Mtr4 RNA helicase structures and interactions
Mtr4 is a Ski2-like RNA helicase that plays a central role in RNA surveillance and degradation pathways as an activator of the RNA exosome. Multiple crystallographic and cryo-EM studies over the past 10 years have revealed important insight into the Mtr4 structure and interactions with protein and nucleic acid binding partners. These structures place Mtr4 at the center of a dynamic process that recruits RNA substrates and presents them to the exosome. In this review, we summarize the available Mtr4 structures and highlight gaps in our current understanding.
<!>Mtr4 Architecture<!>Helicase Core<!>N-terminus<!>Arch<!>Overview of Mtr4 structures and complexes<!>Mtr4 (without protein cofactors)<!>FRH<!>Adaptor complexes<!>TRAMP<!>NEXT<!>The Arch Interaction Motif (AIM)<!>Inhibitor complex<!>Exosome complexes<!>Arch dynamics<!>RNA Interactions<!>Unwinding mechanism<!>Protein-protein interactions<!>Outlook
<p>Nuclear RNA processing and surveillance is a complex process that utilizes multiple proteins and protein assemblies to identify and eventually degrade targeted RNA substrates. These complex assemblies perform essential tasks, including maturation of ribosomal RNA and turnover of aberrant or unneeded RNAs. Numerous cellular, biochemical, and biophysical studies have addressed fundamental questions of how and what RNA substrates are identified by the RNA surveillance machinery, and how targeted substrates are ultimately processed and degraded (reviewed in Chlebowski et al. 2013, Januszyk and Lima 2014, Schmid and Jensen 2019, Schneider and Tollervey 2013).</p><p>The RNA helicase, Mtr4, plays a central role in nuclear RNA surveillance by providing a bridge between substrate targeting and RNA decay. RNA recognition is performed directly by Mtr4 and by various adaptor proteins that recruit Mtr4 to targeted substrates (Falk et al. 2017b, Lingaraju et al. 2019, Thoms et al. 2015). RNA substrates are threaded through Mtr4 and delivered to the RNA exosome, which utilizes 3' exonuclease and endonuclease activities to degrade the RNA (Weick and Lima 2020). While the molecular details of each of these steps are still emerging, structural studies spanning the past decade have expanded our understanding of Mtr4 structure and interactions, providing significant insight into Mtr4 function. These studies reveal a dynamic helicase engaged in multiple complex interactions to facilitate RNA turnover.</p><p>Mtr4 is a member of the Ski2-like family of RNA and DNA helicases, which includes several proteins involved in RNA degradation (Mtr4 and Ski2), mRNA splicing (Brr2), translation (Slh1), and DNA replication/recombination (Hel308) (Caprara 2010, Jankowsky and Fairman-Williams 2010). Ski2-like RNA helicases are ATP-dependent enzymes that process RNA substrates in a 3'–5' direction. They are relatively large helicases (generally > 100 kDa) and typically act as part of larger complexes. Mtr4 and Ski2 have a similar domain architecture (Johnson and Jackson 2013) and both play a central role in eukaryotic RNA surveillance as activators of the RNA exosome, with Mtr4 functioning in the nucleus and Ski2 in the cytosol (Anderson and Parker 1998, Mitchell 2014).</p><p>The nature of the interactions between Mtr4 and interacting partners has been a longstanding question. For the most part, crystal structures of Mtr4 complexes have been limited to structures of Mtr4 bound to small peptide or RNA fragments. Attempts to obtain larger-scale structural information appear to be challenged by the inherently dynamic nature of the Mtr4 structure, as well as by the dynamic nature of the targeted complex interactions. The first crystal structures of Mtr4 from S. cerevisiae were determined ten years ago (Jackson et al. 2010, Weir et al. 2010) and were reviewed previously (Johnson and Jackson 2013). Since that time, several additional structures from S. cerevisiae, N. crassa, and H. sapiens have been determined (Table 1). Aided by advances in cryo-EM methods, impressive structures of Mtr4 bound to the exosome and ribosome have recently emerged (Du et al. 2020, Schuller et al. 2018, Weick et al. 2018). The crystal and cryo-EM structures provide important insight into RNA binding, protein-protein interactions, and conformational dynamics. We begin this review with a description of the overall Mtr4 architecture as established by the original structures, followed by an overview of the more recent structures and a discussion of mechanistic implications arising from these structures.</p><!