The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration SMURF1-UbcH7 K29 ubiquitination of p27 and cell migration.

Ubiquitination is a key regulator of protein stability and function. The multifunctional protein p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin-conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized, and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293, and HELA cells. However, the ubiquitin ligase associated with this stabilization of p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1 but not its close homolog SMURF2, stabilizes p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of p27, we also found that SMURF1, UbcH7, and p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated the colocalization of SMURF1/p27 and UbcH7/p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in the stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.

Estimates of Gaps in Supportive Housing Among Racially and Ethnically Diverse Older Adults with Serious Mental Illness in New York City Boroughs: Manhattan, Bronx, and Brooklyn.

OBJECTIVE: This study aimed to estimate gaps in supportive housing services for racially and ethnically diverse older adults with SMI residing in supportive housing. METHODS: This study had a total of 753 respondents split into two diagnostic groups, the Delusional and Psychotic Disorders Group, and the Mood (Affective) Disorder Group. Demographic and primary ICD diagnosis data (F2x and F3x) were extracted from medical records. Three elements were measured: supportive housing service needs, fall prevention, and activities in daily living and instrumental activities in daily living. Descriptive statistics (i.e., frequencies and percentages) were used in assessing the demographic characteristics of the sample. RESULTS: Respondents had reasonable fall prevention measures in place, did not have challenges carrying out activities in daily living or instrumental activities of daily living and did not need homecare services (n = 515, 68.4%). Respondents needed support managing chronic medical conditions (n = 323, ∼43%). Approximately 57% of the total respondents in this study (n = 426) reported the need for hearing, vision, and dental services. Respondents showed high levels of food insecurity (n = 380, 50.5%). CONCLUSIONS: This is the most extensive study of racially and ethnically diverse older adults with SMI residing in supportive housing. Three areas of unmet need were found: accessing hearing, vision, and dental services, managing chronic health conditions, and food insecurity. These findings can be used to develop new research programs addressing the needs of older adults with SMI and improve late-life circumstances for older adults with SMI.

Assessing the Feasibility, Acceptability, and Preliminary Effectiveness of a School-Aged Program that Supports Physical Activity and Wellness.

The prevalence of anxiety symptoms in children and adolescents aged 4 to 18 years has nearly doubled after the first year of the pandemic. However, only one in five adolescents diagnosed with anxiety is treated. We R H.O.P. E. is a school-based mental health program that includes evidence-based principles designed to engage children and adolescents in anxiety treatment, including wellness and emotional regulation, and the emotional CPR method. We R H.O.P. E. augments traditional services provided by school administrators, school social workers, school teachers, and school nurses. The purpose of this study was to examine the feasibility, acceptability, and preliminary effectiveness of We R H.O.P. E.

Predicting Nonunions in Ankle Fractures Using Quantitative Tibial Hounsfield Samples From Preoperative Computed Tomography: A Multicenter Matched Case Control Study.

The aim of this study is to use tibial Hounsfield unit measurements from preoperative computed tomography scans of ankle fractures to predict delayed union and nonunion. We hypothesize that patients with lower Hounsfield unit averages, an indirect measure of lower bone mineral density, in the distal tibia are more likely to develop delayed union and nonunion complications after ankle fracture surgery. Patient data from January 2010 to January 2020 were retrospectively analyzed from 2 institutions. Exposure cases of delayed union or nonunion that had preoperative computed tomography were compared to 5 controls matched for sex, age, and classification. 3 measurements were taken from the tibia on axial computed tomography and averaged to create a summative measure for overall bone health. Statistical analysis was used to analyze the relationship between the groups. 19 exposure patients were compared to 95 control patients. There were 16 females and 3 males in the exposure group aged from 30 to 88 years. Average follow-up was 1.6 years. The average exposure and control Hounsfield measurements were 186 (161-210) and 258 (248-269), respectively. Significant differences were found for all measured averages. This is the first study to our knowledge relating preoperative tibial Hounsfield measurements to healing rates of ankle fractures. Measurements taken from any of the 3 sites or the average could be an indicator of overall bone health. Using this technique on preoperative imaging will help surgeons adjust their perioperative planning for patients at higher risk for delayed union and nonunion.

Adaptation of an NLP system to a new healthcare environment to identify social determinants of health.

Social determinants of health (SDoH) are increasingly important factors for population health, healthcare outcomes, and care delivery. However, many of these factors are not reliably captured within structured electronic health record (EHR) data. In this work, we evaluated and adapted a previously published NLP tool to include additional social risk factors for deployment at Vanderbilt University Medical Center in an Acute Myocardial Infarction cohort. We developed a transformation of the SDoH outputs of the tool into the OMOP common data model (CDM) for re-use across many potential use cases, yielding performance measures across 8 SDoH classes of precision 0.83 recall 0.74 and F-measure of 0.78.

