Development of a core outcome set for physiotherapy trials in adults with bronchiectasis (the COS-PHyBE study): A Delphi study and consensus meeting.

Background: Physiotherapy is recommended for bronchiectasis management, but there is disparity in evidence supporting its use. This is partly because of inconsistency and poor reporting of outcomes in available studies. A Core Outcome Set (COS) may improve trial consistency and decrease reporting bias. This study aimed to develop a COS for physiotherapy clinical trials in adults with bronchiectasis. Methods: A comprehensive list of outcomes was developed using a systematic review and qualitative semi-structured interviews with patients with bronchiectasis and physiotherapists.An international two-round online Delphi survey was conducted. Outcomes scored 7-9 (crucial) by ≥ 70 % of participants and 1-3 (not that important) by ≤ 15 % of participants from each stakeholder in the Likert scale were nominated for inclusion in the COS. Nominated outcomes and those considered crucial by only one of the stakeholders' groups were further discussed and voted in an international consensus meeting. Results: A list of 137 outcomes was generated; 104 from literature and 33 from interviews. A Delphi survey containing 59 outcomes was completed by 171 participants from 20 countries. After the consensus meeting, representatives agreed on seven outcomes: health-related quality of life, respiratory symptoms, physical functioning, emotional and psychological functioning, fatigue, adherence to treatment, and functional exercise capacity. Conclusion: A minimum set of seven outcomes are recommended to be included as measurements in future trials evaluating physiotherapy interventions for bronchiectasis.

A Paradigm Shift for the Nursing Education Model: A Scoping Review.

BACKGROUND: The Nursing Education Model (NEM) introduced a framework for education reform. This study examined the applications of NEM in education to determine whether modifications to NEM were warranted. METHOD: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Scoping Reviews process for scoping reviews was used. Online databases and searches identified peer-reviewed articles published from 2010-to June 2023, and 71 articles were identified that met the inclusion and exclusion criteria. RESULTS: Six themes were derived from the findings: (1) learner-centered curricula; (2) instructional methods; (3) inclusive classrooms; (4) pedagogical creativity; (5) bridge theory-clinical divide; and (6) multiple units of analysis. CONCLUSION: The findings support the application of the NEM to promote a paradigm shift in instructional content, methods, and evaluation of outcomes. Based on use of the model identified in the literature, future modifications to NEM may be indicated, based on continued evidence. [J Nurs Educ. 2024;63(3):141-147.].

The disordered N-terminal tail of SARS-CoV-2 Nucleocapsid protein forms a dynamic complex with RNA.

The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA binding domain (RBD) and its flanking disordered N-terminal domain (NTD) tail, using single-molecule Förster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs.

Excluded Volume and Weak Interactions in Crowded Solutions Modulate Conformations and RNA Binding of an Intrinsically Disordered Tail.

The cellular milieu is a solution crowded with a significant concentration of different components (proteins, nucleic acids, metabolites, etc.). Such a crowded environment affects protein conformations, dynamics, and interactions. Intrinsically disordered proteins and regions are particularly sensitive to these effects. Here, we investigate the impact on an intrinsically disordered tail that flanks a folded domain, the N-terminal domain, and the RNA-binding domain of the SARS-CoV-2 nucleocapsid protein. We mimic the crowded environment of the cell using polyethylene glycol (PEG) and study its impact on protein conformations using single-molecule Förster resonance energy transfer. We found that high-molecular-weight PEG induces a collapse of the disordered N-terminal tail, whereas low-molecular-weight PEG induces a chain expansion. Our data can be explained by accounting for two opposing contributions: favorable interactions between the protein and crowder molecules and screening of excluded volume interactions. We further characterized the interaction between protein and RNA in the presence of crowding agents. While for all PEG molecules tested, we observed an increase in the binding affinity, the trend is not monotonic as a function of the degree of PEG polymerization. This points to the role of nonspecific protein-PEG interactions on binding in addition to the entropic effects due to crowding. To separate the enthalpic and entropic components of the effects, we investigated the temperature dependence of the association constants in the absence and presence of crowders. Finally, we compared the effects of crowding across mutations in the disordered region and found that the threefold difference in association constants for two naturally occurring variants of the SARS-CoV-2 nucleocapsid protein is reduced to almost identical affinities in the presence of crowders. Overall, our data provide new insights into understanding and modeling the contribution of crowding effects on disordered regions, including the impact of interactions between proteins and crowders and their interplay when binding a ligand.

