End Processing Machinery Research Articles

A CPF-like phosphatase module links transcription termination to chromatin silencing

The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3' processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3' end-processing machinery. LD has been shown to co-associate invivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.

Molecular basis of human poly(A) polymerase recruitment by mPSF.

3' end processing of most eukaryotic precursor-mRNAs (pre-mRNAs) is a crucial cotranscriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3' end processing machinery, the four-subunit mammalian polyadenylation specificity factor (mPSF) recognizes the polyadenylation signal (PAS) in the pre-mRNA and recruits the poly(A) polymerase α (PAPOA) to it. To shed light on the molecular mechanisms of PAPOA recruitment to mPSF, we used a combination of cryogenic-electron microscopy (cryo-EM) single-particle analysis, computational structure prediction, and in vitro biochemistry to reveal an intricate interaction network. A short linear motif in the mPSF subunit FIP1 interacts with the structured core of human PAPOA, with a binding mode that is evolutionarily conserved from yeast to human. In higher eukaryotes, however, PAPOA contains a conserved C-terminal motif that can interact intramolecularly with the same residues of the PAPOA structured core used to bind FIP1. Interestingly, using biochemical assay and cryo-EM structural analysis, we found that the PAPOA C-terminal motif can also directly interact with mPSF at the subunit CPSF160. These results show that PAPOA recruitment to mPSF is mediated by two distinct intermolecular connections and further suggest the presence of mutually exclusive interactions in the regulation of 3' end processing.

A direct interaction between CPF and RNA Pol II links RNA 3′ end processing to transcription

Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, and protein phosphatase activities, which cleave and polyadenylate pre-mRNAs and dephosphorylate RNA Pol II to control transcription. Exactly how the RNA 3' end processing machinery is coupled to transcription remains unclear. Here, we combine invitro reconstitution, structural studies, and genome-wide analyses to show that yeast CPF physically and functionally interacts with RNA Pol II. Surprisingly, CPF-mediated dephosphorylation promotes the formation of an RNA Pol II stalk-to-stalk homodimer invitro. This dimer is compatible with transcription but not with the binding of transcription elongation factors. Disruption of the dimerization interface in cells causes transcription defects, including altered RNA Pol II abundance on protein-coding genes, tRNA genes, and intergenic regions. We hypothesize that RNA Pol II dimerization may provide a mechanistic basis for the allosteric model of transcription termination.

Clp1, an overexpressed subunit of CF IA and insights into its molecular role using biophysical approaches

In eukaryotes, pre-messenger RNA (pre-mRNA) 3′-end processing is vital for gene expression and mRNA biogenesis; any awry in this pre-mRNA processing has detrimental effects and causes various diseases in humans. Pre-mRNA processing is coordinated via complex multi-protein machinery involving over 20 protein factors in Saccharomyces cerevisiae. Endonucleolytic cleavage at the pre-mRNA 3′-end followed by poly(A) tail addition at the newly generated 3′-end requires CF IA, CPF, CF IB and other sequence elements. Among these essential proteins, Clp1 of the CF IA sub-unit is indispensable for the effective functioning of 3′-end processing machinery. Clp1, a conserved protein, is proposed to interlink 3′-end processing, transcriptional elongation and transcription termination. However, experimental evidence defining the precise roles played by Clp1, its interacting partners from other subunits and information about its exact mechanism of action during pre-mRNA processing is lacking. The current study details on the Clp1 N-terminal domain's (1–100 amino acids referred to as Clp11–100) successful cloning, expression and characterization using numerous biophysical techniques like circular dichroism and mass spectrometry etc. This information would open new frontiers to decipher the structure and molecular role of Clp1 during pre-mRNA maturation.

The Arabidopsis pre-mRNA 3' end processing related protein FIP1 promotes seed dormancy via the DOG1 and ABA pathways.

Seed dormancy is an important adaptive trait to prevent germination occurring at an inappropriate time. The mechanisms governing seed dormancy and germination are complex. Here, we report that FACTOR INTERACTING WITH POLY(A) POLYMERASE 1 (FIP1), a component of the pre-mRNA 3' end processing machinery, is involved in seed dormancy and germination processes in Arabidopsis thaliana. FIP1 is mainly expressed in seeds and the knockout of FIP1 causes reduced seed dormancy, indicating that FIP1 positively influences seed dormancy. Meanwhile, fip1 mutants are insensitive to exogenous ABA during seed germination and early seedling establishment. The terms 'seed maturation' and 'response to ABA stimulus' are significantly enriched in a gene ontology analysis based on genes differentially expressed between fip1-1 and the wild type. Several of these genes, including ABI5, DOG1 and PYL12, show significantly decreased transcript levels in fip1. Genetic analysis showed that either cyp707a2 or dog1-5 partially, but in combination completely, represses the reduced seed dormancy of fip1, indicating that the double mutant cyp707a2 dog1-5 is epistatic to fip1. Moreover, FIP1 is required for CFIM59, another component of pre-mRNA 3' end processing machinery, to govern seed dormancy and germination. Overall, we identified FIP1 as a regulator of seed dormancy and germination that plays a crucial role in governing these processes through the DOG1 and ABA pathways.

