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Significance Defects in RNA quality control pathways manifest as disease because they function to selectively remove aberrant or defective species to ensure cellular homeostasis. The RNA exosome and TRAMP complexes encompass key components of the RNA surveillance machinery. Here we show that exoribonuclease activities of Rrp6-associated RNA exosomes protect stable RNA species from TRAMP-mediated polyadenylation and degradation, and that each catalytic activity of the RNA exosome-TRAMP complex contributes to substrate discrimination and degradation of less stable RNA species. Our results highlight a previously unappreciated role for Rrp6 in protecting stable RNA species from degradation and suggest additional mechanistic analogies between RNA and protein quality control pathways with respect to proofreading mechanisms that protect stable species while targeting unstable species for destruction., Quality control requires discrimination between functional and aberrant species to selectively target aberrant substrates for destruction. Nuclear RNA quality control in Saccharomyces cerevisiae includes the TRAMP complex that marks RNA for decay via polyadenylation followed by helicase-dependent 3′ to 5′ degradation by the RNA exosome. Using reconstitution biochemistry, we show that polyadenylation and helicase activities of TRAMP cooperate with processive and distributive exoribonuclease activities of the nuclear RNA exosome to protect stable RNA from degradation while selectively targeting and degrading less stable RNA. Substrate discrimination is lost when the distributive exoribonuclease activity of Rrp6 is inactivated, leading to degradation of stable and unstable RNA species. These data support a proofreading mechanism in which deadenylation by Rrp6 competes with Mtr4-dependent degradation to protect stable RNA while selectively targeting and degrading unstable RNA.

International audience; In eukaryotic cells, aberrant mRNPs with processing and packaging defects are targeted co-transcriptionally by a surveillance system that triggers their nuclear retention and ultimately the degradation of their mRNA component by the 3'-5' activity of the exosome-associated exonuclease Rrp6. This mRNP quality control process is stimulated by the NNS complex (Nrd1-Nab3-Sen1), which otherwise mediates termination, processing, and decay of ncRNAs. The process involves also the exosome co-activator TRAMP complex (Trf4-Air2-Mtr4). Here, we describe a genome-wide approach to visualize the dynamic movement and coordination of these quality control components over the yeast chromosomes upon perturbation of mRNP biogenesis. The method provides valuable information on how the surveillance system is precisely coordinated both physically and functionally with the transcription machinery to detect the faulty events during perturbation of mRNP biogenesis. The overview shows also that the gathering of the quality control components over affected mRNA genes takes place at the expense of their commitment to be recruited at ncRNA genomic features, provoking termination and processing defects of ncRNAs.

Background: The activation of peroxisome proliferator-activated receptor alpha (PPARα) has been shown to reprogram tumor metabolism and exhibits great potential for treating anti-oral tumorigenesis. Methods: In this study, we used a pathway-based strategy to explore possible functional pathways involved in the anticancer activity of PPARα in oral cancer cells through next-generation sequencing (NGS) and bioinformatic approaches. Results: We found that 3919 genes were upregulated and 1060 genes were downregulated through PPARα activation. These genes were mainly involved in the proteasomal, mRNA surveillance, spliceosomal, RNA transport, and RNA degradation pathways, as indicated by GO and KEGG enrichment analysis. Importantly, a total of 13 upregulated genes in the RNA degradation pathway were identified including 3 core exosome factor genes (RRP43, RRP42, and CSL4), 2 TRAMP complex genes (TRF4 and Mtr4), 2 exosome cofactor genes (RRP6 and MPP6), 2 CCR4-NOT complex genes (CNOT2 and CNOT3), 2 Ski complex genes (SKI2 and Ski3), 1 decapping complex gene (EDC4), and 1 gene involved in 5’ exoribonuclease activity (XRN1). Conclusion: Our findings suggest that the activation of PPARα to upregulate the RNA degradation pathway might provide a new strategy for oral cancer treatment.

The RNA exosome is a macromolecular machine that degrades a large variety of RNAs from their 3'-end. It comprises the major 3'-to-5' exonuclease in the cell, completely degrades erroneous and overly abundant RNAs, and is also involved in the precise processing of RNAs. To degrade transcripts both specifically and efficiently the exosome functions together with compartment-specific cofactors. In the yeast S. cerevisiae, the exosome associates with the Ski complex in the cytoplasm and with Mtr4 alone or with Mtr4 as part of the TRAMP complex in the nucleus. Here we describe how to produce, purify, and assemble the Ski and TRAMP complexes from S. cerevisiae.

The RNA exosome processes a wide variety of RNA and mediates RNA maturation, quality control and decay. In marked contrast to its high processivity in vivo, the purified exosome exhibits only weak activity on RNA substrates in vitro. Its activity is regulated by several auxiliary proteins, and protein complexes. In budding yeast, the activity of exosome is enhanced by the polyadenylation complex referred to as TRAMP. TRAMP oligoadenylates precursors and aberrant forms of RNAs to promote their trimming or complete degradation by exosomes. This chapter provides protocols for the purification of TRAMP and exosome complexes from yeast and the in vitro evaluation of exosome activation by the TRAMP complex. The protocols can be used for different purposes, such as the assessment of the role of individual subunits, protein domains or particular mutations in TRAMP-exosome RNA processing in vitro.

Summary The RNA-degrading exosome mediates the processing and decay of many cellular transcripts. In the yeast nucleus, the ubiquitous 10-subunit exosome core complex (Exo-9–Rrp44) functions with four conserved cofactors (Rrp6, Rrp47, Mtr4, and Mpp6). Biochemical and structural studies to date have shed insights into the mechanisms of the exosome core and its nuclear cofactors, with the exception of Mpp6. We report the 3.2-Å resolution crystal structure of a S. cerevisiae Exo-9–Mpp6 complex, revealing how linear motifs in the Mpp6 middle domain bind Rrp40 via evolutionary conserved residues. In particular, Mpp6 binds near a tryptophan residue of Rrp40 that is mutated in human patients suffering from pontocerebellar hypoplasia. Using biochemical assays, we show that Mpp6 is required for the ability of Mtr4 to extend the trajectory of an RNA entering the exosome core, suggesting that it promotes the channeling of substrates from the nuclear helicase to the processive RNase., Graphical Abstract, Highlights • Yeast Mpp6 is stably bound to the nuclear exosome core both in vivo and in vitro • The Mpp6 middle domain binds the Rrp40 exosome subunit with conserved interactions • Mpp6 enhances the ability of the Mtr4 helicase to channel RNA into the exosome core • The pontocerebellar W238R mutation in human EXOSC3 affects the hMPP6-binding site, Falk et al. provide insights into the structure and function of the nuclear RNA exosome. The authors elucidate how the nuclear cofactor Mpp6 is recruited to the exosome core complex and show that it facilitates the threading of RNA substrates from the Mtr4 helicase to the Rrp44 RNase.