Reconciliation Between Lateral X-ray and Axial Computed Tomography Regarding Posterior Malleolus Fracture Anatomy and Medial Malleolus Involvement: A Pilot Study.

Configuration of a posterior malleolus fracture has significant variation based on mechanism of injury and concomitant ankle injuries. Radiographs obtained during early workup of ankle trauma play a pivotal role in closed reduction, surgical planning and preoperative management. Preoperative computed tomography helps distinguish fracture pathoanatomy. The purpose of this study is to relate measurements from traditional lateral radiographs with measurements on axial computed tomography. Imaging from a total of 22 patients treated at our institution from January 2008 to 2018 were evaluated. Two raters measured posterior malleolus fracture height and articular surface length on lateral radiographs, as well as medial-lateral width and anterior-posterior depth on axial computed tomography using calibrated imaging software. Posterior malleolar fractures with medial extension were recorded. Pearson correlations were calculated for all pairwise combinations of measurements. Lateral height and axial width were positively correlated. There was found to be an association between taller lateral height, and separately, wider axial width with presence of medial extension. Based on the correlations found between the measurements as well as the independent associations found with presence of medial extension, we suggest posterior-medial incision be evaluated as a potential approach in taller fractures as noted on lateral radiograph.

Engineering altered protein-DNA recognition specificity.

Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein-protein interactions, modifying protein-DNA interactions is more difficult. This may be related to the structural features of protein-DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein-DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.

Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule.

The acetylcholine-activated inward rectifier potassium current ( IKACh) is constitutively active in persistent atrial fibrillation (AF). We tested the hypothesis that the blocking of IKACh with the small molecule chloroquine terminates persistent AF. We used a sheep model of tachypacing-induced, persistent AF, molecular modeling, electrophysiology, and structural biology approaches. The 50% inhibition/inhibitory concentration of IKACh block with chloroquine, measured by patch clamp, was 1 µM. In optical mapping of sheep hearts with persistent AF, 1 µM chloroquine restored sinus rhythm. Molecular modeling suggested that chloroquine blocked the passage of a hydrated potassium ion through the intracellular domain of Kir3.1 (a molecular correlate of IKACh) by interacting with residues D260 and F255, in proximity to I228, Q227, and L299. 1H 15N heteronuclear single-quantum correlation of purified Kir3.1 intracellular domain confirmed the modeling results. F255, I228, Q227, and L299 underwent significant chemical-shift perturbations upon drug binding. We then crystallized and solved a 2.5 Å X-ray structure of Kir3.1 with F255A mutation. Modeling of chloroquine binding to the mutant channel suggested that the drug's binding to the pore becomes off centered, reducing its ability to block a hydrated potassium ion. Patch clamp validated the structural and modeling data, where the F255A and D260A mutations significantly reduced IKACh block by chloroquine. With the use of numerical and structural biology approaches, we elucidated the details of how a small molecule could block an ion channel and exert antiarrhythmic effects. Chloroquine binds the IKACh channel at a site formed by specific amino acids in the ion-permeation pathway, leading to decreased IKACh and the subsequent termination of AF.-Takemoto, Y., Slough, D. P., Meinke, G., Katnik, C., Graziano, Z. A., Chidipi, B., Reiser, M., Alhadidy, M. M., Ramirez, R., Salvador-Montañés, O., Ennis, S., Guerrero-Serna, G., Haburcak, M., Diehl, C., Cuevas, J., Jalife, J., Bohm, A., Lin,Y.-S., Noujaim, S. F. Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule.

sST2 as a novel biomarker for the prediction of in-hospital mortality after coronary artery bypass grafting.

Objectives: Soluble suppression of tumorigenicity 2 (sST2) biomarker is an emerging predictor of adverse clinical outcomes, but its prognostic value for in-hospital mortality after coronary artery bypass grafting (CABG) is not well understood. This study measured the association between operative sST2 levels and in-hospital mortality after CABG. Methods: A prospective cohort of 1560 CABG patients were analyzed from the Northern New England Cardiovascular Disease Study Group Biomarker Study. The primary outcome was in-hospital mortality after CABG surgery (n = 32). Results: After risk adjustment, patients in the third tercile of pre-, post- and pre-to-postoperative sST2 values experienced significantly greater odds of in-hospital death compared to patients in the first tercile of sST2 values. The addition of both postoperative and pre-to-postoperative sST2 biomarker significantly improved ability to predict in-hospital mortality status following CABG surgery, compared to using the EuroSCORE II mortality model alone, (c-statistic: 0.83 [95% CI: 0.75, 0.92], p value 0.0213) and (c-statistic: 0.83 [95% CI: 0.75, 0.92], p value 0.0215), respectively. Conclusion: sST2 values are associated with in-hospital mortality after CABG surgery and postoperative and pre-to-post operative sST2 values improve prediction. Our findings suggest that sST2 can be used as a biomarker to identify adult patients at greatest risk of in-hospital death after CABG surgery.