An examination of the metal ion content in the active sites of human endonucleases CPSF73 and INTS11

Human cleavage and polyadenylation specificity factor (CPSF)73 (also known as CPSF3) is the endoribonuclease that catalyzes the cleavage reaction for the 3'-end processing of pre-mRNAs. The active site of CPSF73 is located at the interface between a metallo-β-lactamase domain and a β-CASP domain. Two metal ions are coordinated by conserved residues, five His and two Asp, in the active site, and they are critical for the nuclease reaction. The metal ions have long been thought to be zinc ions, but their exact identity has not been examined. Here we present evidence from inductively coupled plasma mass spectrometry and X-ray diffraction analyses that a mixture of metal ions, including Fe, Zn, and Mn, is present in the active site of CPSF73. The abundance of the various metal ions is different in samples prepared from different expression hosts. Zinc is present at less than 20% abundance in a sample expressed in insect cells, but the sample is active in cleaving a pre-mRNA substrate in a reconstituted canonical 3'-end processing machinery. Zinc is present at 75% abundance in a sample expressed in human cells, which has comparable endonuclease activity. We also observe a mixture of metal ions in the active site of the CPSF73 homolog INTS11, the endonuclease for Integrator. Taken together, our results provide further insights into the role of metal ions in the activity of CPSF73 and INTS11 for RNA 3'-end processing.

Mechanistic insights into RNA surveillance by the canonical poly(A) polymerase Pla1 of the MTREC complex

The S. pombe orthologue of the human PAXT connection, Mtl1-Red1 Core (MTREC), is an eleven-subunit complex that targets cryptic unstable transcripts (CUTs) to the nuclear RNA exosome for degradation. It encompasses the canonical poly(A) polymerase Pla1, responsible for polyadenylation of nascent RNA transcripts as part of the cleavage and polyadenylation factor (CPF/CPSF). In this study we identify and characterise the interaction between Pla1 and the MTREC complex core component Red1 and analyse the functional relevance of this interaction in vivo. Our crystal structure of the Pla1-Red1 complex shows that a 58-residue fragment in Red1 binds to the RNA recognition motif domain of Pla1 and tethers it to the MTREC complex. Structure-based Pla1-Red1 interaction mutations show that Pla1, as part of MTREC complex, hyper-adenylates CUTs for their efficient degradation. Interestingly, the Red1-Pla1 interaction is also required for the efficient assembly of the fission yeast facultative heterochromatic islands. Together, our data suggest a complex interplay between the RNA surveillance and 3’-end processing machineries.

The Chromatin Landscape around DNA Double-Strand Breaks in Yeast and Its Influence on DNA Repair Pathway Choice

DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5'-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors, or by post-translational modifications of histones, thus increasing chromatin flexibility and the accessibility of repair enzymes to the DNA. Here, we review histone post-translational modifications occurring around a DSB in the yeast Saccharomyces cerevisiae and their role in DSB repair, with particular attention to DSB repair pathway choice.

Macrophage differentiation is marked by increased abundance of the mRNA 3' end processing machinery, altered poly(A) site usage, and sensitivity to the level of CstF64.

Regulation of mRNA polyadenylation is important for response to external signals and differentiation in several cell types, and results in mRNA isoforms that vary in the amount of coding sequence or 3' UTR regulatory elements. However, its role in differentiation of monocytes to macrophages has not been investigated. Macrophages are key effectors of the innate immune system that help control infection and promote tissue-repair. However, overactivity of macrophages contributes to pathogenesis of many diseases. In this study, we show that macrophage differentiation is characterized by shortening and lengthening of mRNAs in relevant cellular pathways. The cleavage/polyadenylation (C/P) proteins increase during differentiation, suggesting a possible mechanism for the observed changes in poly(A) site usage. This was surprising since higher C/P protein levels correlate with higher proliferation rates in other systems, but monocytes stop dividing after induction of differentiation. Depletion of CstF64, a C/P protein and known regulator of polyadenylation efficiency, delayed macrophage marker expression, cell cycle exit, attachment, and acquisition of structural complexity, and impeded shortening of mRNAs with functions relevant to macrophage biology. Conversely, CstF64 overexpression increased use of promoter-proximal poly(A) sites and caused the appearance of differentiated phenotypes in the absence of induction. Our findings indicate that regulation of polyadenylation plays an important role in macrophage differentiation.