RNAi is a conserved mechanism in which small RNAs induce silencing of complementary targets. How Argonaute-bound small RNAs are targeted for degradation is not well understood. We show that the adenyl-transferase Cid14, a member of the TRAMP complex, and the uridyl-transferase Cid16 add non-templated nucleotides to Argonaute-bound small RNAs in fission yeast. The tailing of Argonaute-bound small RNAs recruits the 3′–5′ exonuclease Rrp6 to degrade small RNAs. Failure in degradation of Argonaute-bound small RNAs results in accumulation of ‘noise' small RNAs on Argonaute and targeting of diverse euchromatic genes by RNAi. To protect themselves from uncontrolled RNAi, cid14Δ cells exploit the RNAi machinery and silence genes essential for RNAi itself, which is required for their viability. Our data indicate that surveillance of Argonaute-bound small RNAs by Cid14/Cid16 and the exosome protects the genome from uncontrolled RNAi and reveal a rapid RNAi-based adaptation to stress conditions., While RNA interference is a highly conserved mechanism of gene regulation, how Argonaute-bound small RNAs are targeted for degradation is not well understood. Here the authors show that Cid14 and Cid16 target Argonaute-bound small RNAs for degradation and protect the genome from uncontrolled RNAi activity.

RNA surveillance via the nuclear exosome requires cofactors such as the helicase SKIV2L2 to process and degrade certain noncoding RNAs. This research aimed to characterize the phenotype associated with RNAi knockdown of Skiv2l2 in two murine cancer cell lines: Neuro2A and P19. SKIV2L2 depletion in Neuro2A and P19 cells induced changes in gene expression indicative of cell differentiation and reduced cellular proliferation by 30%. Propidium iodide-based cell-cycle analysis of Skiv2l2 knockdown cells revealed defective progression through the G2/M phase and an accumulation of mitotic cells, suggesting SKIV2L2 contributes to mitotic progression. Since SKIV2L2 targets RNAs to the nuclear exosome for processing and degradation, we identified RNA targets elevated in cells depleted of SKIV2L2 that could account for the observed twofold increase in mitotic cells. Skiv2l2 knockdown cells accumulated replication-dependent histone mRNAs, among other RNAs, that could impede mitotic progression and indirectly trigger differentiation.

Pontocerebellar hypoplasia type 1b (PCH1b) is an autosomal recessive disorder that causes cerebellar hypoplasia and spinal motor neuron degeneration, leading to mortality in early childhood. PCH1b is caused by mutations in the RNA exosome subunit gene, EXOSC3. The RNA exosome is an evolutionarily conserved complex, consisting of nine different core subunits, and one or two 3′-5′ exoribonuclease subunits, that mediates several RNA degradation and processing steps. The goal of this study is to assess the functional consequences of the amino acid substitutions that have been identified in EXOSC3 in PCH1b patients. To analyze these EXOSC3 substitutions, we generated the corresponding amino acid substitutions in the Saccharomyces cerevisiae ortholog of EXOSC3, Rrp40. We find that the rrp40 variants corresponding to EXOSC3-G31A and -D132A do not affect yeast function when expressed as the sole copy of the essential Rrp40 protein. In contrast, the rrp40-W195R variant, corresponding to EXOSC3-W238R in PCH1b patients, impacts cell growth and RNA exosome function when expressed as the sole copy of Rrp40. The rrp40-W195R protein is unstable, and does not associate efficiently with the RNA exosome in cells that also express wild-type Rrp40. Consistent with these findings in yeast, the levels of mouse EXOSC3 variants are reduced compared to wild-type EXOSC3 in a neuronal cell line. These data suggest that cells possess a mechanism for optimal assembly of functional RNA exosome complex that can discriminate between wild-type and variant exosome subunits. Budding yeast can therefore serve as a useful tool to understand the molecular defects in the RNA exosome caused by PCH1b-associated amino acid substitutions in EXOSC3, and potentially extending to disease-associated substitutions in other exosome subunits.

The RNA exosome is fundamental for the degradation of RNA in eukaryotic nuclei. Substrate targeting is facilitated by its co-factor Mtr4p/hMTR4, which links to RNA-binding protein adaptors. One example is the trimeric human nuclear exosome targeting (NEXT) complex, which is composed of hMTR4, the Zn-finger protein ZCCHC8, and the RNA-binding factor RBM7. NEXT primarily targets early and unprocessed transcripts, which demands a rationale for how the nuclear exosome recognizes processed RNAs. Here, we describe the poly(A) tail exosome targeting (PAXT) connection, which comprises the ZFC3H1 Zn-knuckle protein as a central link between hMTR4 and the nuclear poly(A)-binding protein PABPN1. Individual depletion of ZFC3H1 and PABPN1 results in the accumulation of common transcripts that are generally both longer and more extensively polyadenylated than NEXT substrates. Importantly, ZFC3H1/PABPN1 and ZCCHC8/RBM7 contact hMTR4 in a mutually exclusive manner, revealing that the exosome targets nuclear transcripts of different maturation status by substituting its hMTR4-associating adaptors. © 2016 Elsevier Inc.