Crystal structure of an engineered, HIV-specific recombinase for removal of integrated proviral DNA.

As part of the HIV infection cycle, viral DNA inserts into the genome of host cells such that the integrated DNA encoding the viral proteins is flanked by long terminal repeat (LTR) regions from the retrovirus. In an effort to develop novel genome editing techniques that safely excise HIV provirus from cells, Tre, an engineered version of Cre recombinase, was designed to target a 34-bp sequence within the HIV-1 LTR (loxLTR). The sequence targeted by Tre lacks the symmetry present in loxP, the natural DNA substrate for Cre. We report here the crystal structure of a catalytically inactive (Y324F) mutant of this engineered Tre recombinase in complex with the loxLTR DNA substrate. We also report that 17 of the 19 amino acid changes relative to Cre contribute to the altered specificity, even though many of these residues do not contact the DNA directly. We hypothesize that some mutations increase the flexibility of the Cre tetramer and that this, along with flexibility in the DNA, enable the engineered enzyme and DNA substrate to adopt complementary conformations.

Biomarkers improve prediction of 30-day unplanned readmission or mortality after paediatric congenital heart surgery.

OBJECTIVE: To evaluate the association between novel pre- and post-operative biomarker levels and 30-day unplanned readmission or mortality after paediatric congenital heart surgery. METHODS: Children aged 18 years or younger undergoing congenital heart surgery (n = 162) at Johns Hopkins Hospital from 2010 to 2014 were enrolled in the prospective cohort. Collected novel pre- and post-operative biomarkers include soluble suppression of tumorgenicity 2, galectin-3, N-terminal prohormone of brain natriuretic peptide, and glial fibrillary acidic protein. A model based on clinical variables from the Society of Thoracic Surgery database was developed and evaluated against two augmented models. RESULTS: Unplanned readmission or mortality within 30 days of cardiac surgery occurred among 21 (13%) children. The clinical model augmented with pre-operative biomarkers demonstrated a statistically significant improvement over the clinical model alone with a receiver-operating characteristics curve of 0.754 (95% confidence interval: 0.65-0.86) compared to 0.617 (95% confidence interval: 0.47-0.76; p-value: 0.012). The clinical model augmented with pre- and post-operative biomarkers demonstrated a significant improvement over the clinical model alone, with a receiver-operating characteristics curve of 0.802 (95% confidence interval: 0.72-0.89; p-value: 0.003). CONCLUSIONS: Novel biomarkers add significant predictive value when assessing the likelihood of unplanned readmission or mortality after paediatric congenital heart surgery. Further exploration of the utility of these novel biomarkers during the pre- or post-operative period to identify early risk of mortality or readmission will aid in determining the clinical utility and application of these biomarkers into routine risk assessment.

Structural Based Analyses of the JC Virus T-Antigen F258L Mutant Provides Evidence for DNA Dependent Conformational Changes in the C-Termini of Polyomavirus Origin Binding Domains.

The replication of human polyomavirus JCV, which causes Progressive Multifocal Leukoencephalopathy, is initiated by the virally encoded T-antigen (T-ag). The structure of the JC virus T-ag origin-binding domain (OBD) was recently solved by X-ray crystallography. This structure revealed that the OBD contains a C-terminal pocket, and that residues from the multifunctional A1 and B2 motifs situated on a neighboring OBD molecule dock into the pocket. Related studies established that a mutation in a pocket residue (F258L) rendered JCV T-ag unable to support JCV DNA replication. To establish why this mutation inactivated JCV T-ag, we have solved the structure of the F258L JCV T-ag OBD mutant. Based on this structure, it is concluded that the structural consequences of the F258L mutation are limited to the pocket region. Further analyses, utilizing the available polyomavirus OBD structures, indicate that the F258 region is highly dynamic and that the relative positions of F258 are governed by DNA binding. The possible functional consequences of the DNA dependent rearrangements, including promotion of OBD cycling at the replication fork, are discussed.