Physiotherapist perspectives of airway clearance techniques in bronchiectasis.

INTRODUCTION: Our understanding regarding the personalization of airway clearance techniques (ACTs) in bronchiectasis is limited. OBJECTIVE: This study aimed to determine physiotherapist perceptions regarding the prescription of ACTs in inpatients and outpatients with bronchiectasis. METHODS: A single-center qualitative study using semi-structured interviews of physiotherapists who treated individuals with bronchiectasis was undertaken. All interviews were audio recorded and transcribed verbatim. Data was analyzed using the thematic framework approach described by Braun and Clark. NVivo 12 software assisted with coding and thematic analysis of the interview transcripts. Findings were summarized into major conceptual themes. Participant demographic data was also obtained. RESULTS: Eleven physiotherapists participated in the interviews. Central to all themes was the complexity of physiotherapy clinical decision-making regarding ACT prescription. Main themes included: organizational factors (i.e. workload, scope of service, access to resources/ACTs); patient-related factors (i.e. symptom severity, finances, disease-specific knowledge, social commitments, clinical setting, and perceived benefit); and physiotherapist/profession-related factors (i.e. clinical experience, access to professional support and education, awareness of evidence of ACTs, and evaluating ACT effectiveness). CONCLUSION: Physiotherapists regularly and routinely prescribe ACTs for individuals with bronchiectasis allowing for a multitude of competing factors. These factors should be considered by physiotherapists to enhance the personalized prescription of ACTs and may help promote patient adherence to ACTs to improve outcomes.

Patient perspectives of airway clearance techniques in bronchiectasis.

INTRODUCTION: While airway clearance techniques (ACTs) are recommended for individuals with bronchiectasis, data suggests the use of and adherence to ACTs is poor. OBJECTIVE: This study aimed to identify patient perceptions regarding ACTs, the barriers and facilitators to ACTs, and factors affecting adherence. METHODS: A multi-center qualitative study using in-depth semi-structured interviews of individuals with bronchiectasis was undertaken. All interviews were audio recorded and transcribed verbatim. Data was analyzed using the thematic framework approach described by Braun and Clark. NVIVO™ 12 software assisted with coding and thematic analysis of the interview transcripts. Data saturation was achieved when no new common themes were identified. Findings were summarized into major conceptual themes. Participant demographic data was also obtained. RESULTS: Twenty-four participants participated in semi-structured interviews. The main facilitators to using ACTs included a perceived health and quality of life benefit, a tailored approach to ACTs and the use of self-management strategies. Main barriers included lack of time and motivation, lack of access to resources, and a lack of perceived health benefit. A number of factors were identified by participants that may help promote adherence including combining and trialing different ACTs, receiving regular ACT reviews and education from physiotherapists, and having good social support. CONCLUSION: To assist the personalized prescription of ACTs, these facilitators and barriers should be considered by clinicians to help promote adherence and improve patient outcomes.

Benchmarking service provision, scope of practice, and skill mix for physiotherapists in adult cystic fibrosis care delivery.