The eukaryotic RNA exosome is an essential, multi-subunit complex that catalyzes RNA turnover, maturation, and quality control processes. Its non-catalytic donut-shaped core includes 9 subunits that associate with the 3' to 5' exoribonucleases Rrp6, and Rrp44/Dis3, a subunit that also catalyzes endoribonuclease activity. Although recent structures and biochemical studies of RNA bound exosomes from S. cerevisiae revealed that the Exo9 central channel guides RNA to either Rrp6 or Rrp44 using partially overlapping and mutually exclusive paths, several issues related to RNA recruitment remain. Here, we identify activities for the highly basic Rrp6 C-terminal tail that we term the 'lasso' because it binds RNA and stimulates ribonuclease activities associated with Rrp44 and Rrp6 within the 11-subunit nuclear exosome. Stimulation is dependent on the Exo9 central channel, and the lasso contributes to degradation and processing activities of exosome substrates in vitro and in vivo. Finally, we present evidence that the Rrp6 lasso may be a conserved feature of the eukaryotic RNA exosome.

The RNA exosome is a highly conserved ribonuclease endowed with 3'-5' exonuclease and endonuclease activities. The multisubunit complex resides in both the nucleus and the cytoplasm, with varying compositions and activities between the two compartments. While the cytoplasmic exosome functions mostly in mRNA quality control pathways, the nuclear RNA exosome partakes in the 3'-end processing and complete decay of a wide variety of substrates, including virtually all types of noncoding (nc) RNAs. To handle these diverse tasks, the nuclear exosome engages with dedicated cofactors, some of which serve as activators by stimulating decay through oligoA addition and/or RNA helicase activities or, as adaptors, by recruiting RNA substrates through their RNA-binding capacities. Most nuclear exosome cofactors contain the essential RNA helicase Mtr4 (MTR4 in humans). However, apart from Mtr4, nuclear exosome cofactors have undergone significant evolutionary divergence. Here, we summarize biochemical and functional knowledge about the nuclear exosome and exemplify its cofactor variety by discussing the best understood model organisms-the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and human cells.

A polyA (pA) tail is an essential modification added to the 3′ ends of a wide range of RNAs at different stages of their metabolism. Here, we describe the main sources of polyadenylation and outline their underlying biochemical interactions within the nuclei of budding yeast Saccharomyces cerevisiae , human cells and, when relevant, the fission yeast Schizosaccharomyces pombe . Polyadenylation mediated by the S. cerevisiae Trf4/5 enzymes, and their human homologues PAPD5/7, typically leads to the 3′-end trimming or complete decay of non-coding RNAs. By contrast, the primary function of canonical pA polymerases (PAPs) is to produce stable and nuclear export-competent mRNAs. However, this dichotomy is becoming increasingly blurred, at least in S. pombe and human cells, where polyadenylation mediated by canonical PAPs may also result in transcript decay. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

The exosome complex is a major eukaryotic exoribonuclease that requires the SKI complex for its activity in the cytoplasm. In yeast, the Ski7 protein links both complexes, whereas a functional equivalent of the Ski7 has remained unknown in the human genome. Proteomic analysis revealed that a previously uncharacterized short splicing isoform of HBS1L (HBS1LV3) is the long-sought factor linking the exosome and SKI complexes in humans. In contrast, the canonical HBS1L variant, HBS1LV1, which acts as a ribosome dissociation factor, does not associate with the exosome and instead interacts with the mRNA surveillance factor PELOTA. Interestingly, both HBS1LV1 and HBS1LV3 interact with the SKI complex and HBS1LV1 seems to antagonize SKI/exosome supercomplex formation. HBS1LV3 contains a unique C-terminal region of unknown structure, with a conserved RxxxFxxxL motif responsible for exosome binding and may interact with the exosome core subunit RRP43 in a way that resembles the association between Rrp6 RNase and Rrp43 in yeast. HBS1LV3 or the SKI complex helicase (SKI2W) depletion similarly affected the transcriptome, deregulating multiple genes. Furthermore, half-lives of representative upregulated mRNAs were increased, supporting the involvement of HBS1LV3 and SKI2W in the same mRNA degradation pathway, essential for transcriptome homeostasis in the cytoplasm.

In yeast, the Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex acts as a cofactor for the nuclear exosome to promote degradation of various RNAs. However, the corresponding machinery in mammals is less characterized. We analyzed the interactions of the human TRAMP-like proteins, PAPD5, ZCCHC7, and MTR4, with the nuclear exosome. PAPD5 and ZCCHC7 exhibited mutual interactions in presence of the exosome catalytic subunit RRP6, whereas MTR4 was dispensable for their assembly. Furthermore, the human TRAMP-like proteins were involved in the RRP6-catalyzed turnover of pre-rRNA 5'ETS fragments. These results suggest the significant role for RRP6 in the assembly of TRAMP-like proteins during nucleolar RNA surveillance.

RNA degradation is tightly regulated to selectively target aberrant RNAs, including viral RNA, but this regulation is incompletely understood. Through RNAi screening in Drosophila cells, we identified the 3′-to-5′ RNA exosome and two components of the exosome cofactor TRAMP (Trf4/5–Air1/2–Mtr4 polyadenylation) complex, dMtr4 and dZcchc7, as antiviral against a panel of RNA viruses. We extended our studies to human orthologs and found that the exosome as well as TRAMP components hMTR4 and hZCCHC7 are antiviral. While hMTR4 and hZCCHC7 are normally nuclear, infection by cytoplasmic RNA viruses induces their export, forming a cytoplasmic complex that specifically recognizes and induces degradation of viral mRNAs. Furthermore, the 3′ untranslated region (UTR) of bunyaviral mRNA is sufficient to confer virus-induced exosomal degradation. Altogether, our results reveal that signals from viral infection repurpose TRAMP components to a cytoplasmic surveillance role where they selectively engage viral RNAs for degradation to restrict a broad range of viruses.

