The Human ZC3H3 and RBM26/27 Proteins Are Critical for PAXT-mediated Nuclear RNA Decay

Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26 and RBM27 along with the known PAXT-associated protein, PABPN1. The zinc-finger protein ZC3H3 interacts directly with MTR4-ZFC3H1 and loss of any of the newly identified PAXT components results in the accumulation of PAXT substrates. Collectively, our results establish new factors involved in the turnover of nuclear pA+ RNA and suggest that these are limiting for PAXT activity. RNA from HeLa cells was analysed by next generation sequencing upon depletion of EGFP(control), ZC3H3 and co-depletion of RBM26+RBM27. Total RNA was collected for each condition in triplicates. Triplicate ZC3H3 depletion was carried out together with triplicate EGFP depletion (batchZC3H3) and RBM26+RBM27 depletions were performed in 2 separate batches (batch RBM with 2 samples; and batch RBM2 with 1 sample) where each batch contained the same number of EGFP controls.