Assembly mechanism of Integrator's RNA cleavage module.

The modular Integrator complex is a transcription regulator that is essential for embryonic development. It attenuates coding gene expression via premature transcription termination and performs 3'-processing of non-coding RNAs. For both activities, Integrator requires endonuclease activity that is harbored by an RNA cleavage module consisting of INTS4-9-11. How correct assembly of Integrator modules is achieved remains unknown. Here, we show that BRAT1 and WDR73 are critical biogenesis factors for the human cleavage module. They maintain INTS9-11 inactive during maturation by physically blocking the endonuclease active site and prevent premature INTS4 association. Furthermore, BRAT1 facilitates import of INTS9-11 into the nucleus, where it is joined by INTS4. Final BRAT1 release requires locking of the mature cleavage module conformation by inositol hexaphosphate (IP6). Our data explain several neurodevelopmental disorders caused by BRAT1, WDR73, and INTS11 mutations as Integrator assembly defects and reveal that IP6 is an essential co-factor for cleavage module maturation.

CCAR1 promotes DNA repair via alternative splicing.

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

Basis of gene-specific transcription regulation by the Integrator complex.

The Integrator complex attenuates gene expression via the premature termination of RNA polymerase II (RNAP2) at promoter-proximal pausing sites. It is required for stimulus response, cell differentiation, and neurodevelopment, but how gene-specific and adaptive regulation by Integrator is achieved remains unclear. Here, we identify two sites on human Integrator subunits 13/14 that serve as binding hubs for sequence-specific transcription factors (TFs) and other transcription effector complexes. When Integrator is attached to paused RNAP2, these hubs are positioned upstream of the transcription bubble, consistent with simultaneous TF-promoter tethering. The TFs co-localize with Integrator genome-wide, increase Integrator abundance on target genes, and co-regulate responsive transcriptional programs. For instance, sensory cilia formation induced by glucose starvation depends on Integrator-TF contacts. Our data suggest TF-mediated promoter recruitment of Integrator as a widespread mechanism for targeted transcription regulation.

Targeted high-throughput mutagenesis of the human spliceosome reveals its in vivo operating principles.

The spliceosome is a staggeringly complex machine, comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire human spliceosome and investigated the mutants using the U2 snRNP/SF3b inhibitor, pladienolide B. Hypersensitive substitutions define functional sites in the U1/U2-containing A complex but also in components that act as late as the second chemical step after SF3b is dissociated. Viable resistance substitutions map not only to the pladienolide B-binding site but also to the G-patch domain of SUGP1, which lacks orthologs in yeast. We used these mutants and biochemical approaches to identify the spliceosomal disassemblase DHX15/hPrp43 as the ATPase ligand for SUGP1. These and other data support a model in which SUGP1 promotes splicing fidelity by triggering early spliceosome disassembly in response to kinetic blocks. Our approach provides a template for the analysis of essential cellular machines in humans.

Take a break: Transcription regulation and RNA processing by the Integrator complex.

The metazoan-specific Integrator complex is a >1.5 MDa machinery that interacts with RNA polymerase II (RNAP2) to attenuate coding gene transcription by early termination close to transcription start sites. Using a highly related mechanism, Integrator also performs the initial 3'-end processing step for many non-coding RNAs. Its transcription regulation functions are essential for cell differentiation and response to external stimuli. Recent studies revealed that the complex incorporates phosphatase PP2A to counteract phosphorylation reactions that are required for transcription elongation. Structures of Integrator bound to RNAP2 explain the basis for its recruitment to promoter proximal RNAP2 by recognition of its paused state. Furthermore, several studies indicate that Integrator's cleavage activity is regulated at multiple levels through activators, modifications, and small molecules.

Sequence-specific RNA recognition by an RGG motif connects U1 and U2 snRNP for spliceosome assembly.

