Isolation and Bioinformatic Analyses of Small RNAs Interacting with Germ Cell-Specific Argonaute in Rice

The small noncoding RNAs in plants are categorized into two major classes, 21-nucleotides (nt) micro RNA (miRNA) and 21- or 24-nt small-interfering RNA (siRNA). ARGONAUTE (AGO) proteins associate with small RNAs and play central roles in transcriptional and posttranscriptional gene regulation. In plants, AGO1–miRNA complexes mainly regulate developmental processes, and AGO4–siRNA complexes suppress the activity of transposons and exogenous viral infections via RNA-directed DNA methylation. In many animal species, the PIWI-subfamily AGOs interact with PIWI-interacting RNAs (piRNAs), which are most commonly 24–34 nt, and function to tame transposons and to regulate mRNA translation and stability in the germline. The rice protein MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1) is a plant AGO member that has roles specific to development and maintenance of germ cells before meiosis. MEL1-binding small RNAs are mainly 21 nt, have a 5′-terminal cytosine, and are distinct from animal piRNAs. In this chapter, we describe methods for RNA-immunoprecipitation (RNA-IP) using a specific antibody that recognizes MEL1 and subsequent purification of MEL1-associating small RNAs from the IP fraction. We also introduce the bioinformatic procedures including mapping, annotation, and identifying small RNA clusters on the rice genome.