Summary The RNA exosome complex associates with nuclear and cytoplasmic cofactors to mediate the decay, surveillance, or processing of a wide variety of transcripts. In the cytoplasm, the conserved core of the exosome (Exo10) functions together with the conserved Ski complex. The interaction of S. cerevisiae Exo10 and Ski is not direct but requires a bridging cofactor, Ski7. Here, we report the 2.65 Å resolution structure of S. cerevisiae Exo10 bound to the interacting domain of Ski7. Extensive hydrophobic interactions rationalize the high affinity and stability of this complex, pointing to Ski7 as a constitutive component of the cytosolic exosome. Despite the absence of sequence homology, cytoplasmic Ski7 and nuclear Rrp6 bind Exo10 using similar surfaces and recognition motifs. Knowledge of the interacting residues in the yeast complexes allowed us to identify a splice variant of human HBS1-Like as a Ski7-like exosome-binding protein, revealing the evolutionary conservation of this cytoplasmic cofactor., Graphical Abstract, Highlights • The yeast exosome binds Ski7 with low nanomolar affinity and extensive interactions • The Ski7 exosome-binding domain folds upon recognizing Csl4, Mtr3, and Rrp43 subunits • Ski7 and Rrp6 lack sequence homology but form a similar interface with the exosome • The exosome interface residues of yeast Ski7 are conserved in human Hbs1L isoform 3, Kowalinski et al. (2016) show that the yeast exosome core complex recognizes the cytoplasmic cofactor Ski7 and the nuclear cofactor Rrp6 similarly. Through structural analyses, they identify a splice variant of HSB1-Like as the long-sought Ski7-like exosome binding cofactor in humans.

The RNA exosome complex is the most versatile RNA-degradation machine in eukaryotes. The exosome has a central role in several aspects of RNA biogenesis, including RNA maturation and surveillance. Moreover, it is emerging as an important player in regulating the expression levels of specific mRNAs in response to environmental cues and during cell differentiation and development. Although the mechanisms by which RNA is targeted to (or escapes from) the exosome are still not fully understood, general principles have begun to emerge, which we discuss in this Review. In addition, we introduce and discuss novel, previously unappreciated functions of the nuclear exosome, including in transcription regulation and in the maintenance of genome stability.

Nuclear VCP-like 2 (NVL2) is a chaperone-like nucleolar ATPase of the AAA (ATPase associated with diverse cellular activities) family, which exhibits a high level of amino acid sequence similarity with the cytosolic AAA-ATPase VCP/p97. These proteins generally act on macromolecular complexes to stimulate energy-dependent release of their constituents. We previously showed that NVL2 interacts with RNA processing/degradation machinery containing an RNA helicase MTR4/DOB1 and an exonuclease complex, nuclear exosome, and involved in the biogenesis of 60S ribosomal subunits. These observations implicate NVL2 as a remodeling factor for the MTR4-exosome complex during the maturation of pre-ribosomal particles. Here, we used a proteomic screen and identified a WD repeat-containing protein 74 (WDR74) as a factor that specifically dissociates from this complex depending on the ATPase activity of NVL2. WDR74 shows weak amino acid sequence similarity with the yeast ribosome biogenesis protein Nsa1 and is co-localized with NVL2 in the nucleolus. Knockdown of WDR74 decreases 60S ribosome levels. Taken together, our results suggest that WDR74 is a novel regulatory protein of the MTR4-exsosome complex whose interaction is regulated by NVL2 and is involved in ribosome biogenesis.

SummaryThe exosome regulates the processing, degradation, and surveillance of a plethora of RNA species. However, little is known about how the exosome recognizes and is recruited to its diverse substrates. We report the identification of adaptor proteins that recruit the exosome-associated helicase, Mtr4, to unique RNA substrates. Nop53, the yeast homolog of the tumor suppressor PICT1, targets Mtr4 to pre-ribosomal particles for exosome-mediated processing, while a second adaptor Utp18 recruits Mtr4 to cleaved rRNA fragments destined for degradation by the exosome. Both Nop53 and Utp18 contain the same consensus motif, through which they dock to the “arch” domain of Mtr4 and target it to specific substrates. These findings show that the exosome employs a general mechanism of recruitment to defined substrates and that this process is regulated through adaptor proteins.

The eukaryotic exosome is a conserved RNA-degrading complex that functions in RNA surveillance, turnover and processing. How the same machinery can either completely degrade or precisely trim RNA substrates has long remained unexplained. Here we report the crystal structures of a yeast nuclear exosome containing the 9-subunit core, the 3'-5' RNases Rrp44 and Rrp6, and the obligate Rrp6-binding partner Rrp47 in complex with different RNAs. The combined structural and biochemical data of this 12-subunit complex reveal how a single-stranded RNA can reach the Rrp44 or Rrp6 active sites directly or can bind Rrp6 and be threaded via the central channel towards the distal RNase Rrp44. When a bulky RNA is stalled at the entrance of the channel, Rrp6-Rrp47 swings open. The results suggest how the same molecular machine can coordinate processive degradation and partial trimming in an RNA-dependent manner by a concerted swinging mechanism of the two RNase subunits.

SummaryWe have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3′ regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3′ regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function.

Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3'-5' turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The 'ratchet helix' is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates.

Nuclear RNA exosomes catalyze a range of RNA processing and decay activities that are coordinated in part by cofactors, including Mpp6, Rrp47, and the Mtr4 RNA helicase. Mpp6 interacts with the nine-subunit exosome core, while Rrp47 stabilizes the exoribonuclease Rrp6 and recruits Mtr4, but it is less clear if these cofactors work together. Using biochemistry with Saccharomyces cerevisiae proteins, we show that Rrp47 and Mpp6 stimulate exosome-mediated RNA decay, albeit with unique dependencies on elements within the nuclear exosome. Mpp6-exosomes can recruit Mtr4, while Mpp6 and Rrp47 each contribute to Mtr4-dependent RNA decay, with maximal Mtr4-dependent decay observed with both cofactors. The 3.3 Å structure of a twelve-subunit nuclear Mpp6 exosome bound to RNA shows the central region of Mpp6 bound to the exosome core, positioning its Mtr4 recruitment domain next to Rrp6 and the exosome central channel. Genetic analysis reveals interactions that are largely consistent with our model. DOI: http://dx.doi.org/10.7554/eLife.29062.001

The archaeal exosome, a protein complex responsible for phosphorolytic degradation and tailing of RNA, has an RNA-binding platform containing Rrp4, Csl4, and DnaG. Aiming to detect novel interaction partners of the exosome, we copurified Nop5, which is a part of an rRNA methylating ribonucleoprotein complex, with the exosome of Sulfolobus solfataricus grown to a late stationary phase. We demonstrated the capability of Nop5 to bind to the exosome with a homotrimeric Rrp4-cap and to increase the proportion of polyadenylated RNAin vitro, suggesting that Nop5 is a dual-function protein. Since tailing of RNA probably serves to enhance RNA degradation, association of Nop5 with the archaeal exosome in the stationary phase may enhance tailing and degradation of RNA as survival strategy.

