The exosome‐binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase

Abstract

The exosome is a conserved multi-subunit ribonuclease complex that functions in 3 0 end processing, turnover and surveillance of nuclear and cytoplasmic RNAs. In the yeast nucleus, the 10-subunit core complex of the exosome (Exo-10) physically and functionally interacts with the Rrp6 exoribonuclease and its associated cofactor Rrp47, the helicase Mtr4 and Mpp6. Here, we show that binding of Mtr4 to Exo-10 in vitro is dependent upon both Rrp6 and Rrp47, whereas Mpp6 binds directly and independently of other cofactors. Crystallographic analyses reveal that the N-terminal domains of Rrp6 and Rrp47 form a highly intertwined structural unit. Rrp6 and Rrp47 synergize to create a composite and conserved surface groove that binds the N-terminus of Mtr4. Mutation of conserved residues within Rrp6 and Mtr4 at the structural interface disrupts their interaction and inhibits growth of strains expressing a C-terminal GFP fusion of Mtr4. These studies provide detailed structural insight into the interaction between the Rrp6-Rrp47 complex and Mtr4, revealing an important link between Mtr4 and the core exosome.

Exosome Cofactors Connect Transcription Termination to RNA Processing by Guiding Terminated Transcripts to the Appropriate Exonuclease within the Nuclear Exosome

“…A recent report that Mpp6 directly binds to the core exosome independently of Rrp6 may also reflect a role of Mpp6 in Dis3-mediated RNA degradation (23). On the other hand, binding of Trf4 to the Nrd1 CID would exclude Mpp6 and lead to Rrp6-dependent processing of the transcripts via Trf4-Rrp6 and/or Trf4-Mtr4-Rrp6/Rrp47 interactions (19,23). Consistently, deletion of MPP6 increases the interaction of Nrd1 with Rrp6 (Fig.…”

“…In this sense Mpp6 has been previously proposed to stimulate the activity of Dis3 (21). A recent report that Mpp6 directly binds to the core exosome independently of Rrp6 may also reflect a role of Mpp6 in Dis3-mediated RNA degradation (23). On the other hand, binding of Trf4 to the Nrd1 CID would exclude Mpp6 and lead to Rrp6-dependent processing of the transcripts via Trf4-Rrp6 and/or Trf4-Mtr4-Rrp6/Rrp47 interactions (19,23).…”

“…Other known cofactors of the nuclear exosome are Rrp47 (C1D in humans) and Mpp6 (20,21), which preferentially bind to structured and pyrimidine-rich RNAs, respectively (21,22). Rrp47 directly interacts with the PMC2NT domain of Rrp6 (22) and forms a composite surface for recruiting Mtr4 (23), suggesting that Rrp47 and TRAMP may be functionally linked to the activity of Rrp6. Mpp6 is a nuclear exo-some-associated RNA-binding protein involved in 5.8S rRNA maturation in humans (24), and roles in RNA surveillance and degradation of non-coding RNAs have been reported in yeast (21).…”

Structural insights into the interaction of the nuclear exosome helicase Mtr4 with the preribosomal protein Nop53

“…Cytoplasmic Exo-10 is constitutively bound to the adaptor protein Ski7, which in turn recruits the Ski2-Ski3-Ski8 helicase complex (Brown et al 2000;Araki et al 2001;Halbach et al 2013;Kowalinski et al 2016). Nuclear Exo-10 is constitutively bound to Rrp6-Rrp47 and together with Mpp6 recruits the Mtr4 helicase (Schilders et al 2005;Makino et al 2013aMakino et al , 2015Schuch et al 2014;Wasmuth et al 2014;Zinder et al 2016). These cofactors are conserved from yeast to human and are required for the specific functions of the exosome complex in either subcellular compartment (Sloan et al 2012;Chlebowski et al 2013;Kowalinski et al 2016).…”

“…The nuclear and cytoplasmic exosome-associated helicases share a similar overall architecture (Johnson and Jackson 2013;Ozgur et al 2015). The N-terminal regions of Mtr4 and Ski2 are intrinsically unstructured and interact with other cofactors (Rrp6-Rrp47 and Ski3, respectively) in either a transient or stable manner (Halbach et al 2013;Schuch et al 2014). The helicase regions contain the DExH core typical of processive 3 ′ -5 ′ RNA-dependent ATPases as well as a specialized insertion generally known as the "arch" domain for its characteristic curved structure (Jackson et al 2010;Weir et al 2010;Halbach et al 2012;Johnson and Jackson 2013).…”