><p>The first Mtr4 structures include an apo structure (Jackson et al. 2010) and a structure of Mtr4 bound to a short poly(A) RNA (Weir et al. 2010). These structures showed that the overall domain organization of Mtr4 includes a four domain helicase core, a largely unstructured N-terminus, and a unique "arch" domain that spans one face of the helicase core (Figure 1).</p><!><p>The helicase core is a ring-like structure composed of two RecA-like domains (RecA1 and RecA2), a winged helix domain, and a helical bundle domain. This core structure is the fundamental structural feature of all Ski2-like helicases (Johnson and Jackson 2013). Conserved sequence motifs are located along the interface of the RecA domains, and contain residues associated with RNA binding, and nucleotide binding and hydrolysis (Jankowsky and Fairman-Williams 2010). The helical bundle domain also interacts with RNA substrates and appears to influence Mtr4 substrate preferences (Taylor et al. 2014). The winged helix is positioned at a junction between the RecA domains, the helical bundle, and the arch. Although the role of this domain in Mtr4 is not well understood, it is poised to act as a central hub around which the other domains move (Johnson and Jackson 2013).</p><!><p>The N-terminus of Mtr4 varies in length (ranging from 128 amino acids (aa) in H. sapiens to 169 aa in N. crassa) and has limited sequence conservation. Immediately adjacent to RecA1 is a ~ 40aa species dependent beta or beta-loop-beta structure that extends the β-sheet of RecA1 and spans the interface of RecA1 and RecA2. The remainder of the N-terminus is unstructured and is generally not visible in the existing structures. Consequently, this unstructured region is often removed for structural studies.</p><!><p>While the helicase core is common to all Ski2-like helicases, the arch is unique to the helicases that activate the exosome (Mtr4 and Ski2). It derives its name from the arch like appearance observed in the original structures. The domain is a large insertion in the winged helix, composed of antiparallel helices ("stalks") with a β-barrel structure on the distal end called a KOW (Kyrpides–Ouzounis–Woese) domain. The structure of the arch is figuratively described as being composed of an arm and forearm (stalks), and fist (KOW).</p><!><p>Table 1 provides a summary of the current Mtr4 structures and complexes. These structures include adaptor complexes, an inhibitor complex, and exosome-bound complexes. Here we briefly describe general features and observations arising from these structures.</p><!><p>In addition to the original Mtr4 structures from S. cerevisiae, similar crystal structures of Mtr4 without protein cofactors have now been determined from N. crassa, and H. sapiens. The N. crassa structures are described below.</p><!><p>FRH (Frequency Interacting Helicase) is an Mtr4 homolog found in N. crassa. In addition to its assumed role in RNA surveillance, FRH is a central component of the circadian clock (Baker et al. 2012, Cheng et al. 2005), a function that doesn't require helicase activity (Hurley et al. 2013). In this role, FRH binds to a negative clock element FRQ, to form the FRQ-FRH complex (FFC). The FCC further binds the positive transcriptional regulators of the White Collar complex (WCC) White Collar 1 and White Collar 2 (WC-1 and WC-2), thus disrupting transcription of clock genes.</p><p>The FRH structures include three apo structures in different crystal forms and a structure bound to a short RNA oligo and ADP (Conrad et al. 2016, Morales et al. 2018). Overall, the FRH structure closely resembles Mtr4. Small differences are observed in the N-terminus, where only 1 strand extends off the β-sheet of RecA1 instead of the two observed in scMtr4. In the nucleotide binding site, F179, which stacks with the adenosine base in scMtr4, is displaced in FRH and doesn't interact with ADP (Conrad et al. 2016). This displacement appears to be a crystallographic artifact since F179 adopts an scMtr4-like conformation in the trigonal crystal form (Morales et al. 2018).</p><p>A "small cell" orthorhombic apo structure shows an unusually compressed arch domain, which appears to result from a compressed unit cell (Conrad et al. 2016). Conversely, the trigonal apo form adopts an open conformation where the KOW is rotated 70° and displaced by 30 Å compared to the orthorhombic small cell structure (Morales et al. 2018). This open conformation is stabilized by formation of a crystallographic FRH dimer in the crystal (see "arch dynamics" below). Despite the dramatic differences in arch conformation observed between the various FRH structures, the helicase core remains largely unchanged.</p><!