Homo sapiens mRNA, which shows an m1A/A ratio of ~0.02%. We develop the m1A-ID-seq technique, based on m1A immunoprecipitation and the inherent ability of m1A to stall reverse tran- scription, as a means for transcriptome-wide m1A profiling. m1A-ID-seq identifies 901 m1A peaks (from 600 genes) in mRNA and noncoding RNA and reveals a prominent feature, enrichment in the 5' untranslated region of mRNA transcripts, that is dis- tinct from the pattern for N6-methyladenosine, the most abundant internal mammalian mRNA modification. Moreover, m1A in mRNA is reversible by ALKBH3, a known DNA/RNA demethylase. Lastly, we show that m1A methylation responds dynamically to stimuli, and we identify hundreds of stress-induced m1A sites. Collectively, our approaches allow comprehensive analysis of m1A modification and provide tools for functional studies of potential epigenetic regulation via the reversible and dynamic m1A methylation.
Project description:Given the interest in the COVID mRNA vaccines, we sought to investigate how the RNA modification N1-methylpseudouridine (and its related modification, pseudouridine) is read by ribosomes and reverse transcriptases. By looking at reverse transcriptase data, we can gain information on how the modification affects duplex stability, which may have important consequences for the tRNA-mRNA interactions found in the ribosome.
Project description:Aicardi-Goutières syndrome (AGS) is a genetically heterogeneous encephalopathy whose pathology is linked to an abnormal type I interferon response induced by self-derived nucleic acids. Data indicate that endogenous retroelements represent one source of interferon-stimulatory self-nucleic acid. No effective therapies are available for this disorder. In this pilot study involving patients with AGS due to mutations in TREX1, RNASEH2A, RNASEH2B or SAMHD1 three nucleoside analogue reverse transcriptase inhibitors (RTIs) were administered over 12 months. Transcription profiling was done by RNA-seq.