Cre Recombinase and Other Tyrosine Recombinases.

Tyrosine-type site-specific recombinases (T-SSRs) have opened new avenues for the predictable modification of genomes as they enable precise genome editing in heterologous hosts. These enzymes are ubiquitous in eubacteria, prevalent in archaea and temperate phages, present in certain yeast strains, but barely found in higher eukaryotes. As tools they find increasing use for the generation and systematic modification of genomes in a plethora of organisms. If applied in host organisms, they enable precise DNA cleavage and ligation without the gain or loss of nucleotides. Criteria directing the choice of the most appropriate T-SSR system for genetic engineering include that, whenever possible, the recombinase should act independent of cofactors and that the target sequences should be long enough to be unique in a given genome. This review is focused on recent advancements in our mechanistic understanding of simple T-SSRs and their application in developmental and synthetic biology, as well as in biomedical research.

Targeting Liver Fibrosis with a Cell-penetrating Protease-activated Receptor-2 (PAR2) Pepducin.

Chronic liver inflammation and fibrosis in nonalcoholic steatohepatitis can lead to cirrhosis and liver failure for which there are currently no approved treatments. Protease-activated receptor-2 (PAR2) is an emerging new target expressed on liver stellate cells and hepatocytes that regulates the response to liver injury and inflammation. Here, we identified a pepducin to block the deleterious actions of PAR2 in promoting liver fibrosis. Non-alcoholic fatty liver disease and early fibrosis were induced by the methionine-choline-deficient diet in mice. Fibrotic liver disease was induced by administering carbon tetrachloride for 8 weeks. Mice were treated with the pepducin PZ-235 either from onset of the experiment or after fibrosis was established. Hepatic fibrosis, collagen content, inflammatory cytokines, steatosis, triglycerides, and NAFLD activity score were assessed as primary outcome parameters depending on the model. The activity of the PAR2 pepducin on cultured stellate cell activation and hepatocyte reactive oxygen species production was evaluated. PZ-235 significantly suppressed liver fibrosis, collagen deposition, inflammatory cytokines, NAFLD activity score, steatosis, triglycerides, aspartate transaminase, alanine transaminase, and stellate cell proliferation by up to 50-100%. The PAR2 inhibitor afforded significant protective effects against hepatocellular necrosis and attenuated PAR2-mediated reactive oxygen species production in hepatocytes. PZ-235 was distributed to liver and other mouse tissues and was found to form a well structured α-helix that closely resembles the juxtamembrane helical region of the analogous TM6 and third intracellular region of the intact receptor that is critical for coupling to internal G proteins. The ability of PZ-235 to effectively suppress fibrosis, hepatocellular necrosis, reactive oxygen species production, steatosis, and inflammation indicates the potential for PAR2 pepducin inhibitors to be broadly efficacious in the treatment of liver fibrosis.

Gα13 Switch Region 2 Relieves Talin Autoinhibition to Activate αIIbß3 Integrin.

Integrins function as bi-directional signaling transducers that regulate cell-cell and cell-matrix signals across the membrane. A key modulator of integrin activation is talin, a large cytoskeletal protein that exists in an autoinhibited state in quiescent cells. Talin is a large 235-kDa protein composed of an N-terminal 45-kDa FERM (4.1, ezrin-, radixin-, and moesin-related protein) domain, also known as the talin head domain, and a series of helical bundles known as the rod domain. The talin head domain consists of four distinct lobes designated as F0-F3. Integrin binding and activation are mediated through the F3 region, a critically regulated domain in talin. Regulation of the F3 lobe is accomplished through autoinhibition via anti-parallel dimerization. In the anti-parallel dimerization model, the rod domain region of one talin molecule binds to the F3 lobe on an adjacent talin molecule, thus achieving the state of autoinhibition. Platelet functionality requires integrin activation for adherence and thrombus formation, and thus regulation of talin presents a critical node where pharmacological intervention is possible. A major mechanism of integrin activation in platelets is through heterotrimeric G protein signaling regulating hemostasis and thrombosis. Here, we provide evidence that switch region 2 (SR2) of the ubiquitously expressed G protein (Gα13) directly interacts with talin, relieves its state of autoinhibition, and triggers integrin activation. Biochemical analysis of Gα13 shows SR2 binds directly to the F3 lobe of talin's head domain and competes with the rod domain for binding. Intramolecular FRET analysis shows Gα13 can relieve autoinhibition in a cellular milieu. Finally, a myristoylated SR2 peptide shows demonstrable decrease in thrombosis in vivo Altogether, we present a mechanistic basis for the regulation of talin through Gα13.

