A streamlined protocol for the detection of mRNA–sRNA interactions using AMT-crosslinking in vitro

Abstract

Until recently, RNA–RNA interactions were mainly identified by crosslinking RNAs with interacting proteins, RNA proximity ligation and deep sequencing. Recently, AMT-based direct RNA crosslinking was established. Yet, several steps of these procedures are rather inefficient, reducing the output of identified interaction partners. To increase the local concentration of RNA ends, interacting RNAs are often fragmented. However, the resulting 2′,3′-cyclic phosphate and 5′-OH ends are not accepted by T4 RNA ligase and have to be converted to 3′-OH and 5′-phosphate ends. Using an artificial mRNA/sRNA pair, we optimized the workflow downstream of the crosslinking reaction in vitro. The use of a tRNA ligase allows direct fusion of 2′,3′-cyclic phosphate and 5′-OH RNA ends.

Keywords

• Arabidopsis thaliana tRNA ligase (AtRNL)
• M-MuLV reverse transcriptase
• RevertAid transcriptase
• RNA
• tRNA 2′-phosphotransferase 1 (Tpt1)