><p>To deal with the diversity of RNA substrates that Mtr4 must identify and deliver to the exosome, Mtr4 enlists multiple adaptor proteins/complexes to target specific substrates (Schmid and Jensen 2019). The Mtr4-adaptor complexes for which structures have been determined are described below.</p><!><p>The S. cerevisiae TRAMP complex is a three-protein complex composed of Mtr4, a poly(A) polymerase (Trf4 or Trf5), and a noncatalytic protein containing five Zn-knuckles (Air1 or Air2) (Lacava et al. 2005, Vanacova et al. 2005, Wyers et al. 2005). Variations in the composition of the complex are related to substrate specificity and localization in the nucleus (Delan-Forino et al. 2020). For example, Trf4-Air2-Mtr4 primarily targets ncRNA classes (CUTs, SUTs, and XUTs), antisense RNA, and RNAPIII transcripts, while Trf5-Air1-Mtr4 targets pre-rRNA spacer regions (ETS, and ITS) and protein-coding genes. TRAMP adds a short poly(A) tail to RNA substrates using the polymerase activity of Trf4/Trf5. Mtr4, which exhibits a preference for polyadenylated substrates, facilitates delivery of the polyadenylated RNA to the exosome. Complex formation enhances Mtr4 activity (Jia et al. 2012) and modulates Trf4 activity (Jia et al. 2011). The molecular mechanisms underlying these effects on activity have not been delineated.</p><p>In order to better understand the assembly of the TRAMP complex, Mtr4 was crystallized with a hybrid peptide containing known interacting regions of Air2 (residues 1–62) and Trf4 (residues 111–160) (Falk et al. 2014). In the resulting structure, short segments of Trf4 (121–127) and Air2 (19–52) are observed wrapping around the helicase core. Trf4 forms a short β-strand that extends the β-sheet of RecA2. Mutation of interacting residues disrupted binding in vitro and produced a severe slow growth phenotype in vivo. A similar disruption of binding was observed for the analogous Trf5/Air1 peptide, suggesting that both complexes interact with Mtr4 in a similar manner.</p><p>Interestingly, the N-terminal portion of the Air2 peptide (residues 6–18) reaches across to a neighboring Mtr4 molecule in the crystal lattice and contacts the KOW. Air2 binds the KOW in the same region as other adaptor proteins (see Arch Interaction Motif discussion below). This interaction may not be required for TRAMP assembly since the complex still forms when the arch is removed from Mtr4 (Falk et al. 2014).</p><p>The architecture of the full TRAMP complex is not well understood. A structure of the central and catalytic domains of Trf4 bound to two Zn knuckles has been determined (Hamill et al. 2010). However, the Mtr4 interacting regions of Trf4 and Air2 lie in low-complexity regions not observed in the Trf4-Air2 structure. Additional structural data is needed to understand the interplay between the TRAMP components, including the RNA path through the complex.</p><!><p>The human NEXT (nuclear exosome targeting) complex is another three-protein complex that is involved in the surveillance and turnover of aberrant and noncoding RNAs (Lubas et al. 2015, Lubas et al. 2011). The NEXT complex may also play a role in resolving DNA:RNA hybrids and R-loops, and has been shown to resolve DNA:RNA hybrids and R-loops in vitro (Puno and Lima 2018). NEXT consists of Mtr4, an RNA binding protein (RBM7), and a scaffold zinc-knuckle protein (ZCCHC8). Complex assembly increases the Mtr4 unwinding rate 30-fold. An optimal in vitro substrate contains a 3'poly(A) tail with an upstream poly(U) sequence, with selectivity for the poly(U) sequence coming from RBM7 (Puno and Lima 2018).</p><p>ZCCHC8 interacts with Mtr4 through both the N-terminus (1–282) and the C-terminus (659–707). However, only the C-terminal domain (CTD) of ZCCHC8 is needed to stimulate helicase activity of Mtr4. To better understand the ZCCHC8 CTD-Mtr4 interaction, ZCCHC8 CTD was crystallized with the helicase core of Mtr4. The crystal structure shows that ZCCHC8 interacts with all four domains of Mtr4 helicase core. ZCCHC8 wraps around the bottom of the helicase core, extending from the WH and HB junction to the interface between the RecA domains. Notably, this interface does not overlap with the Trf4/Air2 interactions in the helicase core. Mutations were made at multiple points along the ZCCHC8-Mtr4 interface to probe for effects on unwinding activity. Only mutations associated with RecA2 were found to have an effect on unwinding (Puno and Lima 2018).</p><!><p>Several adaptor proteins recruit Mtr4 to substrates generated during ribosomal processing. For example, Nop53 recruits Mtr4 to 7S pre-rRNA, which promotes processing by the exosome. Utp18 recruits Mtr4 to the 5' ETS, which results in complete degradation of the RNA. Nop53 and Utp18 both recruit Mtr4 through a short sequence motif located in the N-terminus of each protein. The motif interacts directly with the Mtr4 arch and is called the "arch interaction motif," or AIM, with a consensus sequence of LFXΦD (X is any residue and Φ is any hydrophobic residue) (Thoms et al. 2015).