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

Abstract

The yeast Nrd1 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNApII) through its CTD-interacting domain (CID) and also associates with the nuclear exosome, thereby acting as both a transcription termination and RNA processing factor. Previously, we found that the Nrd1 CID is required to recruit the nuclear exosome to the Nrd1 complex, but it was not clear which exosome subunits were contacted. Here, we show that two nuclear exosome cofactors, Mpp6 and Trf4, directly and competitively interact with the Nrd1 CID and differentially regulate the association of Nrd1 with two catalytic subunits of the exosome. Importantly, Mpp6 promotes the processing of Nrd1-terminated transcripts preferentially by Dis3, whereas Trf4 leads to Rrp6-dependent processing. This suggests that Mpp6 and Trf4 may play a role in choosing a particular RNA processing route for Nrd1-terminated transcripts within the exosome by guiding the transcripts to the appropriate exonuclease.The Nrd1-Nab3-Sen1 complex terminates transcription of small non-coding RNAs by RNApII 2 (1-4). Nrd1 and Nab3 are sequence-specific RNA binding proteins, and Sen1 helicase (senataxin in humans) has an ATPase activity that directly dissociates RNApII from the templates (5). Nrd1 also recognizes the serine 5-phosphorylated (Ser(P)-5) CTD of RNApII using its CID (6). Because the Ser(P)-5 CTD is prevalent in the early stage of transcription, the Nrd1 CID-RNApII CTD interaction has been suggested to dictate a regional specificity of Nrd1-Nab3-Sen1-dependent transcription termination (7). Indeed, Nrd1havingtheCIDofRtt103thatrecognizestheSerine2-phosphorylated CTD becomes capable of triggering RNApII termination at regions where Nrd1-Nab3 binding sites and serine 2-phosphorylated CTD are co-localized, satisfyingly confirming this model (8).The RNAs generated via Nrd1-Nab3-Sen1-dependent termination are trimmed or degraded by the exosome, mediated by Nrd1 complex interactions with this 3Ј-5Ј exonuclease (9). Intriguingly, swapping or deletion of the Nrd1 CID reduced the interaction between Nrd1 and the exosome (8), indicating that the Nrd1 CID also plays an important role in coupling termination and RNA processing by recruiting the exosome.The nuclear exosome consists of the core exosome and a nuclear-specific subunit Rrp6 (PM/Scl100 in humans) that functions in RNA 3Ј-end processing using 3Ј-5Ј exoribonuclease activity (10 -13). The core exosome is a catalytically inactive barrel-shaped complex composed of nine subunits (Exo-9: RNase pleckstrin homology-like proteins (Rrp41/42/43/45/46 and Mtr3) and S1/KH domain proteins (Rrp4/40 and Csl4)) as well as Dis3 (also known as Rrp44), which is a 3Ј-5Ј exo/endonuclease. Located at the bottom of Exo-9, Dis3 trims or degrades the RNA substrates passed through the central pore of . In contrast, Rrp6 sits on top of the Exo-9 S1/KH ring above the central channel, and the RNAs traverse the S1/KH ring and enter into the active site of Rrp6 for degradation (15,16).The TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex is ...

Targeting RNA for processing or destruction by the eukaryotic RNA exosome and its cofactors

“…Cross-linking experiments coupled to mass spectrometry showed that it contacts the S1/KH protein Rrp40 in the yeast complex (Shi et al 2015), and competition binding experiments at high concentrations reported low-affinity interactions between its C-terminal region and the Rrp6 catalytic domain (Kim et al 2016). Human Mpp6 interacts directly with Mtr4 (Chen et al 2001), supporting a role in recruitment of Mtr4 or Mtr4 complexes in higher eukaryotes.…”

Identification of three sequence motifs in the transcription termination factor Sen1 that mediate direct interactions with Nrd1

“…Nrd1 also contains a CID that has a preference for Ser5P C-terminal domain (CTD) of Pol II (7,8), while Sen1 has a preference for Pol II Ser2P CTD (9). NNS couples tightly with the TRAMP complex for 3¢-end trimming of ncRNAs and degradation of unstable RNAs (10)(11)(12)(13), which is mediated by the interaction between Nrd1 CID and Trf4, a subunit of the TRAMP complex (14,15). The Nrd1 CID also interacts with Mpp6, a cofactor of the nuclear exosome (15).…”

Termination of non-coding transcription in yeast relies on both a CTD-interaction domain and a CTD-mimic in Sen1

“…This is due to an alternative 31 conformation of the C-terminal region of Sen1 NIM that can be accommodated in the binding 32 pocket of Nrd1 CID. We suggest that similar extended conformation exists also in the case of 33 the CTD mimic found in Mpp6 (Kim et al, 2016) as its DLDK C-terminal motif (figure 2E) has structurally related CTD-Interaction domains. Of course, such protein regions should be 1 present in the appropriate protein context (i.e.…”

“…Interaction Motif. A subsequent report described a second NIM in Mpp6, an exosome cofactor 19 (Kim et al, 2016). During the course of our previous work, we discovered that the CID is also 20 required for the interaction between Nrd1 and Sen1 ( figure 1A), an observation that was 21 reported in an independent study (Heo et al, 2013).…”