The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The RNA exosome-associated EXOSC10 protein is a distributive, 3'-5' exoribonuclease. The following protocol describes an approach to monitor the ribonucleolytic activity of affinity-purified EXOSC10-containing RNA exosomes, originating from HEK-293 cells, as reported in (Domanski et al., 2016) and further detailed in the companion bio-protocol to this one (Domanski and LaCava, 2017).

The exosome is a conserved multiprotein complex essential for RNA processing and degradation. The nuclear exosome is a key factor for pre-rRNA processing through the activity of its catalytic subunits, Rrp6 and Rrp44. In Saccharomyces cerevisiae, Rrp6 is exclusively nuclear and has been shown to interact with exosome cofactors. With the aim of analyzing proteins associated with the nuclear exosome, in this work, we purified the complex with Rrp6-TAP, identified the co-purified proteins by mass spectrometry, and found karyopherins to be one of the major groups of proteins enriched in the samples. By investigating the biological importance of these protein interactions, we identified Srp1, Kap95, and Sxm1 as the most important karyopherins for Rrp6 nuclear import and the nuclear localization signals recognized by them. Based on the results shown here, we propose a model of multiple pathways for the transport of Rrp6 to the nucleus.

The eukaryotic RNA exosome is an essential and conserved protein complex that can degrade or process RNA substrates in the 3′-to-5′ direction. Since its discovery nearly two decades ago, studies have focused on determining how the exosome, along with associated cofactors, achieves the demanding task of targeting particular RNAs for degradation and/or processing in both the nucleus and cytoplasm. In this review, we highlight recent advances that have illuminated roles for the RNA exosome and its cofactors in specific biological pathways, alongside studies that attempted to dissect these activities through structural and biochemical characterization of nuclear and cytoplasmic RNA exosome complexes.

The nuclear exosome and the associated RNA helicase Mtr4 participate in the processing of several ribonucleoprotein particles (RNP), including the maturation of the large ribosomal subunit (60S). S. cerevisiae Mtr4 interacts directly with Nop53, a ribosomal biogenesis factor present in late pre-60S particles containing precursors of the 5.8S rRNA. The Mtr4–Nop53 interaction plays a pivotal role in the maturation of the 5.8S rRNA, providing a physical link between the nuclear exosome and the pre-60S RNP. An analogous interaction between Mtr4 and another ribosome biogenesis factor, Utp18, directs the exosome to an earlier preribosomal particle. Nop53 and Utp18 contain a similar Mtr4-binding motif known as the arch-interacting motif (AIM). Here, we report the 3.2 Å resolution crystal structure of S. cerevisiae Mtr4 bound to the interacting region of Nop53, revealing how the KOW domain of the helicase recognizes the AIM sequence of Nop53 with a network of hydrophobic and electrostatic interactions. The AIM-interacting residues are conserved in Mtr4 and are not present in the related cytoplasmic helicase Ski2, rationalizing the specificity and versatility of Mtr4 in the recognition of different AIM-containing proteins. Using nuclear magnetic resonance (NMR), we show that the KOW domain of Mtr4 can simultaneously bind an AIM-containing protein and a structured RNA at adjacent surfaces, suggesting how it can dock onto RNPs. The KOW domains of exosome-associated helicases thus appear to have evolved from the KOW domains of ribosomal proteins and to function as RNP-binding modules in the context of the nuclear exosome.

Centrally positioned in nuclear RNA metabolism, the exosome deals with virtually all transcript types. This 3'-5' exo- and endo-nucleolytic degradation machine is guided to its RNA targets by adaptor proteins that enable substrate recognition. Recently, the discovery of the 'Poly(A) tail exosome targeting (PAXT)' connection as an exosome adaptor to human nuclear polyadenylated transcripts has relighted the interest of poly(A) binding proteins (PABPs) in both RNA productive and destructive processes.

The TRAMP complex is involved in the nuclear surveillance and turnover of noncoding RNAs and intergenic transcripts. TRAMP is associated with the nuclear exosome and consists of a poly(A)polymerase subcomplex (Trf4-Air2) and a helicase (Mtr4). We found that N-terminal low-complexity regions of Trf4 and Air2 bind Mtr4 in a cooperative manner. The 2.4 Å resolution crystal structure of the corresponding ternary complex reveals how Trf4 and Air2 wrap around the DExH core of the helicase. Structure-based mutations on the DExH core impair binding to Trf4 and Air2, and also to Trf5 and Air1. The combination of structural, biochemical, and biophysical data suggests that the poly(A)polymerase core of Trf4-Air2 is positioned below the base of the helicase, where the unwound 3' end of an RNA substrate is expected to emerge. The results reveal conceptual similarities between the two major regulators of the exosome, the nuclear TRAMP and cytoplasmic Ski complexes.