</p><p>The AIM-Mtr4 interaction was observed by co-crystallizing scMtr4 with a short peptide containing the AIM of scNop53 (Falk et al. 2017b). The resulting structure shows the AIM sequence bound in a shallow pocket on the surface of the KOW (Figure 2A). The Nop53 peptide binds in an extended conformation, forming a β-strand that stacks in an antiparallel manner with the central β-sheet of the KOW. Multiple electrostatic and hydrophobic interactions define the binding site, including a salt bridge between the aspartate of the AIM sequence and a strictly conserved R774 in the KOW. The importance of this interaction is confirmed by an R774E mutation, which completely disrupts the Mtr4-Nop53 interaction.</p><p>Other adaptor proteins utilize non-canonical AIM sequences to recruit Mtr4 (Lingaraju et al. 2019). The nuclear VCP-like (NVL) protein is a metazoan ribosomal biogenesis factor. The Mtr4-interacting sequence of NVL, GWFIDKTP, does not perfectly match the canonical LFXΦD AIM sequence, although it is generally hydrophobic and also contains an aspartate. The sequence is referred to as a "tryptophan centered arch interacting motif," or W-AIM, because mutation of the tryptophan significantly disrupts binding. Despite the sequence differences between the AIM and W-AIM, the crystal structure of hsMtr4 bound to the W-AIM peptide of NVL shows that the W-AIM binds in the same location and conformation as the Nop53 AIM, including formation of a salt-bridge between the W-AIM aspartate and R743 (the human equivalent of the yeast R774) (Figure 2B).</p><p>Interestingly, the Air2 peptide in the TRAMP structure binds to the KOW in the same region and orientation as Nop53 and NVL using a sequence that differs slightly from the canonical AIM motif but retains the aspartate and general hydrophobic character (Figure 2B) (Falk et al. 2014). The NEXT component, ZCCHC8, also interacts with the Mtr4 KOW through non-canonical AIM motifs located in the N-terminus of the protein (Lingaraju et al. 2019). Although no structures of the ZCCHC8-KOW interaction are available, mutagenesis of the "cysteine-centered" FCLDKLG (C-AIM) motif of ZCCHC8 disrupts binding similar to that observed with W-AIM, suggesting a similar binding mode. Interestingly, a second "isoleucine-centered" IEEF (I-AIM) sequence located upstream of the C-AIM interacts with a different region of the KOW, demonstrating that some adaptor proteins utilize multiple KOW binding sites in recruiting Mtr4.</p><p>Sequence and structural alignment of the various AIM motifs leads to a revised consensus sequence of xωxxD(x)1/2G/P, where ω represents an aromatic residue (Figure 2B) (Lingaraju et al. 2019). The C-terminal glycine or proline residue allows the peptide to bend as it exits the AIM binding site. While there is broad variability in AIM sequences, each peptide interacts with the conserved arginine (hsArg743/scArg774) located at the center of the AIM binding site. Notably, this same residue in FRH (R806) is required for interaction with the While Collar complex (Shi et al. 2010), suggesting that FRH may exploit AIM-like interactions for both exosome and clock functions.</p><!><p>Mtr4 and exosome activity can be regulated by the human NRDE2 protein, which associates with Mtr4 and inhibits exosome function, thereby promoting export of mRNAs to the cytosol (Wang et al. 2019). Depletion of NRDE2 results in a global reduction of mRNA and lncRNA levels, and an overall increase of Mtr4 association with these RNAs. Immunoprecipitation of Mtr4 associated proteins shows a significant reduction of Mtr4-exosome interaction upon overexpression of NRDE2.</p><p>Mtr4 was co-crystallized with the Mtr4 interacting domain (MID) of NRDE2, comprising residues 163–266. In the structure, NRDE2 forms extensive interactions with the KOW, and then extends down to the HB and around the core to the RecA2 domain. Consequently, the arch is locked down into a tightly closed conformation. NRDE2 utilizes an AIM-like motif and binds in the same manner as the AIM containing adaptor proteins (Figure 2B). NRDE2 binding would prevent adaptor proteins from interacting with the KOW. Along the helicase core, NRDE2 occupies the same binding sites as Trf4 and Air2, suggesting that NRDE2 binding may prevent binding of adaptor complexes to the core as well. Curiously, NRDE2 does not bind in a region that is expected to directly impact exosome interactions (see discussion below). In addition, Mtr4 unwinding activity is enhanced in the presence of NRDE2, even though the observed conformation of the KOW and NRDE2 appears to block the expected RNA entry site into the core (Wang et al. 2019).