Sensitive and quantitative probing of pseudouridine modification in mRNA and long noncoding RNA.

Journal Article (Journal Article)

Pseudouridine (Ψ) is the most abundant RNA modification in cellular RNA present in tRNA/rRNA/snRNA and also in mRNA and long noncoding RNA (lncRNA). Elucidation of Ψ function in mRNA/lncRNA requires mapping and quantitative assessment of its modification fraction at single-base resolution. The most widely used Ψ mapping method for mRNA/lncRNA relies on its reaction with N -Cyclohexyl-N '-(2-morpholinoethyl)carbodiimide (CMC), forming an adduct with the Ψ base in RNA that is detectable by reverse transcription (RT) stops. However, this method has not produced consistent Ψ maps in mRNAs; furthermore, available protocols do not lend confidence to the estimation of Ψ fraction at specific sites, which is a crucial parameter for investigating the biological relevance of mRNA modifications. Here we develop a quantitative RT-PCR based method that can detect and quantify the modification fraction of target Ψ sites in mRNA/lncRNA, termed C MC-RT and l igation a ssisted P CR analysis of Ψ modification (CLAP). The method still relies on RT stop at a CMC-Ψ site, but uses site-specific ligation and PCR to generate two distinct PCR products in the same sample, corresponding to the modified and unmodified site, that are visualized by gel electrophoresis. CLAP not only requires a small amount of cellular RNA to validate Ψ sites but also determines the Ψ fraction semiquantitatively at target sites in mRNA/lncRNA. We determined the Ψ status of four mRNA sites and one lncRNA site whose modification fractions range from 30% to 84% in three human cell lines. Our method enables precise mapping and assessment of Ψ modification levels in low abundance cellular RNAs.

Full Text

Duke Authors

Cited Authors

  • Zhang, W; Eckwahl, MJ; Zhou, KI; Pan, T

Published Date

  • September 2019

Published In

Volume / Issue

  • 25 / 9

Start / End Page

  • 1218 - 1225

PubMed ID

  • 31227565

Pubmed Central ID

  • PMC6800517

Electronic International Standard Serial Number (EISSN)

  • 1469-9001

International Standard Serial Number (ISSN)

  • 1355-8382

Digital Object Identifier (DOI)

  • 10.1261/rna.072124.119

Language

  • eng