The vast majority of the mammalian genome has the potential to expressnoncoding RNA (ncRNA). The 11-subunit RNA exosome complex is the main source of cellular 3′–5′ exoribonucleolytic activity and potentially regulates the mammalian noncoding transcriptome1. Here we generated a mouse model in which the essential subunit Exosc3 of the RNA exosome complex can be conditionally deleted. Exosc3-deficient B cells lack the ability to undergo normal levels of class switch recombination and somatic hypermutation, two mutagenic DNA processes used to generate antibody diversity via the B-cell mutator protein activation-induced cytidine deaminase (AID)2,3. The transcriptome of Exosc3-deficient B cells has revealed the presence of many novel RNA exosome substrate ncRNAs. RNA exosome substrate RNAs include xTSS-RNAs, transcription start site (TSS)-associated antisense transcripts that can exceed 500 base pairs in length and are transcribed divergently from cognate coding gene transcripts. xTSS-RNAs are most strongly expressed at genes that accumulate AID-mediated somatic mutations and/or are frequent translocation partners of DNA double-strand breaks generated at Igh in B cells4,5. Strikingly, translocations near TSSs or within gene bodies occur over regions of RNA exosome substrate ncRNA expression. These RNA exosome-regulated, antisense-transcribed regions of the B-cell genome recruit AID and accumulate single-strand DNA structures containing RNA–DNA hybrids. We propose that RNA exosome regulation of ncRNA recruits AID to single-strand DNA-forming sites of antisense and divergent transcription in the B-cell genome, thereby creating a link between ncRNA transcription and overall maintenance of B-cell genomic integrity.

The exosome ribonuclease complex functions in both the limited trimming of the 3′-ends of nuclear substrates during RNA processing events and the complete destruction of nuclear and cytoplasmic RNAs. The two RNases of the eukaryotic exosome, Rrp44 (rRNA-processing protein 44) and Rrp6, are bound at either end of a catalytically inert cylindrical core. RNA substrates are threaded through the internal channel of the core to Rrp44 by RNA helicase components of the nuclear TRAMP complex (Trf4–Air2–Mtr4 polyadenylation complex) or the cytoplasmic Ski (superkiller) complex. Recent studies reveal that Rrp44 can also associate directly with substrates via channel-independent routes. Although the substrates of the exosome are known, it is not clear whether specific substrates are restricted to one or other pathway. Data currently available support the model that processed substrates are targeted directly to the catalytic subunits, whereas at least some substrates that are directed towards discard pathways must be threaded through the exosome core.

The eukaryotic RNA exosome processes and degrades RNA by directing substrates to the distributive or processive 3′ to 5′ exoribonuclease activities of Rrp6 or Rrp44, respectively. The non-catalytic nine-subunit exosome core (Exo9) features a prominent central channel. Although RNA can pass through the channel to engage Rrp44, it is not clear how RNA is directed to Rrp6 or whether Rrp6 uses the central channel. Here we report a 3.3 A crystal structure of a ten-subunit RNA exosome complex from Saccharomyces cerevisiae composed of the Exo9 core and Rrp6 bound to single-stranded poly(A) RNA. The Rrp6 catalytic domain rests on top of the Exo9 S1/KH ring above the central channel, the RNA 3′ end is anchored in the Rrp6 active site, and the remaining RNA traverses the S1/KH ring in an opposite orientation to that observed in a structure of a Rrp44-containing exosome complex. Solution studies with human and yeast RNA exosome complexes suggest that the RNA path to Rrp6 is conserved and dependent on the integrity of the S1/KH ring. Although path selection to Rrp6 or Rrp44 is stochastic in vitro, the fate of a particular RNA may be determined in vivo by the manner in which cofactors present RNA to the RNA exosome. The exosome complex contains two catalytic subunits which degrade RNA in either a distributive (Rrp6) or a processive (Rrp44) manner—previous structures indicated how RNA could be directed to Rrp44, but the path taken to Rrp6 was unclear; here the location of the Rrp6 catalytic domain and the RNA 3′ end are determined and it is found that the RNA lies in an opposite orientation from that of the Rrp44-containing exosome structure, suggesting that the fate of an RNA may be influenced by the manner in which cofactors present it. The exosome complex contains two catalytic subunits that degrade RNA in either a distributive (Rrp6) or a processive (Rrp44) manner. Previous structures indicated how RNA could be directed to Rrp44, but the path taken to Rrp6 was unclear. Christopher Lima and colleagues have now solved the crystal structure of a yeast ten-subunit exosome complex (lacking Rrp44) bound to single-stranded RNA. The location of the Rrp6 catalytic domain and the RNA 3' end are determined, but more importantly, it is found that the RNA lies in an orientation opposite that from the Rrp44-containing exosome structure. This result suggests that the fate of an RNA may be a stochastic decision based on how the RNA binds to the complex.

The RNA-degrading exosome in archaea is structurally very similar to the nine-subunit core of the essential eukaryotic exosome and to bacterial polynucleotide phosphorylase (PNPase). In contrast to the eukaryotic exosome, PNPase and the archaeal exosome exhibit metal ion-dependent, phosphorolytic activities and synthesize heteropolymeric RNA tails in addition to the exoribonucleolytic RNA degradation in 3' → 5' direction. The archaeal nine-subunit exosome consists of four orthologs of eukaryotic exosomal subunits: the RNase PH-domain-containing subunits Rrp41 and Rrp42 form a hexameric ring with three active sites, whereas the S1-domain-containing subunits Rrp4 and Csl4 form an RNA-binding trimeric cap on the top of the ring. In vivo, this cap contains Rrp4 and Csl4 in variable amounts. Rrp4 confers poly(A) specificity to the exosome, whereas Csl4 is involved in the interaction with the archaea-specific subunit of the complex, the homolog of the bacterial primase DnaG. The archaeal DnaG is a highly conserved protein and its gene is present in all sequenced archaeal genomes, although the exosome was lost in halophilic archaea and some methanogens. In exosome-containing archaea, DnaG is tightly associated with the exosome. It functions as an additional RNA-binding subunit with poly(A) specificity in the reconstituted exosome of Sulfolobus solfataricus and enhances the degradation of adenine-rich transcripts in vitro. Not only the RNA-binding cap but also the hexameric Rrp41-Rrp42 ring alone shows substrate selectivity and prefers purines over pyrimidines. This implies a coevolution of the exosome and its RNA substrates resulting in 3'-ends with different affinities to the exosome.