</p><!><p>The eukaryotic RNA exosome is 10-subunit complex (Exo-10) made up of a 9-protein non-enzymatic barrel-like core and a 3'-exo/endonuclease (Rrp44/Dis3/EXOSC11) located at the bottom of the core. RNA substrates enter the top of the core and thread through the complex to reach Rrp44 (Januszyk and Lima 2014). Three additional co-factors, Rrp6/EXOSC10 (a 3' exonuclease), Rrp47/C1D and Mpp6, associate with the top of the exosome core and help facilitate Mtr4 engagement (Falk et al. 2017a, Januszyk and Lima 2014, Schuch et al. 2014). The N-terminal regions of Rrp6 and Rrp47 intertwine to form a composite binding surface for a short, conserved helix at the N-terminus of Mtr4 (scMtr4 residues 6–11) (Schuch et al. 2014). Two Cryo-EM structures, one from S. cerevisiae and one from H. sapiens, reveal even more extensive interactions between Mtr4 and the exosome, demonstrating how Mtr4 engages with the exosome to deliver RNA for degradation (Schuller et al. 2018, Weick et al. 2018).</p><p>The S. cerevisiae structure is an Mtr4-exosome complex stalled on a maturing ribosome (Schuller et al. 2018). The complex was isolated by combining endogenous 7S-containing pre-60S particles (captured using a tagged form of Nop53 that recruits Mtr4 to the ribosome) with recombinantly expressed Exo-14n (Exo-10 + a catalytically inactive Rrp6, Rrp47, Mpp6, and Mtr4). This strategy allowed Mtr4 and the exosome to engage the 7S rRNA, trimming it down to the 5.8S + 30 rRNA using the exonuclease activity of Rrp44. Rrp6 would normally further process the rRNA, but the inactive mutant of Rrp6 caused stalling on the substrate. The structure shows Mtr4 positioned between the ribosome and the exosome with the 3' extension of the 5.8S +30 rRNA channeled through Mtr4 into the exosome core and the Rrp44 active site (Figure 3). (Recent Cryo-EM structures of the 90S preribosome similarly show Mtr4 wedged between the ribosome and the exosome, although no refined coordinates of Mtr4 and the exosome are currently available (Du et al. 2020).) Mtr4 binds to the exosome "edge-on" with RecA1 and the helical bundle contacting the top of the core. Strikingly, the arch adopts an extended conformation that places the KOW 90 Å away from the core. In this conformation, Mtr4 interacts with the ribosome in a bidentate conformation where the KOW faces domain I of the 25S rRNA while RecA2 and the helical bundle contact domain V of the 25S rRNA. The arms of the arch are stabilized by interactions with Rrp6 and Rrp47. Deletion of the arch results in a severe slow growth phenotype in S. cerevisiae that is attributed to defects in rRNA processing (Jackson et al. 2010). Specifically, 5.8S processing by Rrp6 is impaired in an archless Mtr4 mutant. Engagement of the arch by Rrp6 in the yeast Mtr4-exosome complex provides a structural rationale for this observation (Schuller et al. 2018).</p><p>The human Mtr4-exosome structure was reconstituted using recombinantly expressed components (Weick et al. 2018). In order to stabilize the complex, an RNA:DNA hybrid substrate was designed that allowed Mtr4 to translocate along the RNA strand until it reached the DNA portion of the substrate, at which point translocation stalled. The structure shows the duplex region approaching the helicase core with the single stranded RNA overhang threading through Mtr4, following the same path observed in the yeast structure. As the RNA exits the bottom of the helicase core, it enters the top of the exosome core and extends to Rrp44 at the bottom of the exosome. Mtr4 engages the exosome through conserved surfaces in a region also known to bind Rrp6. Mpp6 stabilizes interactions with the core by binding along the interface of the RecA domains. Notably, the arch is disordered in the overall reconstruction. However, 3D classifications revealed two subclasses with the arch in either an open or closed conformation. In the closed conformation, the KOW appears to engage with the RNA:DNA duplex.</p><!