title: 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 (Psi) 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.
authors: Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637, USA.
authors: Qing
authors: Q
authors: Dai
authors: Robert
authors: R
authors: Fong
authors: Mridusmita
authors: M
authors: Saikia
authors: David
authors: D
authors: Stephenson
authors: Yi-tao
authors: YT
authors: Yu
authors: Tao
authors: T
authors: Pan
authors: Joseph A
authors: JA
authors: Piccirilli
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D011088:DNA Ligases; D009843:Oligoribonucleotides / Q000737:chemistry; D011560:Pseudouridine / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D015003:Yeasts / Q000235:genetics
chemical_list: D009843:Oligoribonucleotides; D012331:RNA, Fungal; D011560:Pseudouridine; C010223:N-methyladenosine; D011088:DNA Ligases; D000241:Adenosine