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.

Mechanistic insights into mRNA 3'-end processing.

The polyadenosine (poly(A)) tail found on the 3'-end of almost all eukaryotic mRNAs is important for mRNA stability and regulation of translation. mRNA 3'-end processing occurs co-transcriptionally and involves more than 20 proteins to specifically recognize the polyadenylation site, cleave the pre-mRNA, add a poly(A) tail, and trigger transcription termination. The polyadenylation site (PAS) defines the end of the 3'-untranslated region (3'-UTR) and, therefore, selection of the cleavage site is a critical event in regulating gene expression. Integrated structural biology approaches including biochemical reconstitution of multi-subunit complexes, cross-linking mass spectrometry, and structural analyses by X- ray crystallography and single-particle electron cryo-microscopy (cryoEM) have enabled recent progress in understanding the molecular mechanisms of the mRNA 3'-end processing machinery. Here, we describe new molecular insights into pre-mRNA recognition, cleavage and polyadenylation.

Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins.

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

Author Correction: Structural basis of AAUAAA polyadenylation signal recognition by the human CPSF complex.

In the version of this article initially published online, an incorrect accession code PDB 6FN9 was introduced in Methods, in the 'Model building' section, line 2. This has been corrected to PDB 6F9N. The error has been corrected in the PDF and HTML versions of this article.

Structural basis of AAUAAA polyadenylation signal recognition by the human CPSF complex.

Mammalian mRNA biogenesis requires specific recognition of a hexanucleotide AAUAAA motif in the polyadenylation signals (PAS) of precursor mRNA (pre-mRNA) transcripts by the cleavage and polyadenylation specificity factor (CPSF) complex. Here we present a 3.1-Å-resolution cryo-EM structure of a core CPSF module bound to the PAS hexamer motif. The structure reveals the molecular interactions responsible for base-specific recognition, providing a rationale for mechanistic differences between mammalian and yeast 3' polyadenylation.

Structural insights into the assembly and polyA signal recognition mechanism of the human CPSF complex.

3' polyadenylation is a key step in eukaryotic mRNA biogenesis. In mammalian cells, this process is dependent on the recognition of the hexanucleotide AAUAAA motif in the pre-mRNA polyadenylation signal by the cleavage and polyadenylation specificity factor (CPSF) complex. A core CPSF complex comprising CPSF160, WDR33, CPSF30 and Fip1 is sufficient for AAUAAA motif recognition, yet the molecular interactions underpinning its assembly and mechanism of PAS recognition are not understood. Based on cross-linking-coupled mass spectrometry, crystal structure of the CPSF160-WDR33 subcomplex and biochemical assays, we define the molecular architecture of the core human CPSF complex, identifying specific domains involved in inter-subunit interactions. In addition to zinc finger domains in CPSF30, we identify using quantitative RNA-binding assays an N-terminal lysine/arginine-rich motif in WDR33 as a critical determinant of specific AAUAAA motif recognition. Together, these results shed light on the function of CPSF in mediating PAS-dependent RNA cleavage and polyadenylation.

USP4 inhibits SMAD4 monoubiquitination and promotes activin and BMP signaling.

SMAD4 is a common intracellular effector for TGF-ß family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin-specific protease (USP) 4 strongly induces activin/BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand-induced regulatory (R)-SMAD-SMAD4 complex formation. Whereas the interaction of the negative regulator c-SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c-SKI-SMAD2 complex and triggers c-SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF-ß family signaling. An increase in monoubiquitinated SMAD4 in USP4-depleted mouse embryonic stem cells (mESCs) decreased both the BMP- and activin-induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin- and BMP-mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity.

A conserved two-step binding for the UAF1 regulator to the USP12 deubiquitinating enzyme.

Regulation of deubiquitinating enzyme (DUB) activity is an essential step for proper function of cellular ubiquitin signals. UAF1 is a WD40 repeat protein, which binds and activates three important DUBs, USP1, USP12 and USP46. Here, we report the crystal structure of the USP12-Ub/UAF1 complex at a resolution of 2.8Å and of UAF1 at 2.3Å. In the complex we find two potential sites for UAF1 binding, analogous to what was seen in a USP46/UAF1 complex. In line with these observed dual binding states, we show here that USP12/UAF1 complex has 1:2 stoichiometry in solution, with a two-step binding at 4nM and 325nM respectively. Mutagenesis studies show that the fingers sub-domain of USP12 interacts with UAF1 to form the high affinity interface. Our activation studies confirm that the high affinity binding is important for activation while the second UAF1 binding does not affect activation. Nevertheless, we show that this two step binding is conserved in the well-studied USP12 paralog, USP1. Our results highlight the interfaces essential for regulation of USP12 activity and show a conserved second binding of UAF1 which could be important for regulatory functions independent of USP12 activity.

