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.