Construction of Specific Parallel Amplification of RNA Ends (SPARE) libraries for the systematic identification of plant microRNA processing intermediates

Abstract

MicroRNAs (miRNAs) are small RNAs that derive from endogenous precursors harboring foldback structures. Plant miRNA precursors are quite variable in their size and shape. Still, the miRNA processing machinery, consisting of DICER-LIKE1 (DCL1) and accessory proteins recognize structural features on the precursors to cleave them at specific places releasing the mature miRNAs. The identification of miRNA processing intermediates in plants has mostly relied on a modified 5′ RACE method, designed to detect the 5′ end of uncapped RNAs. However, this method is time consuming and is, therefore, only practical for the analysis of a handful miRNAs. Here, we present a modification of this approach in order to perform genome-wide analysis of miRNA processing intermediates. Briefly, a reverse transcription is performed with a mixture of specific primers designed against all known miRNA precursors. miRNA processing intermediates are then specifically amplified to generate a library and subjected to deep sequencing. This method, called SPARE (Specific Parallel Amplification of 5′ RNA Ends) allows the identification of processing intermediates for most of the Arabidopsis miRNAs. The results enable the determination of the DCL1 processing direction and the cleavage sites introduced by miRNA processing machinery in the precursors. The SPARE method can be easily adapted to detect miRNA-processing intermediates in other systems.