In mammals, the structural basis for the interaction between U1 and U2 small nuclear ribonucleoproteins (snRNPs) during the early steps of splicing is still elusive. The binding of the ubiquitin-like (UBL) domain of SF3A1 to the stem-loop 4 of U1 snRNP (U1-SL4) contributes to this interaction. Here, we determined the 3D structure of the complex between the UBL of SF3A1 and U1-SL4 RNA. Our crystallography, NMR spectroscopy, and cross-linking mass spectrometry data show that SF3A1-UBL recognizes, sequence specifically, the GCG/CGC RNA stem and the apical UUCG tetraloop of U1-SL4. In vitro and in vivo mutational analyses support the observed intermolecular contacts and demonstrate that the carboxyl-terminal arginine-glycine-glycine-arginine (RGGR) motif of SF3A1-UBL binds sequence specifically by inserting into the RNA major groove. Thus, the characterization of the SF3A1-UBL/U1-SL4 complex expands the repertoire of RNA binding domains and reveals the capacity of RGG/RG motifs to bind RNA in a sequence-specific manner.

Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution.

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.

Regulation of DEAH-box RNA helicases by G-patch proteins.

RNA helicases of the DEAH/RHA family form a large and conserved class of enzymes that remodel RNA protein complexes (RNPs) by translocating along the RNA. Driven by ATP hydrolysis, they exert force to dissociate hybridized RNAs, dislocate bound proteins or unwind secondary structure elements in RNAs. The sub-cellular localization of DEAH-helicases and their concomitant association with different pathways in RNA metabolism, such as pre-mRNA splicing or ribosome biogenesis, can be guided by cofactor proteins that specifically recruit and simultaneously activate them. Here we review the mode of action of a large class of DEAH-specific adaptor proteins of the G-patch family. Defined only by their eponymous short glycine-rich motif, which is sufficient for helicase binding and stimulation, this family encompasses an immensely varied array of domain compositions and is linked to an equally diverse set of functions. G-patch proteins are conserved throughout eukaryotes and are even encoded within retroviruses. They are involved in mRNA, rRNA and snoRNA maturation, telomere maintenance and the innate immune response. Only recently was the structural and mechanistic basis for their helicase enhancing activity determined. We summarize the molecular and functional details of G-patch-mediated helicase regulation in their associated pathways and their involvement in human diseases.

INTS10-INTS13-INTS14 form a functional module of Integrator that binds nucleic acids and the cleavage module.

The Integrator complex processes 3'-ends of spliceosomal small nuclear RNAs (snRNAs). Furthermore, it regulates transcription of protein coding genes by terminating transcription after unstable pausing. The molecular basis for Integrator's functions remains obscure. Here, we show that INTS10, Asunder/INTS13 and INTS14 form a separable, functional Integrator module. The structure of INTS13-INTS14 reveals a strongly entwined complex with a unique chain interlink. Unexpected structural homology to the Ku70-Ku80 DNA repair complex suggests nucleic acid affinity. Indeed, the module displays affinity for DNA and RNA but prefers RNA hairpins. While the module plays an accessory role in snRNA maturation, it has a stronger influence on transcription termination after pausing. Asunder/INTS13 directly binds Integrator's cleavage module via a conserved C-terminal motif that is involved in snRNA processing and required for spermatogenesis. Collectively, our data establish INTS10-INTS13-INTS14 as a nucleic acid-binding module and suggest that it brings cleavage module and target transcripts into proximity.

Structural basis for DEAH-helicase activation by G-patch proteins.

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA-protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch-mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.

USP16 counteracts mono-ubiquitination of RPS27a and promotes maturation of the 40S ribosomal subunit.

Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors. We demonstrate that USP16 constitutes a component of late cytoplasmic pre-40S subunits that promotes the removal of ubiquitin from an internal lysine of ribosomal protein RPS27a/eS31. USP16 deletion leads to late 40S subunit maturation defects, manifesting in incomplete processing of 18S rRNA and retarded recycling of late-acting ribosome biogenesis factors, revealing an unexpected contribution of USP16 to the ultimate step of 40S synthesis. Finally, ubiquitination of RPS27a appears to depend on active translation, pointing at a potential connection between 40S maturation and protein synthesis.

