Pseudouridine RNA modification detection and quantification by RT-PCR

Pseudourine (Psi) is the most abundant cellular RNA modification, present in tRNA, rRNA, snRNA, mRNA, long noncoding RNA (lncRNA), and others. Psi sites and fractions are dynamically regulated in stress response and across development stages. Although high throughput Psi sequencing methods based on N-Cyclohexyl-N'-(2-morpholinoethyl)carbodiimide (CMC) reaction are available for Psi detection transcriptome-wide, a simple method for the analysis of specific, targeted Psi sites and their fraction quantitation is needed to better investigate Psi function. Here, we describe an RT-PCR and gel electrophoresis based method that can sensitively and quantitatively assess Psi at single-nucleotide resolution in mRNA/lncRNA, termed CMC-RT and ligation assisted PCR analysis of Psi modification (CLAP). The principle of the CMC-method is the reverse transcription stop induced by the CMC-Psi adduct. In CLAP, CMC reaction is first carried out with the RNA sample. Reverse transcription using a non-processive RT produces two cDNA products for each RNA transcript, one with the 3' end at the Psi site, the other read-through product from the unmodified RNA. Using splint oligonucleotide assisted site-specific ligation, these two cDNA products are then visualized on a gel as two distinct PCR products in the same lane corresponding to the Psi-modified and unmodified target site. CLAP validates Psi sites identified by high throughput sequencing, quantifies Psi levels in mRNA and lncRNA, and enables convenient and rapid investigation on the function and mechanism of the Psi modification.

Automated summary

In this study, a RT-PCR and gel electrophoresis based method called CLAP was developed to sensitively and quantitatively assess Psi modification in mRNA/lncRNA. The method visualizes both Psi-modified and unmodified target sites and validates high throughput sequencing results.