Birth of a poly(A) tail: mechanisms and control of mRNA polyadenylation.

During their synthesis in the cell nucleus, most eukaryotic mRNAs undergo a two-step 3'-end processing reaction in which the pre-mRNA is cleaved and released from the transcribing RNA polymerase II and a polyadenosine (poly(A)) tail is added to the newly formed 3'-end. These biochemical reactions might appear simple at first sight (endonucleolytic RNA cleavage and synthesis of a homopolymeric tail), but their catalysis requires a multi-faceted enzymatic machinery, the cleavage and polyadenylation complex (CPAC), which is composed of more than 20 individual protein subunits. The activity of CPAC is further orchestrated by Poly(A) Binding Proteins (PABPs), which decorate the poly(A) tail during its synthesis and guide the mRNA through subsequent gene expression steps. Here, we review the structure, molecular mechanism, and regulation of eukaryotic mRNA 3'-end processing machineries with a focus on the polyadenylation step. We concentrate on the CPAC and PABPs from mammals and the budding yeast, Saccharomyces cerevisiae, because these systems are the best-characterized at present. Comparison of their functions provides valuable insights into the principles of mRNA 3'-end processing.

UBE3D Regulates mRNA 3'-End Processing and Maintains Adipogenic Potential in 3T3-L1 Cells.

We have previously described the role of an essential Saccharomyces cerevisiae gene, important for cleavage and polyadenylation 1 (IPA1), in the regulation of gene expression through its interaction with Ysh1, the endonuclease subunit of the mRNA 3'-end processing complex. Through a similar mechanism, the mammalian homolog ubiquitin protein ligase E3D (UBE3D) promotes the migratory and invasive potential of breast cancer cells, but its role in the regulation of gene expression during normal cellular differentiation has not previously been described. In this study, we show that CRISPR/Cas9-mediated knockout of Ube3d in 3T3-L1 cells blocks their ability to differentiate into mature adipocytes. Consistent with previous studies in other cell types, Ube3d knockout leads to decreased levels of CPSF73 and global changes in cellular mRNAs indicative of a loss of 3'-end processing capacity. Ube3d knockout cells also display decreased expression of known preadipogenic markers. Overexpression of either UBE3D or CPSF73 rescues the differentiation defect and partially restores protein levels of these markers. These results support a model in which UBE3D is necessary for the maintenance of the adipocyte-committed state via its regulation of the mRNA 3'-end processing machinery.

U1 snRNP proteins promote proximal alternative polyadenylation sites by directly interacting with 3' end processing core factors.

In eukaryotic cells, both alternative splicing and alternative polyadenylation (APA) play essential roles in the gene regulation network. U1 small ribonucleoprotein particle (U1 snRNP) is a major component of spliceosome, and U1 snRNP complex can suppress proximal APA sites through crosstalking with 3' end processing factors. However, here we show that both knockdown and overexpression of SNRPA, SNRPC, SNRNP70, and SNRPD2, the U1 snRNP proteins, promote the usage of proximal APA sites at the transcriptome level. SNRNP70 can drive the phase transition of PABPN1 from droplet to aggregate, which may reduce the repressive effects of PABPN1 on the proximal APA sites. Additionally, SNRNP70 can also promote the proximal APA sites by recruiting CPSF6, suggesting that the function of CPSF6 on APA is related with other RNA-binding proteins and cell context-dependent. Consequently, these results reveal that, on the contrary to U1 snRNP complex, the free proteins of U1 snRNP complex can promote proximal APA sites through the interaction with 3' end processing machinery.

Fip1 is a multivalent interaction scaffold for processing factors in human mRNA 3' end biogenesis.

3' end formation of most eukaryotic mRNAs is dependent on the assembly of a ~1.5 MDa multiprotein complex, that catalyzes the coupled reaction of pre-mRNA cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) constitutes the core of the 3' end processing machinery onto which the remaining factors, including cleavage stimulation factor (CstF) and poly(A) polymerase (PAP), assemble. These interactions are mediated by Fip1, a CPSF subunit characterized by high degree of intrinsic disorder. Here, we report two crystal structures revealing the interactions of human Fip1 (hFip1) with CPSF30 and CstF77. We demonstrate that CPSF contains two copies of hFip1, each binding to the zinc finger (ZF) domains 4 and 5 of CPSF30. Using polyadenylation assays we show that the two hFip1 copies are functionally redundant in recruiting one copy of PAP, thereby increasing the processivity of RNA polyadenylation. We further show that the interaction between hFip1 and CstF77 is mediated via a short motif in the N-terminal 'acidic' region of hFip1. In turn, CstF77 competitively inhibits CPSF-dependent PAP recruitment and 3' polyadenylation. Taken together, these results provide a structural basis for the multivalent scaffolding and regulatory functions of hFip1 in 3' end processing.

