Purification and In Vitro Analysis of the Exosome Cofactors Nrd1-Nab3 and Trf4-Air2.

In many eukaryotic organisms from yeast to human, the exosome plays an important role in the control of pervasive transcription and in non-coding RNA (ncRNA) processing and quality control by trimming precursor RNAs and degrading aberrant transcripts. In Saccharomyces cerevisiae this function is enabled by the interaction of the exosome with several cofactors: the Nrd1-Nab3 heterodimer and the Trf4-Air2-Mtr4 (TRAMP4) complex. Nrd1 and Nab3 are RNA binding proteins that recognize specific motifs enriched in the target ncRNAs, whereas TRAMP4 adds polyA tails at the 3' end of transcripts and stimulates RNA degradation by the exosome. This chapter provides protocols for the purification of recombinant forms of these exosome cofactors and for the in vitro assessment of their activity.