The eukaryotic RNA exosome is an essential multi-subunit ribonuclease complex that contributes to the degradation or processing of nearly every class of RNA in both the nucleus and cytoplasm. Its nine-subunit core shares structural similarity to phosphorolytic exoribonucleases such as bacterial PNPase. PNPase and the RNA exosome core feature a central channel that can accommodate single stranded RNA although unlike PNPase, the RNA exosome core is devoid of ribonuclease activity. Instead, the core associates with Rrp44, an endoribonuclease and processive 3′→5′ exoribonuclease, and Rrp6, a distributive 3′→5′ exoribonuclease. Recent biochemical and structural studies suggest that the exosome core is essential because it coordinates Rrp44 and Rrp6 recruitment, RNA can pass through the central channel, and the association with the core modulates Rrp44 and Rrp6 activities.

The evolutionarily conserved Lsm1-7-Pat1 complex is the most critical activator of mRNA decapping in eukaryotic cells and plays many roles in normal decay, AU-rich element-mediated decay, and miRNA silencing, yet how Pat1 interacts with the Lsm1-7 complex is unknown. Here, we show that Lsm2 and Lsm3 bridge the interaction between the C-terminus of Pat1 (Pat1C) and the Lsm1-7 complex. The Lsm2-3-Pat1C complex and the Lsm1-7-Pat1C complex stimulate decapping in vitro to a similar extent and exhibit similar RNA-binding preference. The crystal structure of the Lsm2-3-Pat1C complex shows that Pat1C binds to Lsm2-3 to form an asymmetric complex with three Pat1C molecules surrounding a heptameric ring formed by Lsm2-3. Structure-based mutagenesis revealed the importance of Lsm2-3-Pat1C interactions in decapping activation in vivo. Based on the structure of Lsm2-3-Pat1C, a model of Lsm1-7-Pat1 complex is constructed and how RNA binds to this complex is discussed.

Getting rid of faulty mRNA The cell monitors the health of its mRNAs, destroying those that are faulty or damaged. Destruction by the exosome complex prevents them from being used to synthesize deranged and potentially dangerous proteins. Schmidt et al. determined the structure of the Ski helicase complex, which guides RNAs to the exosome complex destruction machinery in association with a mRNAbound ribosome. The end of the mRNA is threaded from the ribosome into the heart of the helicase, whence the message would be channeled into the maw of the exosome complex. Science , this issue p. 1431

The yeast exosome is a conserved multiprotein complex essential for RNA processing and degradation. The complex is formed by a nine-subunit core that associates with two hydrolytic 3'-5' exoribonucleases. Although catalytically inert, the assembly of this nine-subunit core seems to be essential for the exosome activity, as mutations in regions that do not directly bind RNA or are not in the active sites of the exonucleases impair the function of the complex. Previously isolated mutations in the exosome core subunit Rrp43p have been shown to negatively affect the function of the complex. With the aim of investigating the effect of these mutations on the complex stability and activity, Rrp43p and its mutant forms were purified by means of the TAP method. Mass spectrometry analyses showed that lower amounts of the exosome subunits are copurified with the mutant Rrp43p proteins. Additionally, by decreasing the stability of the exosome, other nonspecific protein interactions are favored (the data have been deposited to the ProteomeXchange with identifier PXD000580). Exosome copurified with mutant Rrp43p exhibited increased exonuclease activity, suggesting higher dissociation constants for these mutant complexes. Therefore, data reported here indicate that complexes containing a mutant Rrp43p exhibit decreased stability and provide information on additional protein interactions.

Chromosome transmission fidelity 4 (Ctf4) is a conserved protein required for DNA replication. In this report, interactions between human Ctf4 (hCtf4) and the replicative helicase containing the cell division cycle 45 (Cdc45)/minichromosome maintenance 2-7 (Mcm2-7)/Go, Ichi, Nii, and San (GINS) (CMG) proteins [human CMG (hCMG) complex] were examined. The hCtf4-CMG complex was isolated following in vitro interaction of purified proteins (hCtf4 plus the hCMG complex), coinfection of Spodoptera frugiperda (Sf9) insect cells with viruses expressing the hCMG complex and hCtf4, and from HeLa cell chromatin after benzonase and immunoprecipitation steps. The stability of the hCtf4-CMG complex depends upon interactions between hCtf4 and multiple components of the hCMG complex. The hCtf4-CMG complex, like the hCMG complex, contains DNA helicase activity that is more salt-resistant than the helicase activity of the hCMG complex. We demonstrate that the hCtf4-CMG complex contains a homodimeric hCtf4 and a monomeric hCMG complex and suggest that the homodimeric hCtf4 acts as a platform linking polymerase α to the hCMG complex. The role of the hCMG complex as the core of the replisome is also discussed.

Highlights • A wide range of in vivo targets for the exosome complex has been established. • RNA polymerase III transcripts have emerged as major substrates. • The human nucleus has spatially localized forms of the exosome, with matching cofactors. • Structural analyses reveal a highly conserved RNA path through the eukaryotic exosome., In eukaryotes, the exosome complex degrades RNA backbones and plays key roles in RNA processing and surveillance. It was predicted that RNA substrates are threaded through a central channel. This pathway is conserved between eukaryotic and archaeal complexes, even though nuclease activity was lost from the nine-subunit eukaryotic core (EXO-9) and transferred to associated proteins. The exosome cooperates with nuclear and cytoplasmic cofactors, including RNA helicases Mtr4 and Ski2, respectively. Structures of an RNA-bound exosome and both helicases revealed how substrates are channeled through EXO-9 to the associated nuclease Rrp44. Recent high-throughput analyses provided fresh insights relating exosome structure to its diverse in vivo functions. They also revealed surprisingly high degradation rates for newly synthesized RNAs, particularly RNA polymerase III transcripts.

