Purification of C/D-Box snoRNP and Fibrillarin-Associated Factors from Arabidopsis thaliana Plants Using GFP-Tagged Proteins.

Fibrillarin is an evolutionarily conserved methyltransferase found in Archaea, algae, yeast, animals, and plants. It catalyzes the 2'-O-methylation (2'-O-Me) of ribosomal RNAs (rRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), and transfer RNAs (tRNAs) as well as the glutamine methylation (Qme) in histone H2A. Fibrillarin interacts with specific accessory proteins and snoRNA guides to perform 2'-O-Me, although it can also act independently in the case of Qme. Additionally, fibrillarin has been reported to exhibit ribonuclease activity and interact with long non-coding RNAs that regulate gene expression. To identify proteins and RNAs that interact with fibrillarin in vivo, we developed a protocol-detailed herein-combining the immunoprecipitation (IP) of a fibrillarin-Yellow Fluorescent Protein (FIB2-YFP) fusion protein in Arabidopsis thaliana with high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.