><p>Comparison of the existing Mtr4 structures highlights substantial conformational changes in the arch domain in all three organisms (Figure 4). In the case of the yeast exosome and NRDE2 bound structures, the arch conformation is stabilized by protein-protein contacts, resulting in a dramatically open (exosome) or closed (NRDE2) conformation. In other structures, crystal contacts stabilize the arch conformation. When crystal contacts or protein-protein interactions are not present, electron density for the arch is generally weak, underscoring the inherent mobility of the arch. Extending the analogy of the arch being composed of an arm, forearm and fist, arch motions can be described as occurring through hinge point rearrangements centered at the "shoulder," "elbow," and "wrist." Notably, while the arch exhibits low conservation overall, strictly conserved residues are found at the center of each hinge point (Morales et al. 2018).</p><!><p>Mtr4-RNA interactions observed in the crystal and cryo-EM structures are summarized in Figure 5. (For clarity, interactions with non-substrate rRNA in the scMtr4-exosome structure are differentiated from other observed Mtr4-RNA interactions.) The structures demonstrate that each of the core domains engage RNA, with extensive interactions through the center of the helicase core along RecA1, RecA2, and the Helical Bundle. The RNA path through Mtr4 begins at the top of the core near the region where the KOW contacts the core in the closed arch structures. In the hsMtr4-exosome structures, the first base pair of the RNA:DNA duplex stacks with aromatic residues located at the base of a β-hairpin that extends upward from RecA2. The single stranded portion of the RNA then binds along the "ratchet helix" of the helical bundle domain, coordinated by backbone interactions with RecA2. The binding observed along this helix is essentially identical in both Mtr4-exosome structures and in the Mtr4 and FRH structures containing short poly(A) oligos. The longer RNA substrates continue past the ratchet helix along RecA1 before exiting out the bottom of the helicase core.</p><p>RNA interactions with the arch are not well defined in available crystal and cryo-EM structures. Electrophoretic mobility shift assays (EMSAs) indicate that the KOW binds larger, structured RNAs, but does not bind ssRNA (Halbach et al. 2012, Weir et al. 2010). An NMR study defined regions in the KOW that are altered upon RNA binding (Falk et al. 2017b). Notably, the RNA binding site is different from the AIM binding site, meaning both interactions could occur simultaneously. The RNA binding surface corresponds to the face of the KOW that approaches the ribosome in the scMtr4-exosome complex. However, the resolution of the cryo-EM structure is insufficient to define interactions at a molecular level. We further note that the observed KOW-RNA interactions are with the 25S rRNA rather than with the 5.8S + 30 rRNA substrate that threads through the helicase core and is processed by the exosome (Schuller et al. 2018). In the case of the hsMtr4-exosome complex, the KOW does engage with the stalled RNA:DNA duplex in a subset of particles, although molecular details are similarly obscure (Weick et al. 2018). Thus, it remains to be determined precisely how or under what circumstances the arch engages with RNA substrates.</p><!><p>Translocation across an RNA strand is understood to lead to resolution of secondary structure through displacement of complementary RNA or DNA strands. The RecA domains of the helicase provide the motor that allows Mtr4 to translocate along an RNA substrate in an ATP-dependent manner (Jankowsky and Fairman-Williams 2010). A detailed molecular mechanism for Mtr4 translocation has not been established. There are, however, structural features that appear to contribute to helicase activity.</p><p>The β-hairpin that extends up from RecA2 and engages the RNA:DNA duplex in the hsMtr4-exosome structure likely acts as a wedge to separate complementary strands. While no strand displacement is observed in the hsMtr4-exosome structure, strand displacement is observed in a structure of the related Ski2-like DNA helicase, Hel308, bound to an unraveling DNA duplex (Buttner et al. 2007). In that structure, the β-hairpin protrudes into the junction of the unwinding DNA, stacking with the first DNA base pair, stabilizing the displaced strand on one side of the hairpin, and directing the translocating strand in the opposite direction.</p><p>In each RNA-bound Mtr4 structure, RNA binds along the ratchet helix of the helical bundle. Conserved residues along this helix interact with the RNA nucleotides, providing an opportunity to read the RNA sequence. Indeed, mutation of one of these residues, a strictly conserved arginine (R1030 in S. cerevisiae), has a modest effect on unwinding activity but eliminates the preference for a poly(A) sequence (Taylor et al. 2014). It has been proposed that the ratchet helix could serve as a ratchet (giving rise to the name of the helix) that moves along the RNA in response to conformational changes in the RecA domains upon ATP binding and hydrolysis (Buttner et al. 2007). More recently, an "expansion" model for RNA translocation has been proposed for DEAH/RHA, Ski2-like, and NS3/NPH-II RNA helicases (He et al. 2017). This model focuses on the observation that 4–5 RNA bases appear to stack along the ratchet helix, wedged between the β hairpin of Rec2A (the β hairpin that is poised to unwind complementary RNA strands) and a second hairpin protruding from RecA1. Conformational changes associated with ATP binding/hydrolysis would facilitate stepping between a 4 nt and 5 nt stack.