Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes.

Premessenger RNA splicing is catalyzed by the spliceosome, a multimegadalton RNA-protein complex that assembles in a highly regulated process on each intronic substrate. Most studies of splicing and spliceosomes have been carried out in human or S. cerevisiae model systems. There exists, however, a large diversity of spliceosomes, particularly in organisms with reduced genomes, that suggests a means of analyzing the essential elements of spliceosome assembly and regulation. In this review, we characterize changes in spliceosome composition across phyla, describing those that are most frequently observed and highlighting an analysis of the reduced spliceosome of the red alga Cyanidioschyzon merolae We used homology modeling to predict what effect splicing protein loss would have on the spliceosome, based on currently available cryo-EM structures. We observe strongly correlated loss of proteins that function in the same process, for example, in interacting with the U1 snRNP (which is absent in C. merolae), regulation of Brr2, or coupling transcription and splicing. Based on our observations, we predict splicing in C. merolae to be inefficient, inaccurate, and post-transcriptional, consistent with the apparent trend toward its elimination in this lineage. This work highlights the striking flexibility of the splicing pathway and the spliceosome when viewed in the context of eukaryotic diversity.

Overcoming barriers to equality, diversity, inclusivity, and sense of belonging in healthcare education: the Underrepresented Groups' Experiences in Osteopathic Training (UrGEnT) mixed methods study.

BACKGROUND: Individuals from minority groups have historically faced social injustices. Those from underrepresented groups have been less likely to access both healthcare services and higher education. Little is known about the experiences of underrepresented students during their undergraduate studies in osteopathy in the UK. The aim of this project was to explore awareness of cultural diversity and beliefs about patients from underrepresented groups in current osteopathic educational environments and evaluate students' preparedness to manage patients from diverse groups. The project also aimed to investigate the educational experiences of students from underrepresented backgrounds during their training and their opinions on changes that could support better levels of recruitment and achievement. The findings were discussed with stakeholders in interactive workshops with the aim to develop recommendations for action and change. METHODS: A transformative action research paradigm informed this mixed methods project. It included: 1/ a survey of students from all seven osteopathic educational providers in the UK using the Multidimensional Cultural Humility Scale (MCHS); 2/ a series of focus groups with students from underrepresented groups (women, students with disabilities, students from minority ethnic backgrounds, and students identifying as LGBTQIA+); and 3/ a workshop forum to discuss findings. RESULTS: A total of 202 participants completed the MCHS and demographic questionnaire and seven focus groups were conducted. A model was developed to describe participants' training experiences comprising two main themes: institutional contextual obstacles (with four sub-themes) and underrepresented students' conceptual understanding of Equity, Diversity and Inclusion (EDI). Recommendations for change identified in the workshops were based on three topics: institutions, staff, and students. CONCLUSION: Our findings confirm conclusions from other institutions that staff education is urgently needed to create and maintain equitable, inclusive environments in osteopathic educational institutions in the UK to support all students, particularly those from underrepresented groups. Institutional EDI processes and policies also need to be clarified or modified to ensure their usefulness, accessibility, and implementation.

Silencing of natural transformation by an RNA chaperone and a multitarget small RNA.

A highly conserved DNA uptake system allows many bacteria to actively import and integrate exogenous DNA. This process, called natural transformation, represents a major mechanism of horizontal gene transfer (HGT) involved in the acquisition of virulence and antibiotic resistance determinants. Despite evidence of HGT and the high level of conservation of the genes coding the DNA uptake system, most bacterial species appear non-transformable under laboratory conditions. In naturally transformable species, the DNA uptake system is only expressed when bacteria enter a physiological state called competence, which develops under specific conditions. Here, we investigated the mechanism that controls expression of the DNA uptake system in the human pathogen Legionella pneumophila We found that a repressor of this system displays a conserved ProQ/FinO domain and interacts with a newly characterized trans-acting sRNA, RocR. Together, they target mRNAs of the genes coding the DNA uptake system to control natural transformation. This RNA-based silencing represents a previously unknown regulatory means to control this major mechanism of HGT. Importantly, these findings also show that chromosome-encoded ProQ/FinO domain-containing proteins can assist trans-acting sRNAs and that this class of RNA chaperones could play key roles in post-transcriptional gene regulation throughout bacterial species.

Radical probing of spliceosome assembly.