The DUSP-Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange.

Ubiquitin-specific protease USP4 is emerging as an important regulator of cellular pathways, including the TGF-ß response, NF-κB signalling and splicing, with possible roles in cancer. Here we show that USP4 has its catalytic triad arranged in a productive conformation. Nevertheless, it requires its N-terminal DUSP-Ubl domain to achieve full catalytic turnover. Pre-steady-state kinetics measurements reveal that USP4 catalytic domain activity is strongly inhibited by slow dissociation of ubiquitin after substrate hydrolysis. The DUSP-Ubl domain is able to enhance ubiquitin dissociation, hence promoting efficient turnover. In a mechanism that requires all USP4 domains, binding of the DUSP-Ubl domain promotes a change of a switching loop near the active site. This 'allosteric regulation of product discharge' provides a novel way of regulating deubiquitinating enzymes that may have relevance for other enzyme classes.

Structural and functional analysis of the three MIF4G domains of nonsense-mediated decay factor UPF2.

Nonsense-mediated decay (NMD) is a eukaryotic quality control pathway, involving conserved proteins UPF1, UPF2 and UPF3b, which detects and degrades mRNAs with premature stop codons. Human UPF2 comprises three tandem MIF4G domains and a C-terminal UPF1 binding region. MIF4G-3 binds UPF3b, but the specific functions of MIF4G-1 and MIF4G-2 are unknown. Crystal structures show that both MIF4G-1 and MIF4G-2 contain N-terminal capping helices essential for stabilization of the 10-helix MIF4G core and that MIF4G-2 interacts with MIF4G-3, forming a rigid assembly. The UPF2/UPF3b/SMG1 complex is thought to activate the kinase SMG1 to phosphorylate UPF1 in vivo. We identify MIF4G-3 as the binding site and in vitro substrate of SMG1 kinase and show that a ternary UPF2 MIF4G-3/UPF3b/SMG1 complex can form in vitro. Whereas in vivo complementation assays show that MIF4G-1 and MIF4G-2 are essential for NMD, tethering assays reveal that UPF2 truncated to only MIF4G-3 and the UPF1-binding region can still partially accomplish NMD. Thus UPF2 MIF4G-1 and MIF4G-2 appear to have a crucial scaffolding role, while MIF4G-3 is the key module required for triggering NMD.

CBC-ARS2 stimulates 3'-end maturation of multiple RNA families and favors cap-proximal processing.

The nuclear cap-binding complex (CBC) stimulates multiple steps in several RNA maturation pathways, but how it functions in humans is incompletely understood. For small, capped RNAs such as pre-snRNAs, the CBC recruits PHAX. Here, we identify the CBCAP complex, composed of CBC, ARS2 and PHAX, and show that both CBCAP and CBC-ARS2 complexes can be reconstituted from recombinant proteins. ARS2 stimulates PHAX binding to the CBC and snRNA 3'-end processing, thereby coupling maturation with export. In vivo, CBC and ARS2 bind similar capped noncoding and coding RNAs and stimulate their 3'-end processing. The strongest effects are for cap-proximal polyadenylation sites, and this favors premature transcription termination. ARS2 functions partly through the mRNA 3'-end cleavage factor CLP1, which binds RNA Polymerase II through PCF11. ARS2 is thus a major CBC effector that stimulates functional and cryptic 3'-end processing sites.

The differential modulation of USP activity by internal regulatory domains, interactors and eight ubiquitin chain types.

Ubiquitin-specific proteases (USPs) are papain-like isopeptidases with variable inter- and intramolecular regulatory domains. To understand the effect of these domains on USP activity, we have analyzed the enzyme kinetics of 12 USPs in the presence and absence of modulators using synthetic reagents. This revealed variations of several orders of magnitude in both the catalytic turnover (k(cat)) and ubiquitin (Ub) binding (K(M)) between USPs. Further activity modulation by intramolecular domains affects both the k(cat) and K(M), whereas the intermolecular activators UAF1 and GMPS mainly increase the k(cat). Also, we provide the first comprehensive analysis comparing Ub chain preference. USPs can hydrolyze all linkages and show modest Ub-chain preferences, although some show a lack of activity toward linear di-Ub. This comprehensive kinetic analysis highlights the variability within the USP family.

Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases.