BACKGROUND: Increasing age, numbers, and complexity of care are potentially impacting physiotherapy service delivery for adults with cystic fibrosis (CF). PURPOSE: This study aimed to describe physiotherapy service provision, scope of practice, and skill mix in a large tertiary adult CF center, and determine if services were meeting clinical practice recommendations. METHODS: A prospective cross-sectional study examined inpatient and outpatient physiotherapy care across a three-month period in a tertiary adult CF center. Physiotherapy services were described by number and skill level of physiotherapists, total hours of activity, and number, type, and duration of each physiotherapy activity. RESULTS: Twenty-two physiotherapists provided care. Respiratory (n = 1058, 38%), and exercise treatments (n = 338, 12%) were the most frequent. Exercise testing (n = 20, 1%), and detailed treatment reviews (n = 79, 3%) occurred infrequently. Time for research was limited. Junior physiotherapists undertook more exercise treatments per day (p < .01), with senior physiotherapists attending outpatient clinics (p < .01). CONCLUSION: A large number of physiotherapists were involved in the delivery of services. Recommended respiratory and exercise treatments were frequently provided; however, other recommended activities occurred infrequently. The impact of increasing age, numbers of patients, and complexity of care may be contributing to demand exceeding supply for physiotherapy services. Future studies are required to determine innovative approaches to address the gaps in clinical practice recommendations.

Evaluation and quality improvement of Doctor of Nursing Practice-Family Nurse Practitioner scholarly projects.

BACKGROUND: The Commission on Collegiate Nursing Education's accreditation standards require nursing programs to demonstrate that students acquire doctoral-level knowledge and competencies beyond that expected at the baccalaureate and/or masters levels. The purpose of this article was to describe a quality improvement (QI) project for Doctor of Nursing Practice-Family Nurse Practitioner (NP) scholarly projects. LOCAL PROBLEM: Nurse practitioner faculty inquired about whether students' scholarly projects were of the quality and rigor expected at the doctoral, rather than masters, level. METHODS: This project was conducted as a QI initiative and was designated as such by the institutional review board. Methods were based on Deming Plan-Do-Study-Act QI process model. INTERVENTIONS: Two doctorally prepared nursing faculty evaluated nine doctoral scholarly projects using the Roush DNP-PCAT instrument. This instrument evaluates 16 components, and key elements within each component, for completeness. The DNP-PCAT tool was evaluated for interrater reliability and content validity in an earlier study. RESULTS: Strengths and weaknesses were identified using quantitative and qualitative data analyses. Floor effects were seen in strategic planning, ethical concerns, data collection, results, discussion, and limitations. Qualitative findings identified weaknesses across all 16 components. Findings were similar to the results reported in a prior study. Results were reviewed and action plans were developed to improve the rigor of scholarly projects. CONCLUSIONS: The nursing faculty plan to incorporate routine evaluation of scholarly projects into their program evaluation. Other NP programs may benefit from instituting a similar process.

Physiotherapy service provision in a specialist adult cystic fibrosis service: A pre-post design study with the inclusion of an allied health assistant.

What is the impact of including an allied health assistant (AHA) role on physiotherapy service delivery in an acute respiratory service? A pragmatic pre-post design study examined physiotherapy services across two 3-month periods: current service delivery [P1] and current service delivery plus AHA [P2]. Clinical and non-clinical activity quantified as number, type and duration (per day) of all staff activity categorised for skill level (AHA, junior, senior). Physiotherapy service delivery increased in P2 compared to P1 (n = 4730 vs n = 3048). Physiotherapists undertook fewer respiratory (p < 0.001) and exercise treatments (p < 0.001) but increased reviews for inpatients (p < 0.001) and at multidisciplinary clinics in P2 (56% vs 76%, p < 0.01). The AHA accounted for 20% of all service provision. AHA activity comprised mainly non-direct clinical care including oversight of respiratory equipment use (e.g. supply, set-up, cleaning, loan audits) and other patient-related administrative tasks associated with delegation handovers, supervision and clinical documentation (72%), delegated supervision of established respiratory (5%) and exercise treatments (10%) and delegated exercise tests (3%). The AHA completed most of the exercise tests (n = 25). AHA non-direct clinical tasks included departmental management activities (11%). No adverse events were reported. AHA inclusion in an acute respiratory care service changed physiotherapy service provision. The AHA completed delegated routine clinical and non-clinical tasks. Physiotherapists increased clinic activity and annual reviews. Including an AHA role offers sustainable options for enhancing physiotherapy service provision in acute respiratory care.

