Transcriptome-wide base-resolution sequencing of pseudouridine in human normal and AD brain tissues Studies in the past several decades have revealed several layers of complex biological regulations through various RNA species. Among these, arguably the most complicated and recently emerging one involves diverse chemical modifications on almost all RNA species. Over 170 types of different post-transcriptional RNA modifications are known to exist in all kingdoms of life. Research in the past 8-9 years revealed prevalent chemical modifications on messenger RNA and regulatory non-coding RNAs. These modifications, in particular those in mammalian systems, can be dynamic and reversible, and can dramatically impact a broad range of biological processes. For example, about 0.4-0.6% of all adenosines in mammalian mRNA are N6-methyladenosine (m6A) modified, corresponding to on average ~3 m6A residues per mRNA molecule. Lack of m6A methylation is lethal in mammals; animal model studies have shown that RNA m6A methylation is essential or critical to almost all stem cell differentiation processes in most tissue types examined so far. Pseudouridine (?), with a ~0.2% of ?/U ratio in most mammalian mRNA, is the second most abundant mammalian mRNA modification. Its abundance is approximately 1/3 of that of m6A, or one ? per mRNA molecule on average in mammals, and roughly 5-10 folds higher than the abundances of other mammalian mRNA modifications. There are 13 annotated potential pseudouridine synthases in the human genome, and mutations of some of these enzymes can lead to neurodegenerative conditions such as Alzheimer's and Parkinson's diseases. Due to a lack of highly sensitive and quantitative sequencing methods previously, we lacked both understanding of the substrates of these pseudouridine synthases and the comprehensive map of ? in mammals, which will help us understand its characteristics and functions. We propose to apply a newly developed sequencing method from our CEGS (Center for dynamic RNA epitranscriptome) and sequence ? at base-resolution in normal and AD human brain tissues. This effort will generate the first reference pseudouridine epitranscriptome in human brain tissues and will examine alternations in human AD tissues. AD fly models will be employed to determine the contribution of epitranscriptome alterations to AD pathogenesis. Enabled by the new technologies developed in our CEGS, this supplement application represents the first study on this important topic.
Alzheimer's Disease (AD) is an age-related progressive neurodegenerative disorder affecting millions of Americans without effective interventions, and the neuropathological hallmark of AD is the gradual accumulation of neurofibrillary tangles formed by abnormal protein aggregation. Posttranscription regulation could play critical roles in neural developmental processes and neuronal functions. We propose to study a relatively abundant modification of mRNA and lncRNA, pseudouridine, and understand its role in AD pathogenesis and how this modification influences gene expression and neurodegeneration.
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