</p><p>The common theme between each of the current models for RNA translocation is a conformational change between RecA1 and RecA2 upon ATP binding and hydrolysis. To date, such conformational changes have not been clearly identified in the Mtr4 structures, despite the existence of structures in apo, RNA-bound, and various nucleotide-bound states. It is unclear whether the lack of observed conformational rearrangements is a function of the methods used to capture these structures, or if other mechanisms are at play.</p><p>The arch domain also appears to play an important role in RNA translocation and unwinding. Arch deletion has minimal effect on helicase activity (Jackson et al. 2010, Taylor et al. 2014). However, combination of the arch deletion with an R1030A mutation completely abolishes helicase activity in vitro and is lethal in vivo (Taylor et al. 2014). Intriguingly, a single molecule study demonstrated that Mtr4 is unable to translocate unless a duplex region is located immediately upstream (Patrick et al. 2017). The RNA sensing mechanism has not been identified, but the arch domain is a compelling candidate because it is uniquely positioned to encounter RNA as it approaches the helicase core. Although Mtr4 appears to require secondary structure in substrates that it engages directly, such a requirement may be overcome by adaptor proteins that mimic the effect of RNA secondary structure or promote conformational changes that allow Mtr4 to engage substrates more liberally. Indeed, adaptor proteins and complexes may function specifically to unlock Mtr4 activity on substrates, such as unstructured extensions on the pre-ribosome, that are otherwise ignored.</p><!><p>Protein interactions with Mtr4 are summarized in Figure 5. Although the number of complex structures is somewhat limited, we can make a few general observations. Adaptor proteins/complexes all engage the KOW, with utilization of the AIM recognition site being a common theme. For ribosomal recruitment factors, the AIM interaction may be the primary interaction. Larger adaptor complexes such as TRAMP and NEXT also engage the helicase core in unique ways that impact helicase activity. Simultaneous binding of the core and the arch by adaptor proteins such as Air2 and ZCCHC8 is expected but has not been directly observed.</p><p>In contrast to the adaptor complexes, the exosome engages all regions except the KOW. Interactions are observed in all four domains of the core, the N-terminus, and the arms of the arch domain. Notably, there is little overlap between exosome binding sites and adaptor binding sites. The one exception is ZCCHC8, which binds along the same surface of the helicase core.</p><p>Interestingly, the exosome inhibitor, NRDE2, which disrupts the interaction between Mtr4 and the exosome (Wang et al. 2019), doesn't compete directly with the exosome as it engages Mtr4. Rather, it binds to the same surface utilized by Trf4-Air2 on the helicase core while simultaneously binding the KOW at the AIM recognition site. How does NRDE2 inhibit exosome interaction? One possibility is that by constraining the arch in a closed conformation, it prevents Rrp6 from binding to the arms in an open conformation. Such an effect may partially mimic an archless mutation, which can lead to nearly complete loss of exosome activity in vitro (Holub et al. 2012).</p><p>It should be noted that simultaneous binding of Air2 or ZCCHC8 to the helicase core and the arch likely requires the arch to adopt a relatively fixed conformation that allows some proximity between the KOW and the helicase core. Thus, we speculate that exosome-induced opening of the arch conformation could affect adaptor protein/complex interactions with Mtr4. In this manner, arch dynamics may be exploited by interacting partners to signal a transition between Mtr4 recruitment of RNA substrates and RNA degradation by the exosome. Some support for such a mechanism may come from the scMtr4-exosome complex where Nop53 was used to recruit Mtr4 and isolate the scMtr4-exosome complex. Nop53 was not observed in the cryo-EM map, suggesting that the Mtr4 recruitment step preceded the observed exosome-engaged conformation.