Balancing Specificity and Promiscuity in Enzyme Evolution: Multidimensional Activity Transitions in the Alkaline Phosphatase Superfamily.

Highly proficient, promiscuous enzymes can be springboards for functional evolution, able to avoid loss of function during adaptation by their capacity to promote multiple reactions. We employ a systematic comparative study of structure, sequence, and substrate specificity to track the evolution of specificity and reactivity between promiscuous members of clades of the alkaline phosphatase (AP) superfamily. Construction of a phylogenetic tree of protein sequences maps out the likely transition zone between arylsulfatases (ASs) and phosphonate monoester hydrolases (PMHs). Kinetic analysis shows that all enzymes characterized have four chemically distinct phospho- and sulfoesterase activities, with rate accelerations ranging from 1011- to 1017-fold for their primary and 109- to 1012-fold for their promiscuous reactions, suggesting that catalytic promiscuity is widespread in the AP-superfamily. This functional characterization and crystallography reveal a novel class of ASs that is so similar in sequence to known PMHs that it had not been recognized as having diverged in function. Based on analysis of snapshots of catalytic promiscuity "in transition", we develop possible models that would allow functional evolution and determine scenarios for trade-off between multiple activities. For the new ASs, we observe largely invariant substrate specificity that would facilitate the transition from ASs to PMHs via trade-off-free molecular exaptation, that is, evolution without initial loss of primary activity and specificity toward the original substrate. This ability to bypass low activity generalists provides a molecular solution to avoid adaptive conflict.

Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset.

The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (ßleavinggroup from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.

Mille viae in eukaryotic mRNA decapping.

Cellular mRNA levels are regulated via rates of transcription and decay. Since the removal of the mRNA 5'-cap by the decapping enzyme DCP2 is generally an irreversible step towards decay, it requires regulation. Control of DCP2 activity is likely effected by two interdependent means: by conformational control of the DCP2-DCP1 complex, and by assembly control of the decapping network, an array of mutually interacting effector proteins. Here, we compare three recent and conformationally distinct crystal structures of the DCP2-DCP1 decapping complex in the presence of substrate analogs and decapping enhancers and we discuss alternative substrate recognition modes for the catalytic domain of DCP2. Together with structure-based insight into decapping network assembly, we propose that DCP2-mediated decapping follows more than one path.

Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis.

Mammalian AATF/Che-1 is essential for embryonic development, however, the underlying molecular mechanism is unclear. By immunoprecipitation of human AATF we discovered that AATF forms a salt-stable protein complex together with neuroguidin (NGDN) and NOL10, and demonstrate that the AATF-NGDN-NOL10 (ANN) complex functions in ribosome biogenesis. All three ANN complex members localize to nucleoli and display a mutual dependence with respect to protein stability. Mapping of protein-protein interaction domains revealed the importance of both the evolutionary conserved WD40 repeats in NOL10 and the UTP3/SAS10 domain in NGDN for complex formation. Functional analysis showed that the ANN complex supports nucleolar steps of 40S ribosomal subunit biosynthesis. All complex members were required for 18S rRNA maturation and their individual depletion affected the same nucleolar cleavage steps in the 5'ETS and ITS1 regions of the ribosomal RNA precursor. Collectively, we identified the ANN complex as a novel functional module supporting the nucleolar maturation of 40S ribosomal subunits. Our data help to explain the described role of AATF in cell proliferation during mouse development as well as its requirement for malignant tumor growth.

Structure of the Dcp2-Dcp1 mRNA-decapping complex in the activated conformation.