Reweighting of molecular simulations with explicit-solvent SAXS restraints elucidates ion-dependent RNA ensembles.

Small-angle X-ray scattering (SAXS) experiments are increasingly used to probe RNA structure. A number of forward models that relate measured SAXS intensities and structural features, and that are suitable to model either explicit-solvent effects or solute dynamics, have been proposed in the past years. Here, we introduce an approach that integrates atomistic molecular dynamics simulations and SAXS experiments to reconstruct RNA structural ensembles while simultaneously accounting for both RNA conformational dynamics and explicit-solvent effects. Our protocol exploits SAXS pure-solute forward models and enhanced sampling methods to sample an heterogenous ensemble of structures, with no information towards the experiments provided on-the-fly. The generated structural ensemble is then reweighted through the maximum entropy principle so as to match reference SAXS experimental data at multiple ionic conditions. Importantly, accurate explicit-solvent forward models are used at this reweighting stage. We apply this framework to the GTPase-associated center, a relevant RNA molecule involved in protein translation, in order to elucidate its ion-dependent conformational ensembles. We show that (a) both solvent and dynamics are crucial to reproduce experimental SAXS data and (b) the resulting dynamical ensembles contain an ion-dependent fraction of extended structures.

The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA.

The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

Novel Alzheimer Disease Risk Loci and Pathways in African American Individuals Using the African Genome Resources Panel: A Meta-analysis.

Importance: Compared with non-Hispanic White individuals, African American individuals from the same community are approximately twice as likely to develop Alzheimer disease. Despite this disparity, the largest Alzheimer disease genome-wide association studies to date have been conducted in non-Hispanic White individuals. In the largest association analyses of Alzheimer disease in African American individuals, ABCA7, TREM2, and an intergenic locus at 5q35 were previously implicated. Objective: To identify additional risk loci in African American individuals by increasing the sample size and using the African Genome Resource panel. Design, Setting, and Participants: This genome-wide association meta-analysis used case-control and family-based data sets from the Alzheimer Disease Genetics Consortium. There were multiple recruitment sites throughout the United States that included individuals with Alzheimer disease and controls of African American ancestry. Analysis began October 2018 and ended September 2019. Main Outcomes and Measures: Diagnosis of Alzheimer disease. Results: A total of 2784 individuals with Alzheimer disease (1944 female [69.8%]) and 5222 controls (3743 female [71.7%]) were analyzed (mean [SD] age at last evaluation, 74.2 [13.6] years). Associations with 4 novel common loci centered near the intracellular glycoprotein trafficking gene EDEM1 (3p26; P = 8.9 × 10-7), near the immune response gene ALCAM (3q13; P = 9.3 × 10-7), within GPC6 (13q31; P = 4.1 × 10-7), a gene critical for recruitment of glutamatergic receptors to the neuronal membrane, and within VRK3 (19q13.33; P = 3.5 × 10-7), a gene involved in glutamate neurotoxicity, were identified. In addition, several loci associated with rare variants, including a genome-wide significant intergenic locus near IGF1R at 15q26 (P = 1.7 × 10-9) and 6 additional loci with suggestive significance (P ≤ 5 × 10-7) such as API5 at 11p12 (P = 8.8 × 10-8) and RBFOX1 at 16p13 (P = 5.4 × 10-7) were identified. Gene expression data from brain tissue demonstrate association of ALCAM, ARAP1, GPC6, and RBFOX1 with brain ß-amyloid load. Of 25 known loci associated with Alzheimer disease in non-Hispanic White individuals, only APOE, ABCA7, TREM2, BIN1, CD2AP, FERMT2, and WWOX were implicated at a nominal significance level or stronger in African American individuals. Pathway analyses strongly support the notion that immunity, lipid processing, and intracellular trafficking pathways underlying Alzheimer disease in African American individuals overlap with those observed in non-Hispanic White individuals. A new pathway emerging from these analyses is the kidney system, suggesting a novel mechanism for Alzheimer disease that needs further exploration. Conclusions and Relevance: While the major pathways involved in Alzheimer disease etiology in African American individuals are similar to those in non-Hispanic White individuals, the disease-associated loci within these pathways differ.

