Immunoprecipitation of MicroRNPs and Directional Cloning of MicroRNAs

MicroRNAs (miRNAs) comprise a class of approx 22-nucleotide (nt) regulatory RNAs, found in plants and animals (1 –8 ). miRNAs contain 5′ phosphates and 3′ hydroxyls and are processed by the Dicer nuclease from one of the stems of longer precursors (pre-miRNAs) that form stem-loop structures (9 –11 ). In animals, pre-miRNAs are themselves processed by the nuclease Drosha from longer transcripts, termed primary miRNAs (pri-miRNAs) (12 ). miRNAs bind to Argonaute proteins and typically associate with additional proteins to form microribonucleoproteins (miRNPs), the effector complexes that mediate translational repression or endonucleolytic cleavage of their cognate mRNAs (6 ,10 , 13 ,14 ). Another class of approx 22-nt RNAs, termed short, interfering RNAs (siRNAs), is inextricably linked to miRNAs (15 ,16 ). siRNAs are processed by Dicer from double-stranded RNAs (dsRNAs), and siRNAs are also bound to Argonaute proteins and might assemble with additional proteins to form complexes termed RNA-induced silencing complexes (RISCs) (10 ). miRNPs and RISCs are functionally equivalent and their core components are Argonaute proteins and miRNAs or siRNAs (17 ). siRNAs might also silence chromatin (9 ,18 ). The development of strategies to clone small RNAs has dramatically accelerated the discovery of miRNAs and our understanding of the function of siRNAs and miRNAs. We describe a method for immunoprecipitation of miRNPs and isolation and cloning of associated small RNAs (6 ). This approach combines techniques developed for the immunoprecipitation of RNPs (19 ,20 ), with techniques developed for the directional cloning of small RNAs (15 ). In principal, this methodology could be used for immunopurification and directional cloning of RNAs from any RNP that contains small RNAs.