The removal of the mRNA 5' cap (decapping) by Dcp2 shuts down translation and commits mRNA to full degradation. Dcp2 activity is enhanced by activator proteins such as Dcp1 and Edc1. However, owing to conformational flexibility, the active conformation of Dcp2 and the mechanism of decapping activation have remained unknown. Here, we report a 1.6-Å-resolution crystal structure of the Schizosaccharomyces pombe Dcp2-Dcp1 heterodimer in an unprecedented conformation that is tied together by an intrinsically disordered peptide from Edc1. In this ternary complex, an unforeseen rotation of the Dcp2 catalytic domain allows residues from both Dcp2 and Dcp1 to cooperate in RNA binding, thus explaining decapping activation by increased substrate affinity. The architecture of the Dcp2-Dcp1-Edc1 complex provides a rationale for the conservation of a sequence motif in Edc1 that is also present in unrelated decapping activators, thus indicating that the presently described mechanism of decapping activation is evolutionarily conserved.

Towards a molecular understanding of microRNA-mediated gene silencing.

MicroRNAs (miRNAs) are a conserved class of small non-coding RNAs that assemble with Argonaute proteins into miRNA-induced silencing complexes (miRISCs) to direct post-transcriptional silencing of complementary mRNA targets. Silencing is accomplished through a combination of translational repression and mRNA destabilization, with the latter contributing to most of the steady-state repression in animal cell cultures. Degradation of the mRNA target is initiated by deadenylation, which is followed by decapping and 5'-to-3' exonucleolytic decay. Recent work has enhanced our understanding of the mechanisms of silencing, making it possible to describe in molecular terms a continuum of direct interactions from miRNA target recognition to mRNA deadenylation, decapping and 5'-to-3' degradation. Furthermore, an intricate interplay between translational repression and mRNA degradation is emerging.

An asymmetric PAN3 dimer recruits a single PAN2 exonuclease to mediate mRNA deadenylation and decay.

The PAN2-PAN3 complex functions in general and microRNA-mediated mRNA deadenylation. However, mechanistic insight into PAN2 and its complex with the asymmetric PAN3 dimer is lacking. Here, we describe crystal structures that show that Neurospora crassa PAN2 comprises two independent structural units: a C-terminal catalytic unit and an N-terminal assembly unit that engages in a bipartite interaction with PAN3 dimers. The catalytic unit contains the exonuclease domain in an intimate complex with a potentially modulatory ubiquitin-protease-like domain. The assembly unit contains a WD40 propeller connected to an adaptable linker. The propeller contacts the PAN3 C-terminal domain, whereas the linker reinforces the asymmetry of the PAN3 dimer and prevents the recruitment of a second PAN2 molecule. Functional data indicate an essential role for PAN3 in coordinating PAN2-mediated deadenylation with subsequent steps in mRNA decay, which lead to complete mRNA degradation.

Structural basis for the Nanos-mediated recruitment of the CCR4-NOT complex and translational repression.

The RNA-binding proteins of the Nanos family play an essential role in germ cell development and survival in a wide range of metazoan species. They function by suppressing the expression of target mRNAs through the recruitment of effector complexes, which include the CCR4-NOT deadenylase complex. Here, we show that the three human Nanos paralogs (Nanos1-3) interact with the CNOT1 C-terminal domain and determine the structural basis for the specific molecular recognition. Nanos1-3 bind CNOT1 through a short CNOT1-interacting motif (NIM) that is conserved in all vertebrates and some invertebrate species. The crystal structure of the human Nanos1 NIM peptide bound to CNOT1 reveals that the peptide opens a conserved hydrophobic pocket on the CNOT1 surface by inserting conserved aromatic residues. The substitutions of these aromatic residues in the Nanos1-3 NIMs abolish binding to CNOT1 and abrogate the ability of the proteins to repress translation. Our findings provide the structural basis for the recruitment of the CCR4-NOT complex by vertebrate Nanos, indicate that the NIMs are the major determinants of the translational repression mediated by Nanos, and identify the CCR4-NOT complex as the main effector complex for Nanos function.

The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1.

The removal of the 5'-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5'-to-3' exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1-DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine-arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5'-to-3' mRNA degradation by XRN1 in human cells.