Here we describe the synthesis and use of a directed hydroxyl radical probe, tethered to a pre-mRNA substrate, to map the structure of this substrate during the spliceosome assembly process. These studies indicate an early organization and proximation of conserved pre-mRNA sequences during spliceosome assembly. This methodology may be adapted to the synthesis of a wide variety of modified RNAs for use as probes of RNA structure and RNA-protein interaction.

Reversibly constraining spliceosome-substrate complexes by engineering disulfide crosslinks.

The spliceosome is a highly dynamic mega-Dalton enzyme, formed in part by assembly of U snRNPs onto its pre-mRNA substrate transcripts. Early steps in spliceosome assembly are challenging to study biochemically and structurally due to compositional and conformational dynamics. We detail an approach to covalently and reversibly constrain or trap non-covalent pre-mRNA/protein spliceosome complexes. This approach involves engineering a single disulfide bond between a thiol-bearing cysteine sidechain and a proximal backbone phosphate of the pre-mRNA, site-specifically modified with an N-thioalkyl moiety. When distance and angle between reactants is optimal, the sidechain will react with the single N-thioalkyl to form a crosslink upon oxidation. We provide protocols detailing how this has been applied successfully to trap an 11-subunit RNA-protein assembly, the human U1 snRNP, in complex with a pre-mRNA.

Cas5d processes pre-crRNA and is a member of a larger family of CRISPR RNA endonucleases.

Small RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in bacteria and archaea are involved in an adaptable and heritable gene-silencing pathway. Resistance to invasive genetic material is conferred by the incorporation of short DNA sequences derived from this material into the genome as CRISPR spacer elements separated by short repeat sequences. Processing of long primary transcripts (pre-crRNAs) containing these repeats by a CRISPR-associated (Cas) RNA endonuclease generates the mature effector RNAs that target foreign nucleic acid for degradation. Here we describe functional studies of a Cas5d ortholog, and high-resolution structural studies of a second Cas5d family member, demonstrating that Cas5d is a sequence-specific RNA endonuclease that cleaves CRISPR repeats and is thus responsible for processing of pre-crRNA. Analysis of the structural homology of Cas5d with the previously characterized Cse3 protein allows us to model the interaction of Cas5d with its RNA substrate and conclude that it is a member of a larger family of CRISPR RNA endonucleases.

Structural model of the p14/SF3b155 · branch duplex complex.

Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14 · bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein-RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14 · duplex interaction must be disrupted before the first step of splicing.

Pre-mRNA splicing: a complex picture in higher definition.

Intron excision from pre-mRNAs of higher eukaryotes requires a transition from splice-site recognition across short exons to organization of the spliceosome across long introns. Recently, insight into this transition has been provided and, in addition, it has been shown that an alternative splicing factor, the polypyrimidine-tract-binding protein, can exert its control on splice-site choice by blocking this key step in the assembly of the splicing machinery.

Characterising the interventions designed to affect the reporting of musculoskeletal imaging: a scoping review protocol using the COM-B model.

INTRODUCTION: Attributing musculoskeletal (MSK) pain to normal and commonly occurring imaging findings, such as tendon, cartilage and spinal disc degeneration, has been shown to increase people's fear of movement, reduce their optimism about recovery and increase healthcare costs. Interventions seeking to reduce the negative effects of MSK imaging reporting have had little effect. To understand the ineffectiveness of these interventions, this study seeks to scope their behavioural targets, intended mechanisms of action and theoretical underpinnings. This information alongside known barriers to helpful reporting can enable researchers to refine or create new more targeted interventions. METHODS AND ANALYSIS: The scoping review will be conducted in accordance with the JBI methodology for scoping reviews and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews. Search terms will be devised by the research team. Searches of MEDLINE, EMBASE, CINAHL, AMED and PsycINFO from inception to current day will be performed. The review will include studies, which have developed or evaluated interventions targeting the reporting of MSK imaging. Studies targeting the diagnosis of serious causes of MSK pain will be excluded. Two independent authors will extract study participant data using predefined extraction templates and intervention details using the Template for Intervention Description and Replication checklist. Interventions will be coded and mapped to the technique, mechanism of action and behavioural target according to the Capability, Opportunity, Motivation-Behaviour (COM-B) model categories. Any explicit models or theories used to inform the selection of interventions will be extracted and coded. The study characteristics, behaviour change techniques identified, behavioural targets according to the COM-B and context specific theories within the studies will be presented in narrative and table form. ETHICS AND DISSEMINATION: The information from this review will be used to inform an intervention design process seeking to improve the communication of imaging results. The results will also be disseminated through a peer-reviewed publication, conference presentations and stakeholder events.

