Comparative Poly(A)+ RNA Interactome Capture of RNA Surveillance Mutants

Abstract

RNA exosome complexes degrade many different RNA substrates. Substrate selection and targeting to the exosome complex rely on cofactors, which bind to the substrate RNA, recruit the exosome complex, and help to remodel the associated ribonucleoprotein particle to facilitate RNA degradation. These cofactors are RNA-binding proteins, but their interaction with RNA may be very transient because the RNAs they are bound to are rapidly turned over by the exosome complex. Hence, the cofactors involved in the degradation of many exosome substrates are unknown. Here, we describe comparative poly(A)+ RNA interactome capture as a method to screen for novel RNA-binding proteins involved in exosome-dependent RNA decay.

For this, we compare the poly(A)+ RNA interactome of wild-type cells to that of RNA surveillance mutants, where the decay of exosome substrates is compromised and occupancy of exosome cofactors on RNA is strongly increased. More specifically, protein–RNA complexes in wild-type and mutant cells are UV–cross-linked in vivo after labeling with the photoactivatable nucleoside analogue 4-thiouracil. Following cell lysis, protein–RNA complexes are selected on oligo d(T) beads, subjected to stringent washes, and eluted in a low salt buffer. After RNase digestion of cross-linked RNA, RNA-binding proteins that are enriched in the mutant samples are identified by quantitative mass spectrometry. Here, we quantitatively compare the RNA–protein interactomes of wild-type and rrp6Δ cells to selectively determine cofactors of the nuclear RNA exosome complex in fission yeast. With minor modifications, the comparative interactome approach can easily be adapted to study a range of different RNA-dependent processes in various cellular systems.