Post-transcriptional RNA modifications can impact the sequence, structure and function of RNA molecules. Over 100 distinct modified nucleotides have been characterized so far in RNA, and RNA modifying enzymes comprise a marked proportion of genomes. However, for certain key modifications their full scope, specific enzymes and biological roles are not well understood - due in part to lack of appropriate transcriptome-wide technologies. This is despite the profound clinical implications observed upon loss/gain of function of many RNA modification enzymes, which can confer diverse genetic syndromes, mental retardation, and infertility. We recently developed Aza-IP, a mechanism-based novel technique that greatly enriches the precise direct targets of RNA cytosine methyltransferases (m5C-RMTs). We tested Aza-IP in HeLa cells, and conducted transcriptome-wide target profiling of two important human m5C-RMTs; DNMT2 and NSUN2. Here, in Aim 1 we apply Aza-IP for target profiling of all eight other m5C-RMTs in both HeLa cells and hESCs.
In Aim 2, we expand the applicability of the mechanism-based enrichment strategy - which can be generally referred to as 'Adduct-IP' - for target profiling of another important class of RNA modifying enzymes; pseudouridine synthases. Our mechanism-based 'Adduct-IP' approaches involve the in-vivo covalent attachment of the enzyme to its substrate. For Aza-IP, 5-azacytidine is transcriptionally incorporated in place of cytosine within all RNAs, leading to covalent attachment of the proper m5C-RMT at its specific target - which is followed by immuno-precipitation, release and cDNA sequencing. Aza-IP specifically enriches the direct targets of m5C- RMTs, and also generates a clear and penetrant C>G transversion 'signature' exclusively at the exact target cytosine. Importantly Aza-IP captures low copy RNA targets and also rare methylation events, which are under the detection limit of other techniques such as RNA bisulfite sequencing. Here, we aim to expand this technique to discover the targets for the remaining eight known human m5C-RMTs. Beyond m5C-RMTs, pseudouridine synthases are another important class of RNA modifying enzymes that isomerize specific uridines in various RNA species into pseudouridine. Pseudouridine is the most abundant modified nucleotide in RNA and is essential for proper structure and function of rRNAs, tRNAs and snRNAs. Although presence of pseudouridine in other RNA species (ncRNAs and mRNAs) is expected, the field is hampered by lack of tools for transcriptome-wide profiling of this modification which would reveal the scope of this modification. Interestingly, pseudouridine synthases can become irreversibly inhibited (through covalent linkage) by the nucleotide analogue 5-flurourdine through a similar mechanism as of 5-aza-C inhibition of m5C-RMTs. Here we aim to apply the Adduct-IP strategy for transcriptome-wide target profiling of selected pseudouridine synthases (such as DKC1), with significant involvement in a genetic disease (dyskeratosis congenita) and also in cancer.
Enzymatic modification of RNA, such as cytosine methylation and pseudouridylation, can greatly change its functional properties. Loss or gain of function of particular human RNA cytosine methyltransferases and pseudouridine synthases have been linked to genetic disorders, cancer, growth defects, brain development defects, mental retardation, infertility and vulnerability to stress conditions. Here, we aim to develop and apply innovative transcriptome-wide profiling technologies to discover the targets of human RNA cytosine methyltransferases and pseudouridine synthases in both human cancer cells and embryonic stem cells - which will provide the necessary foundation for functional analyses on both classes of enzymes.
