Substrate selectivity by the exonuclease Rrp6p

The exoribonuclease Rrp6p is critical for RNA decay in the nucleus. While Rrp6p acts on a large range of diverse substrates, it does not indiscriminately degrade all RNAs. How Rrp6p accomplishes this task is not understood. Here, we measure Rrp6p-RNA binding and degradation kinetics in vitro at single-nucleotide resolution and find an intrinsic substrate selectivity that enables Rrp6p to discriminate against specific RNAs. RNA length and the four 3'-terminal nucleotides contribute most to substrate selectivity and collectively enable Rrp6p to discriminate between different RNAs by several orders of magnitude. The most pronounced discrimination is seen against RNAs ending with CCA-3'. These RNAs correspond to 3' termini of uncharged tRNAs, which are not targeted by Rrp6p in cells. The data show that in contrast to many other proteins that use substrate selectivity to preferentially interact with specific RNAs, Rrp6p utilizes its selectivity to discriminate against specific RNAs. This ability allows Rrp6p to target diverse substrates while avoiding a subset of RNAs.