In Vivo Cross-Linking and Co-Immunoprecipitation Procedure to Analyze Nuclear tRNA Export Complexes in Yeast Cells

Abstract

tRNAs are small noncoding RNAs that are predominantly known for their roles in protein synthesis and also participate in numerous other functions ranging from retroviral replication to apoptosis. In eukaryotic cells, all tRNAs move bidirectionally, shuttling between the nucleus and the cytoplasm. Bidirectional nuclear-cytoplasmic tRNA trafficking requires a complex set of conserved proteins. Here, we describe an in vivo biochemical methodology in Saccharomyces cerevisiae to assess the ability of proteins implicated in tRNA nuclear export to form nuclear export complexes with tRNAs. This method employs tagged putative tRNA nuclear exporter proteins and co-immunoprecipitation of tRNA-exporter complexes using antibody-conjugated magnetic beads. Because the interaction between nuclear exporters and tRNAs may be transient, this methodology employs strategies to effectively trap tRNA-protein complexes in vivo. This pull-down method can be used to verify and characterize candidate proteins and their potential interactors implicated in tRNA nuclear-cytoplasmic trafficking.

Keywords: Co-immunoprecipitation; Formaldehyde-cross-linking; Primary tRNA nuclear export; RT-PCR; RT-qPCR; Western blot; tRNA nuclear re-export; tRNA retrograde nuclear import.