Modulation of mRNA 3'-End Processing and Transcription Termination in Virus-Infected Cells.

Eukaryotic mRNA 3´-end processing is a multi-step process beginning with pre-mRNA transcript cleavage followed by poly(A) tail addition. Closely coupled to transcription termination, 3´-end processing is a critical step in the regulation of gene expression, and disruption of 3´-end processing is known to affect mature mRNA levels. Various viral proteins interfere with the 3´-end processing machinery, causing read-through transcription and altered levels of mature transcripts through inhibition of cleavage and polyadenylation. Thus, disruption of 3´-end processing contributes to widespread host shutoff, including suppression of the antiviral response. Additionally, observed features of read-through transcripts such as decreased polyadenylation, nuclear retention, and decreased translation suggest that viruses may utilize these mechanisms to modulate host protein production and dominate cellular machinery. The degree to which the effects of read-through transcript production are harnessed by viruses and host cells remains unclear, but existing research highlights the importance of host 3´-end processing modulation during viral infection.

RBBP6 activates the pre-mRNA 3' end processing machinery in humans.

3' end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3' end of the mature transcript, which is important for mRNA localization, translation, and stability. Cleavage must therefore be tightly regulated. Here, we reconstituted specific and efficient 3' endonuclease activity of human CPSF with purified proteins. This required the seven-subunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm), and, importantly, the multidomain protein RBBP6. Unlike its yeast homolog Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to humans. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in mRNA processing.

CFI 25 Subunit of Cleavage Factor I is Important for Maintaining the Diversity of 3' UTR Lengths in Arabidopsis thaliana (L.) Heynh.

Cleavage and polyadenylation at the 3' end of the pre-mRNA is essential for mRNA function, by regulating its translatability, stability and translocation to the cytoplasm. Cleavage factor I (CFI) is a multi-subunit component of the pre-mRNA 3' end processing machinery in eukaryotes. Here, we report that plant CFI 25 subunit of CFI plays an important role in maintaining the diversity of the 3' ends of mRNA. The genome of Arabidopsis thaliana (L.) Heynh. contained four genes encoding three putative CFI subunits (AtCFI 25, AtCFI 59 and AtCFI 68), orthologous to the mammalian CFI subunits. There were two CFI 25 paralogs (AtCFI 25a and AtCFI 25b) that shared homology with human CFI 25. Two null alleles of AtCFI 25a displayed smaller rosette leaves, longer stigmatic papilla, smaller anther, earlier flowering and lower fertility compared to wild-type plants. Null alleles of AtCFI 25b, as well as, plants ectopically expressing full-length cDNA of AtCFI 25a, displayed no obvious morphological defects. AtCFI 25a was shown to interact with AtCFI 25b, AtCFI 68 and itself, suggesting various forms of CFI in plants. Furthermore, we show that AtCFI 25a function was essential for maintaining proper diversity of the 3' end lengths of transcripts coding for CFI subunits, suggesting a self-regulation of the CFI machinery in plants. AtCFI 25a was also important to maintain 3' ends for other genes to different extent. Collectively, AtCFI 25a, but not AtCFI 25b, seemed to play important roles during Arabidopsis development by maintaining proper diversity of the 3' UTR lengths.

LigD: A Structural Guide to the Multi-Tool of Bacterial Non-Homologous End Joining.