Most unwanted RNA transcripts in the nucleus of eukaryotic cells, such as splicing-defective pre-mRNAs and spliced-out introns, are rapidly degraded by the nuclear exosome. In budding yeast, a number of these unwanted RNA transcripts, including spliced-out introns, are first recognized by the nuclear exosome cofactor Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex before subsequent nuclear-exosome-mediated degradation. However, it remains unclear when spliced-out introns are recognized by TRAMP, and whether TRAMP may have any potential roles in pre-mRNA splicing. Here, we demonstrated that TRAMP is cotranscriptionally recruited to nascent RNA transcripts, with particular enrichment at intronic sequences. Deletion of TRAMP components led to further accumulation of unspliced pre-mRNAs even in a yeast strain defective in nuclear exosome activity, suggesting a novel stimulatory role of TRAMP in splicing. We also uncovered new genetic and physical interactions between TRAMP and several splicing factors, and further showed that TRAMP is required for optimal recruitment of the splicing factor Msl5p. Our study provided the first evidence that TRAMP facilitates pre-mRNA splicing, and we interpreted this as a fail-safe mechanism to ensure the cotranscriptional recruitment of TRAMP before or during splicing to prepare for the subsequent targeting of spliced-out introns to rapid degradation by the nuclear exosome., link_to_OA_fulltext

The multisubunit RNA exosome complex is a major ribonuclease of eukaryotic cells that participates in the processing, quality control and degradation of virtually all classes of RNA in Eukaryota. All this is achieved by about a dozen proteins with only three ribonuclease activities between them. At first glance, the versatility of the pathways involving the exosome and the sheer multitude of its substrates are astounding. However, after fifteen years of research we have some understanding of how exosome activity is controlled and applied inside the cell. The catalytic properties of the eukaryotic exosome are fairly well described and attention is now drawn to how the interplay between these activities impacts cell physiology. Also, it has become evident that exosome function relies on many auxiliary factors, which are intensely studied themselves. In this way, the focus of exosome research is slowly leaving the test tube and moving back into the cell. The exosome also has an interesting evolutionary history, which is evident within the eukaryotic lineage but only fully appreciated when considering similar protein complexes found in Bacteria and Archaea. Thus, while we keep this review focused on the most comprehensively described yeast and human exosomes, we shall point out similarities or dissimilarities to prokaryotic complexes and proteins where appropriate. The article is divided into three parts. In Part One we describe how the exosome is built and how it manifests in cells of different organisms. In Part Two we detail the enzymatic properties of the exosome, especially recent data obtained for holocomplexes. Finally, Part Three presents an overview of the RNA metabolism pathways that involve the exosome. This article is part of a Special Issue entitled: RNA Decay mechanisms.

Background: The Rrp6·Rrp47 complex is important for RNA processing and degradation events. Results: Rrp6 and Rrp47 are independently imported into the nucleus, and Rrp47 is destabilized in cells lacking Rrp6. Conclusion: Rrp6 binds Rrp47 in the nucleus and protects it from proteolysis. Significance: Nuclear assembly of the Rrp6·Rrp47 complex spatially limits the nuclease complex and controls the expression of Rrp47., Rrp6 is a key catalytic subunit of the nuclear RNA exosome that plays a pivotal role in the processing, degradation, and quality control of a wide range of cellular RNAs. Here we report our findings on the assembly of the complex involving Rrp6 and its associated protein Rrp47, which is required for many Rrp6-mediated RNA processes. Recombinant Rrp47 is expressed as a non-globular homodimer. Analysis of the purified recombinant Rrp6·Rrp47 complex revealed a heterodimer, suggesting that Rrp47 undergoes a structural reconfiguration upon interaction with Rrp6. Studies using GFP fusion proteins show that Rrp6 and Rrp47 are localized to the yeast cell nucleus independently of one another. Consistent with this data, Rrp6, but not Rrp47, is found associated with the nuclear import adaptor protein Srp1. We show that the interaction with Rrp6 is critical for Rrp47 stability in vivo; in the absence of Rrp6, newly synthesized Rrp47 is rapidly degraded in a proteasome-dependent manner. These data resolve independent nuclear import routes for Rrp6 and Rrp47, reveal a structural reorganization of Rrp47 upon its interaction with Rrp6, and demonstrate a proteasome-dependent mechanism that efficiently suppresses the expression of Rrp47 in the absence of Rrp6.

The RNA exosome, a highly conserved protein complex originally discovered in S. cerevisiae, has emerged as a major component of 3′-5′ ribonucleoytic RNA surveillance pathways in both archae and eukaryotes 1–5. The eukaryotic exosome is composed of a 9-subunit core, referred to as exo-9. Six of these proteins are arranged in a hexameric ring with a central channel, while the remaining 3 proteins form a trimeric cap and sit on top of this ring structure 6. In S. cerevisiae, catalytic activity resides in an additional component Dis3/Rrp44 that associates with the catalytically inactive core exosome to form the functional cytoplasmic and nuclear exo-10 complex 7–9. RNA substrates with sufficiently long single-stranded 3′ ends may thread through the core to reach the active site of Dis3; alternatively, RNA may bypass the exosome core channel and enter the active site of Dis3 directly 8, 9. In the nucleus, exo-10 can associate further with the nonessential 3′-5′ exoribonuclease Rrp6 to form the exo-11 complex 10. Rrp6 can also function independently of exo-10 on a subset of RNA targets, since mutants unable to interact with exo-10 still complement some functions of native Rrp6 11. Remarkably, experiments have shown that the exosome functions in degradation of a wide range of substrates, including coding and noncoding RNAs produced by all 3 major RNA polymerases 12–15. Furthermore, the exosome functions in the 3′ end formation of a growing list of transcripts 10, 13, 16–20. Hence, the exosome is not only able to adapt to a multitude of RNA substrates, but its catalytic activities can be modulated, likely by protein cofactors such as TRAMP, to distinguish between processing and degradation. The exosome relies on multiple co-factors, such as the TRAMP complex, for recruitment of its vast repertoire of substrates, and possibly modulation of its activity. Originally discovered in yeast, the TRAMP complex is composed of a noncanonical poly(A) polymerase Trf4 or Trf5, a zinc-knuckle protein Air1 or Air2, and the RNA helicase Mtr4 21–24. TRAMP polyadenylates RNAs destined for Rrp6 and the core exosome, assisting in transcript recognition and exosome activation. In this way, TRAMP plays a critical role in ridding the cell of non-coding transcripts generated through pervasive Pol II transcription, as well as functioning in the biogenesis and turnover of functional coding and non-coding RNAs 16, 19, 25. Extensive research has revealed the critical role the TRAMP plays in determining nuclear RNA fate, though the mechanism used for recognition of RNA substrates and subsequent activation of the exosome remains elusive.