Global Identification of Small RNA Targets in Plants by Sequencing Sliced Ends of Messenger RNAs

Abstract

Small RNAs (microRNAs and other classes of endogenous small interfering RNAs) play important roles in a wide variety of biological processes. However, integration of small RNAs in diverse biological networks relies on the confirmation of their RNA targets. In plants, miRNAs negatively regulate mRNA targets by guiding a cleavage in the complementary site that leaves a 3' polyadenylated RNA possessing monophosphate at its 5' end. This chapter describes a detailed step-by-step protocol for cloning such sliced 3' products in order to identify small RNA targets. Using this protocol, we have identified more than 150 small RNA targets in rice; some are conserved and others are non-conserved targets for rice small RNAs.