The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA.

The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

Measuring airway clearance outcomes in bronchiectasis: a review.

While airway clearance techniques (ACTs) are recommended for individuals with bronchiectasis, many trials have demonstrated inconsistent benefits or failed to reach their primary outcome. This review determined the most common clinical and patient-reported outcome measures used to evaluate the efficacy of ACTs in bronchiectasis. A literature search of five databases using relevant keywords and filtering for studies published in English, up until the end of August 2019, was completed. Studies included randomised controlled trials, using crossover or any other trial design, and abstracts. Studies were included where the control was placebo, no intervention, standard care, usual care or an active comparator. Adults with bronchiectasis not related to cystic fibrosis were included. Extracted data comprised study authors, design, duration, intervention, outcome measures and results. The search identified 27 published studies and one abstract. The most common clinical outcome measures were sputum volume (n=23), lung function (n=17) and pulse oximetry (n=9). The most common patient-reported outcomes were health-related quality of life (measured with St George's Respiratory Questionnaire, n=4), cough-related quality of life (measured with Leicester Cough Questionnaire, n=4) and dyspnoea (measured with Borg/modified Borg scale, n=8). Sputum volume, lung function, dyspnoea and health- and cough-related quality of life appear to be the most common clinical and patient-reported measures of airway clearance treatment efficacy.

Ribosomal Protein L11 Selectively Stabilizes a Tertiary Structure of the GTPase Center rRNA Domain.

The GTPase Center (GAC) RNA domain in bacterial 23S rRNA is directly bound by ribosomal protein L11, and this complex is essential to ribosome function. Previous cocrystal structures of the 58-nucleotide GAC RNA bound to L11 revealed the intricate tertiary fold of the RNA domain, with one monovalent and several divalent ions located in specific sites within the structure. Here, we report a new crystal structure of the free GAC that is essentially identical to the L11-bound structure, which retains many common sites of divalent ion occupation. This new structure demonstrates that RNA alone folds into its tertiary structure with bound divalent ions. In solution, we find that this tertiary structure is not static, but rather is best described as an ensemble of states. While L11 protein cannot bind to the GAC until the RNA has adopted its tertiary structure, new experimental data show that L11 binds to Mg2+-dependent folded states, which we suggest lie along the folding pathway of the RNA. We propose that L11 stabilizes a specific GAC RNA tertiary state, corresponding to the crystal structure, and that this structure reflects the functionally critical conformation of the rRNA domain in the fully assembled ribosome.

Comparing the Performance Characteristics of Different Positive Expiratory Pressure Devices.

BACKGROUND: Positive expiratory pressure (PEP) devices are widely used in clinical settings, yet the performance characteristics of these devices remain relatively unknown. This study compared the performance characteristics of 6 airway clearance devices by varying resistance and flow. METHODS: Mean PEP, peak PEP, oscillation frequency, and amplitude PEP of the Flutter, Pari PEP S, Acapella Choice, Acapella DM, Acapella DH, and Aerobika devices were obtained across flows of 5, 10, 15, 20, 25 and 30 L/min and at low, medium, and high resistance using an experimental apparatus custom-built for this bench study. RESULTS: Performance characteristics of the devices differed across flows and resistance settings (device × flow/resistance interaction; P < .001). At a fixed resistance, increasing flows increased mean PEP produced by the Acapella Choice, Acapella DH, Aerobika, and Pari PEP S. Increasing flow resulted in minimal change in mean PEP produced by the Flutter and Acapella DM. Increasing flow increased peak PEP and amplitude PEP produced by all devices except the Acapella DH and Acapella Choice. Increasing flow maintained or increased oscillation frequency for all devices except the Flutter. At a fixed flow, increasing resistance increased mean PEP produced by all devices except the Acapella Choice. Increasing resistance increased peak PEP produced by the Acapella DM, Aerobika, and Pari PEP S but resulted in minimal change in peak PEP for the Flutter and Acapella Choice. Increasing resistance either maintained or increased oscillation frequency for all devices. Amplitude PEP was either maintained or increased during oscillations when increasing resistance for all devices except the Flutter. CONCLUSIONS: PEP devices produced small but statistically significant variations in performance characteristics across a range of flows and resistance settings. There appear to be flow-dependent and non-flow-dependent devices. Varying flow or resistance typically maintained or increased the production of mean, peak, and amplitude PEP and oscillation frequency.