</p><!><p>The structural information obtained over the past decade has provided us with a detailed understanding of Mtr4 architecture and has defined important interactions with adaptor proteins and with the exosome. The recent cryo-EM structures, in particular, provide snapshots of how Mtr4 presents RNA to an engaged RNA complex. However, we still lack structures that describe how Mtr4 targets RNA substrates, including the interplay between Mtr4 and adaptor proteins that coordinate delivery of RNA substrates to Mtr4 and the exosome.</p><p>Auto-regulation of helicase activity is observed in other Ski2-like helicases. Brr2, for example, contains a long unstructured N-terminal region that binds along the RecA domains and inhibits ATP binding and hydrolysis in the absence of an RNA substrate (Absmeier et al. 2020). The arch domain in Ski2 also represses ATPase activity in the SKI complex (Halbach et al. 2013). It is possible that Mtr4 employs analogous auto-regulatory mechanisms on ATPase activity, although none have been described to date. More broadly speaking, it is clear that protein-protein interactions can affect helicase activity, as observed in the enhancement of Mtr4 activity by the TRAMP and NEXT complexes (Jia et al. 2012, Puno and Lima 2018). The absence of full complex structures makes it difficult to assign a structural basis for this enhancement of activity, and represents an important focus for ongoing studies.</p><p>A recent proteomics study reported extensive posttranslational modification of the exosome and associated co-factors in S. pombe (Telekawa et al. 2018). Strikingly, more than half of all the observed modifications occurred on Mtr4, suggesting that the helicase is a primary point of regulation. Next steps include characterization of the biological and biochemical effects of these modifications, including the impact on substrate interactions and complex formation. Identification of the modifying enzymes may open the door to targeted control of the entire RNA surveillance system.</p><p>The arch domain is at once the most intriguing and the most elusive structural feature of Mtr4. It serves as a docking site for multiple adaptor proteins (Falk et al. 2017b, Lingaraju et al. 2019, Thoms et al. 2015). It also has the ability to interact with RNA, although the molecular details of those interactions are poorly defined (Halbach et al. 2012, Johnson and Jackson 2013, Weir et al. 2010). Indeed, we predict that the arch is responsible for the RNA duplex sensing observed in the single molecule studies (see "RNA interactions – unwinding mechanism"). The dynamic flexibility of the domain is expected to play an important functional role, and represents an important area of focus for future studies. As we suggest in the previous section ("protein-protein interactions"), conformational changes in the arch may facilitate docking of adaptor complexes and the exosome in a mutually exclusive manner. Furthermore, the arch can affect helicase activity (Taylor et al. 2014), although the mechanisms underlying that function are unclear. The arch may simply act to orient RNA substrates as they approach the helicase core. Alternatively, the arch conformation may alter the conformation and/or dynamics of the helicase core as it processes RNA substrates. Resolution of these mechanistic questions may depend on our ability to capture Mtr4 when the arch is fully engaged with adaptor complexes and RNA substrates.</p><p>The dynamic nature of the arch presents a significant challenge to structural characterization. Mobile features are often difficult to capture through crystallization. Cryo-EM holds some promise in this regard, as demonstrated by the hsMtr4-exosome structure where multiple arch conformations were identified in the 3D classifications (Weick et al. 2018). Still, we anticipate that informative structural characterization of arch dynamics and function will require improved understanding and control of conformational states.</p><p>The path forward will likely involve complementary biophysical methods that can define complex interactions and dynamics in a less restricted environment. For example, hydrogen-deuterium exchange (HDX) methods may enable visualization of complex interactions that have evaded crystallographic capture. FRET-based approaches could provide a means of correlating Mtr4 conformation with specific protein or RNA binding states. We look forward to the expanded understanding arising from combined structural and biophysical efforts to further characterize the dynamic and complex interactions underpinning Mtr4 function.</p>