The human ZC3H3 and RBM26/27 proteins are critical for PAXT-mediated nuclear RNA decay

Abstract

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.

A Two-Layered Targeting Mechanism Underlies Nuclear RNA Sorting by the Human Exosome

“…Target recognition and decay by the nucleoplasmic RNA exosome requires particular adaptors: the NEXT complex and the PAXT connection (Lubas et al, 2011;Meola et al, 2016;Ogami et al, 2017;Silla et al, 2020). Previous characterization of NEXT and PAXT activities was predominantly based on an RNA biotype-centric division of substrates based on their genomic location and length, whereas exact substrate properties that provide pathway specificity were left uncharacterized.…”

“…Previous substrate analysis, based on a factor depletion/RNA sequencing (RNA-seq) approach, estimated that NEXT primarily targets short immature RNAs, such as PROMPTs, whereas PAXT targets longer and more processed transcripts . However, this distinction was not absolute because some PROMPTs were also affected by depletion of PAXT components Silla et al, 2020). This blurry partition might reflect targeting of different subsets of PROMPTs by each of the two adaptor complexes or, alternatively, may be due to redundant functions of NEXT and PAXT.…”

“…We recently identified the additional PAXT components PABPN1 and ZC3H3 and the paralogous RBM26 and RBM27 factors as being required for PAXT-mediated RNA decay Silla et al, 2020) and therefore inquired whether additive upregulation of NEXT targets could also be achieved upon inactivation of these PAXT components. Although their individual depletion ( Figure S6F) led to only minor upregulation of interrogated NEXT PROMPTs, co-depletion of any of these factors with ZCCHC8 ( Figure S6F) resulted in an additive increase of NEXT PROMPT levels ( Figure 5E).…”

“…The more complex PAXT connection comprises at its core a stable dimer of MTR4 and the Zn-finger protein ZFC3H1 . Additionally, ZFC3H1-MTR4 associates more transiently with the nuclear poly(A) (pA) binding protein PABPN1, the Zn-finger protein ZC3H3, and presumably one of the two RNA binding protein paralogs RBM26 and RBM27 Silla et al, 2020). Although the exact architecture of an active PAXT connection has not been established, individual depletion of any of the abovementioned PAXT components leads to stabilization of a specific subset of nuclear exosome targets (Silla et al, 2020).…”

RNA-binding protein Mub1 and the nuclear RNA exosome act to fine-tune environmental stress response

“…ZCCHC8 interacts with RNA binding protein RMB7 and helicase MTR4 to form the trimeric Nuclear EXosome Targeting (NEXT) (81,83). ZFC3H1 and ZC3H3 form Poly(A) Tail eXosome Targeting (PAXT) complex with MTR4, poly(A) binding protein PABN1 and RNA binding proteins RMB26/27 (53,82,84,85). In addition to the Zinc-finger domain, ZCCHC8 also contains a hydrophobic region called "arch interacting motif" (AIM) that mediates interaction with MTR4 arch domain and the C-terminal region that stimulates helicase activity of MTR4 (86,87).…”