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics.

G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class.

Insights into the initiation of JC virus DNA replication derived from the crystal structure of the T-antigen origin binding domain.

JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML). PML is a disease that occurs primarily in people who are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV) DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD) of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel "pocket"; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable.

N-glycosylation regulates ADAM8 processing and activation.

The transmembrane ADAM8 (A Disintegrin And Metalloproteinase 8) protein is abundantly expressed in human breast tumors and derived metastases compared with normal breast tissue, and plays critical roles in aggressive Triple-Negative breast cancers (TNBCs). During ADAM8 maturation, the inactive proform dimerizes or multimerizes and autocatalytically removes the prodomain leading to the formation of the active, processed form. ADAM8 is a glycoprotein; however, little was known about the structure or functional role of these sugar moieties. Here, we report that in estrogen receptor (ER)α-negative, but not -positive, breast cancer cells ADAM8 contains N-glycosylation, which is required for its correct processing and activation. Consistently ADAM8 dimers were detected on the surface of ERα-negative breast cancer cells but not on ERα-positive ones. Site-directed mutagenesis confirmed four N-glycosylazhytion sites (Asn-67, Asn-91, Asn-436, and Asn-612) in human ADAM8. The Asn-67 and Asn-91 prodomain sites contained high mannose, whereas complex type N-glycosylation was observed on Asn-436 and Asn-612 in the active and remnant forms. The Asn-91 and Asn-612 sites were essential for its correct processing and cell surface localization, in particular its exit from the Golgi and endoplasmic reticulum, respectively. The N436Q mutation led to decreased ADAM8 stability due to enhanced lysosomal degradation. In contrast, mutation of the Asn-67 site had only modest effects on enzyme stability and processing. Thus, N-glycosylation is essential for processing, localization, stability, and activity of ADAM8.

Polyomavirus large T antigen binds symmetrical repeats at the viral origin in an asymmetrical manner.

Polyomaviruses have repeating sequences at their origins of replication that bind the origin-binding domain of virus-encoded large T antigen. In murine polyomavirus, the central region of the origin contains four copies (P1 to P4) of the sequence G(A/G)GGC. They are arranged as a pair of inverted repeats with a 2-bp overlap between the repeats at the center. In contrast to simian virus 40 (SV40), where the repeats are nonoverlapping and all four repeats can be simultaneously occupied, the crystal structure of the four central murine polyomavirus sequence repeats in complex with the polyomavirus origin-binding domain reveals that only three of the four repeats (P1, P2, and P4) are occupied. Isothermal titration calorimetry confirms that the stoichiometry is the same in solution as in the crystal structure. Consistent with these results, mutation of the third repeat has little effect on DNA replication in vivo. Thus, the apparent 2-fold symmetry within the DNA repeats is not carried over to the protein-DNA complex. Flanking sequences, such as the AT-rich region, are known to be important for DNA replication. When the orientation of the central region was reversed with respect to these flanking regions, the origin was still able to replicate and the P3 sequence (now located at the P2 position with respect to the flanking regions) was again dispensable. This highlights the critical importance of the precise sequence of the region containing the pentamers in replication.

The interaction of Pcf11 and Clp1 is needed for mRNA 3'-end formation and is modulated by amino acids in the ATP-binding site.

Polyadenylation of eukaryotic mRNAs contributes to stability, transport and translation, and is catalyzed by a large complex of conserved proteins. The Pcf11 subunit of the yeast CF IA factor functions as a scaffold for the processing machinery during the termination and polyadenylation of transcripts. Its partner, Clp1, is needed for mRNA processing, but its precise molecular role has remained enigmatic. We show that Clp1 interacts with the Cleavage-Polyadenylation Factor (CPF) through its N-terminal and central domains, and thus provides cross-factor connections within the processing complex. Clp1 is known to bind ATP, consistent with the reported RNA kinase activity of human Clp1. However, substitution of conserved amino acids in the ATP-binding site did not affect cell growth, suggesting that the essential function of yeast Clp1 does not involve ATP hydrolysis. Surprisingly, non-viable mutations predicted to displace ATP did not affect ATP binding but disturbed the Clp1-Pcf11 interaction. In support of the importance of this interaction, a mutation in Pcf11 that disrupts the Clp1 contact caused defects in growth, 3'-end processing and transcription termination. These results define Clp1 as a bridge between CF IA and CPF and indicate that the Clp1-Pcf11 interaction is modulated by amino acids in the conserved ATP-binding site of Clp1.