Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6 -methyladenosine

Abstract

Over 100 chemical types of RNA modifications have been identified in thousands of sites in all three domains of life. Recent data suggest that modifications function synergistically to mediate biological function, and that cells may coordinately modulate modification levels for regulatory purposes. However, this area of RNA biology remains largely unexplored due to the lack of robust, high-throughput methods to quantify the extent of modification at specific sites. Recently, we developed a facile enzymatic ligation-based method for detection and quantitation of methylated 2′-hydroxyl groups within RNA. Here we exploit the principles of molecular recognition and nucleic acid chemistry to establish the experimental parameters for ligation-based detection and quantitation of pseudouridine (Ψ) and N6-methyladenosine (m6A), two abundant modifications in eukaryotic rRNA/tRNA and mRNA, respectively. Detection of pseudouridylation at several sites in the large subunit rRNA derived from yeast demonstrates the feasibility of the approach for analysis of pseudouridylation in biological RNA samples.

Pseudouridines in spliceosomal snRNAs

“…Then, in the 1990s, after stagnating for about 40 years, the field of RNA modification began to pick up steam, as different labs began to develop novel techniques to research these non-canonical nucleotides (Bakin and Ofengand, 1993;Maden et al, 1995;Zhao and Yu, 2004a;Saikia et al, 2006;Dai et al, 2007). Powerful experimental systems, such as the Xenopus oocyte system and the yeast biochemistry and genetics systems, provided new ways to go about research into this area, and scientists took full advantage [reviewed in (Wu et al, 2011b)].…”

m6a RNA Methylation: The Implications for Health and Disease

“…Furthermore, the activity of a second demethylase, ALKBH5 has also been established. This resurgence in interest has also been accelerated by overcoming a major obstacle in the detection of m 6 A [28], the development of novel techniques and adaptations of old ones, reviewed by Chandola et al (2014) [29]. These have allowed a greater parity of the occurrence and fluidity of this epigenetic modification [30].…”

Detection of RNA modifications

“…57,58 The RNA containing the supposed modification serves as a template, onto which two complementary DNA oligos are hybridized. These form a nick opposite to the modification site.…”

“…5) is applicable only to abundant RNA species such as rRNA and tRNA. 71,72 The differential ligation approach by Saika et al 57,58 is, in principle, suited to microchip application but has yet to be successfully implemented. A microarray system based on differential hybridization caused by modifications such as m 1 G, m 2 2 G, m 1 A, m 6 2 A and D was developed by Hiley et al 97 While the chip was tiled to cover the entire known yeast transcriptome, the problem of detection of low abundant, i.e., non-stoichiometric nucleotide modifications is not quite resolved.…”