High-throughput methods to produce a large number of soluble recombinant protein variants are particularly important in the process of determining the three-dimensional structure of proteins and their complexes. Here, we describe a collection of protein expression vectors for ligation-independent cloning, which allow co-expression strategies by implementing different affinity tags and antibiotic resistances. Since the same PCR product can be inserted in all but one of the vectors, this allows efficiency in versatility while screening for optimal expression strategies. We first demonstrate the use of these vectors for protein expression in Escherichia coli, on a set of proteins belonging to the ubiquitin specific protease (USP) Family. We have selected 35 USPs, created 145 different expression constructs into the pETNKI-His-3C-LIC-kan vector, and obtained 38 soluble recombinant proteins for 21 different USPs. Finally, we exemplify the use of our vectors for bacterial co-expression and for expression in insect cells, with USP4 and USP7 respectively. We conclude that our ligation-independent cloning strategy allows for high-throughput screening for the expression of soluble proteins in a variety of vectors in E. coli and in insect cells. In addition, the same vectors can be used for co-expression studies, at least for simple binary complexes. Application in the family of ubiquitin specific proteases led to a number of soluble USPs that are used for functional and crystallization studies.

Unusual bipartite mode of interaction between the nonsense-mediated decay factors, UPF1 and UPF2.

Nonsense-mediated decay (NMD) is a eukaryotic quality control mechanism that degrades mRNAs carrying premature stop codons. In mammalian cells, NMD is triggered when UPF2 bound to UPF3 on a downstream exon junction complex interacts with UPF1 bound to a stalled ribosome. We report structural studies on the interaction between the C-terminal region of UPF2 and intact UPF1. Crystal structures, confirmed by EM and SAXS, show that the UPF1 CH-domain is docked onto its helicase domain in a fixed configuration. The C-terminal region of UPF2 is natively unfolded but binds through separated alpha-helical and beta-hairpin elements to the UPF1 CH-domain. The alpha-helical region binds sixfold more weakly than the beta-hairpin, whereas the combined elements bind 80-fold more tightly. Cellular assays show that NMD is severely affected by mutations disrupting the beta-hairpin binding, but not by those only affecting alpha-helix binding. We propose that the bipartite mode of UPF2 binding to UPF1 brings the ribosome and the EJC in close proximity by forming a tight complex after an initial weak encounter with either element.

The advantages of being locked. Assessing the cleavage of short and long RNAs by locked nucleic acid-containing 8-17 deoxyribozymes.

RNA-cleaving deoxyribozymes can be used for the sequence-specific knockdown of mRNAs. It was previously shown that activity of these deoxyribozymes is enhanced when their substrate-binding arms include some locked nucleic acid (LNA) residues, but the mechanistic basis of this enhancement was not explored. Here we dissected the kinetics and thermodynamics underlying the reaction of LNA-containing 8-17 deoxyribozymes. Four 8-17 constructs were designed to target sequences within the E6 mRNA from human papillomavirus type 16. When one of these deoxyribozymes (DNAzymes) and the corresponding LNA-armed enzyme (LNAzyme) were tested against a minimal RNA substrate, they showed similar rates of substrate binding and similar rates of intramolecular cleavage, but the LNAzyme released its substrate more slowly. The superior thermodynamic stability of the LNAzyme-substrate complex led to improved performances in reactions carried out at low catalyst concentrations. The four DNAzymes and the corresponding LNAzymes were then tested against extended E6 transcripts (>500 nucleotides long). With these structured substrates, the LNAzymes retained full activity, whereas the DNAzymes cleaved extremely poorly, unless they were allowed to pre-anneal to their targets. These results imply that LNAzymes can easily overcome the kinetic barrier represented by local RNA structure and bind to folded targets with a faster association rate as compared with DNAzymes. Such faster annealing to structured targets can be explained by a model whereby LNA monomers favor the initial hybridization to short stretches of unpaired residues ("nucleation"), which precedes disruption of the local mRNA structure and completion of the binding process.

A mutational analysis of the 8-17 deoxyribozyme core.

The 8-17 deoxyribozyme is a small RNA-cleaving DNA enzyme of significant applicative interest. We measured the kinetics of over 60 variants of 8-17, mutated within the "core" region. The data were analyzed according to a conceptual framework in which deleterious substitutions can either decrease the stability of the reaction's transition state, or favor unreactive ground-state conformations. In agreement with earlier in vitro evolution studies, the most severe functional effects were observed upon mutating four conserved residues, whose role was further explored by replacing them with non-standard nucleotides. Removal or modification of individual functional groups on the A6 and G7 bases suggested that these residues are involved in a close-contact interaction and form a network of functionally important hydrogen bonds. Mutagenesis of residues C13 and G14 was less revealing, but argued strongly against a role of C13 as a general acid/base catalyst. The use of non-standard nucleotides also led to the identification of one deoxyribozyme variant that, under some ionic conditions, is substantially more active than the wild-type construct. Finally, the effects of mutations in the intramolecular "core stem" correlated only in part with changes in helical stability, suggesting that a stable stem is required but not sufficient for optimal activity.