Relating exercise-induced desaturation and gas-exchange in pulmonary artery hypertension.

We measured pulmonary gas exchange during six minute walk test (6MWT) in patients with idiopathic (IPAH) and congenital heart disease-related pulmonary hypertension (CHDPH), and determined the relationship between the degree of desaturation and changes in minute ventilation to carbon dioxide production V˙EV˙CO2 and end-tidal partial pressure for carbon dioxide (PetCO2) in both groups. Fifty-two patients (IPAH, n = 28; CHDPH, n = 24) completed 6MWT with simultaneous pulmonary gas exchange. Whilst no significant difference in six minute walk distance was observed between groups (IPAH: 529 ± 89; CHDPH: 476 ± 119 m, p = 0.10), oxygen uptake, carbon dioxide production, and minute ventilation were higher in IPAH than CHDPH (p < 0.01). In addition, CHDPH desaturated to a greater extent than IPAH with a lower post-6MWT SPO2 (IPAH: 92.3 ± 7.9; CHDPH: 73.3 ± 14.9%, p < 0.01). The change in SpO2 correlated to the change in V˙E/V˙CO2 (r=-0.44, p = 0.02) and PetCO2 (r = 0.49, p < 0.01) for CHDPH, but not IPAH. The exercise-induced hypoxic stimulus during 6MWT in CHDPH may be associated with an enhanced ventilatory response.

Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure.

Folding of an RNA from secondary to tertiary structure often depends on divalent ions for efficient electrostatic charge screening (nonspecific association) or binding (specific association). To measure how different divalent cations modify folding kinetics of the 60 nucleotide Ecoli rRNA GTPase center, we combined stopped-flow fluorescence in the presence of Mg2+, Ca2+, or Sr2+ together with time-resolved small angle X-ray scattering (SAXS) in the presence of Mg2+ to observe the folding process. Immediately upon addition of each divalent ion, the RNA undergoes a transition from an extended state with secondary structure to a more compact structure. Subsequently, specific divalent ions modulate populations of intermediates in conformational ensembles along the folding pathway with transition times longer than 10 msec. Rate constants for the five folding transitions act on timescales from submillisecond to tens of seconds. The sensitivity of RNA tertiary structure to divalent cation identity affects all but the fastest events in RNA folding, and allowed us to identify those states that prefer Mg2+ The GTPase center RNA appears to have optimized its folding trajectory to specifically utilize this most abundant intracellular divalent ion.

Molecular principles underlying dual RNA specificity in the Drosophila SNF protein.

The first RNA recognition motif of the Drosophila SNF protein is an example of an RNA binding protein with multi-specificity. It binds different RNA hairpin loops in spliceosomal U1 or U2 small nuclear RNAs, and only in the latter case requires the auxiliary U2A' protein. Here we investigate its functions by crystal structures of SNF alone and bound to U1 stem-loop II, U2A' or U2 stem-loop IV and U2A', SNF dynamics from NMR spectroscopy, and structure-guided mutagenesis in binding studies. We find that different loop-closing base pairs and a nucleotide exchange at the tips of the loops contribute to differential SNF affinity for the RNAs. U2A' immobilizes SNF and RNA residues to restore U2 stem-loop IV binding affinity, while U1 stem-loop II binding does not require such adjustments. Our findings show how U2A' can modulate RNA specificity of SNF without changing SNF conformation or relying on direct RNA contacts.