DNA double-strand breaks are the most lethal form of damage for living organisms. The non-homologous end joining (NHEJ) pathway can repair these breaks without the use of a DNA template, making it a critical repair mechanism when DNA is not replicating, but also a threat to genome integrity. NHEJ requires proteins to anchor the DNA double-strand break, recruit additional repair proteins, and then depending on the damage at the DNA ends, fill in nucleotide gaps or add or remove phosphate groups before final ligation. In eukaryotes, NHEJ uses a multitude of proteins to carry out processing and ligation of the DNA double-strand break. Bacterial NHEJ, though, accomplishes repair primarily with only two proteins–Ku and LigD. While Ku binds the initial break and recruits LigD, it is LigD that is the primary DNA end processing machinery. Up to three enzymatic domains reside within LigD, dependent on the bacterial species. These domains are a polymerase domain, to fill in nucleotide gaps with a preference for ribonucleotide addition; a phosphoesterase domain, to generate a 3′-hydroxyl DNA end; and the ligase domain, to seal the phosphodiester backbone. To date, there are no experimental structures of wild-type LigD, but there are x-ray and nuclear magnetic resonance structures of the individual enzymatic domains from different bacteria and archaea, along with structural predictions of wild-type LigD via AlphaFold. In this review, we will examine the structures of the independent domains of LigD from different bacterial species and the contributions these structures have made to understanding the NHEJ repair mechanism. We will then examine how the experimental structures of the individual LigD enzymatic domains combine with structural predictions of LigD from different bacterial species and postulate how LigD coordinates multiple enzymatic activities to carry out DNA double-strand break repair in bacteria.

Dynamics in Fip1 regulate eukaryotic mRNA 3' end processing.

Cleavage and polyadenylation factor (CPF/CPSF) is a multiprotein complex essential for mRNA 3' end processing in eukaryotes. It contains an endonuclease that cleaves pre-mRNAs, and a polymerase that adds a poly(A) tail onto the cleaved 3' end. Several CPF subunits, including Fip1, contain intrinsically disordered regions (IDRs). IDRs within multiprotein complexes can be flexible, or can become ordered upon interaction with binding partners. Here, we show that yeast Fip1 anchors the poly(A) polymerase Pap1 onto CPF via an interaction with zinc finger 4 of another CPF subunit, Yth1. We also reconstitute a fully recombinant 850-kDa CPF. By incorporating selectively labeled Fip1 into recombinant CPF, we could study the dynamics of Fip1 within the megadalton complex using nuclear magnetic resonance (NMR) spectroscopy. This reveals that a Fip1 IDR that connects the Yth1- and Pap1-binding sites remains highly dynamic within CPF. Together, our data suggest that Fip1 dynamics within the 3' end processing machinery are required to coordinate cleavage and polyadenylation.

U7 deciphered: the mechanism that forms the unusual 3′ end of metazoan replication-dependent histone mRNAs

In animal cells, replication-dependent histone mRNAs end with a highly conserved stem-loop structure followed by a 4- to 5-nucleotide single-stranded tail. This unique 3' end distinguishes replication-dependent histone mRNAs from all other eukaryotic mRNAs, which end with a poly(A) tail produced by the canonical 3'-end processing mechanism of cleavage and polyadenylation. The pioneering studies of Max Birnstiel's group demonstrated nearly 40 years ago that the unique 3' end of animal replication-dependent histone mRNAs is generated by a distinct processing mechanism, whereby histone mRNA precursors are cleaved downstream of the stem-loop, but this cleavage is not followed by polyadenylation. The key role is played by the U7 snRNP, a complex of a ∼60 nucleotide U7 snRNA and many proteins. Some of these proteins, including the enzymatic component CPSF73, are shared with the canonical cleavage and polyadenylation machinery, justifying the view that the two metazoan pre-mRNA 3'-end processing mechanisms have a common evolutionary origin. The studies on U7 snRNP culminated in the recent breakthrough of reconstituting an entirely recombinant human machinery that is capable of accurately cleaving histone pre-mRNAs, and determining its structure in complex with a pre-mRNA substrate (with 13 proteins and two RNAs) that is poised for the cleavage reaction. The structure uncovered an unanticipated network of interactions within the U7 snRNP and a remarkable mechanism of activating catalytically dormant CPSF73 for the cleavage. This work provides a conceptual framework for understanding other eukaryotic 3'-end processing machineries.

Structure of the catalytic core of the Integrator complex

SummaryThe Integrator is a specialized 3′ end-processing complex involved in cleavage and transcription termination of a subset of nascent RNA polymerase II transcripts, including small nuclear RNAs (snRNAs). We provide evidence of the modular nature of the Integrator complex by biochemically characterizing its two subcomplexes, INTS5/8 and INTS10/13/14. Using cryoelectron microscopy (cryo-EM), we determined a 3.5-Å-resolution structure of the INTS4/9/11 ternary complex, which constitutes Integrator’s catalytic core. Our structure reveals the spatial organization of the catalytic nuclease INTS11, bound to its catalytically impaired homolog INTS9 via several interdependent interfaces. INTS4, a helical repeat protein, plays a key role in stabilizing nuclease domains and other components. In this assembly, all three proteins form a composite electropositive groove, suggesting a putative RNA binding path within the complex. Comparison with other 3′ end-processing machineries points to distinct features and a unique architecture of the Integrator’s catalytic module.