Long-term health care use and diagnosis after hospitalization for COVID-19: a retrospective matched cohort study.

BACKGROUND: Knowledge pertaining to the health and health care utilization of patients after recovery from acute COVID-19 is limited. We sought to assess the frequency of new diagnoses of disease and health care use after hospitalization with COVID-19. METHODS: We included all patients hospitalized with COVID-19 in Alberta between Mar. 5 and Dec. 31, 2020. Additionally, 2 matched controls (SARS-CoV-2 negative) per case were included and followed up until Apr. 30, 2021. New diagnoses and health care use were identified from linked administrative health data. Repeated measures were made for the periods 1-30 days, 31-60 days, 61-90 days, 91-180 days, and 180 and more days from the index date. We used multivariable regression analysis to evaluate the association of COVID-19-related hospitalization with the number of physician visits during follow-up. RESULTS: The study sample included 3397 cases and 6658 controls. Within the first 30 days of follow-up, the case group had 37.12% (95% confidence interval [CI] 35.44% to 38.80%) more patients with physician visits, 11.12% (95% CI 9.77% to 12.46%) more patients with emergency department visits and 2.92% (95% CI 2.08% to 3.76%) more patients with hospital admissions than the control group. New diagnoses involving multiple organ systems were more common in the case group. Regression results indicated that recovering from COVID-19-related hospitalization, admission to an intensive care unit, older age, greater number of comorbidities and more prior health care use were associated with increased physician visits. INTERPRETATION: Patients recovered from the acute phase of COVID-19 continued to have greater health care use up to 6 months after hospital discharge. Research is required to further explore the effect of post-COVID-19 conditions, pre-existing health conditions and health-seeking behaviours on health care use.

Blinding and sham control methods in trials of physical, psychological, and self-management interventions for pain (article I): a systematic review and description of methods.

ABSTRACT: Blinding is challenging in randomised controlled trials of physical, psychological, and self-management therapies for pain, mainly because of their complex and participatory nature. To develop standards for the design, implementation, and reporting of control interventions in efficacy and mechanistic trials, a systematic overview of currently used sham interventions and other blinding methods was required. Twelve databases were searched for placebo or sham-controlled randomised clinical trials of physical, psychological, and self-management treatments in a clinical pain population. Screening and data extraction were performed in duplicate, and trial features, description of control methods, and their similarity to the active intervention under investigation were extracted (protocol registration ID: CRD42020206590). The review included 198 unique control interventions, published between 2008 and December 2021. Most trials studied people with chronic pain, and more than half were manual therapy trials. The described control interventions ranged from clearly modelled based on the active treatment to largely dissimilar control interventions. Similarity between control and active interventions was more frequent for certain aspects (eg, duration and frequency of treatments) than others (eg, physical treatment procedures and patient sensory experiences). We also provide an overview of additional, potentially useful methods to enhance blinding, as well as the reporting of processes involved in developing control interventions. A comprehensive picture of prevalent blinding methods is provided, including a detailed assessment of the resemblance between active and control interventions. These findings can inform future developments of control interventions in efficacy and mechanistic trials and best-practice recommendations.

Blinding and sham control methods in trials of physical, psychological, and self-management interventions for pain (article II): a meta-analysis relating methods to trial results.

ABSTRACT: Sham interventions in randomized clinical trials (RCTs) of physical, psychological, and self-management (PPS) therapies for pain are highly variable in design and believed to contribute to poor internal validity. However, it has not been formally tested whether the extent to which sham controls resemble the treatment under investigation consistently affects trial outcomes, such as effect sizes, differential attrition, participant expectancy, and blinding effectiveness. Placebo- or sham-controlled RCTs of PPS interventions of clinical pain populations were searched in 12 databases. The similarity of control interventions to the experimental treatment was rated across 25 features. Meta-regression analyses assessed putative links between employed control interventions, observed effect sizes in pain-related outcomes, attrition, and blinding success. The sample included 198 unique control interventions, dominated by manual therapy and chronic musculoskeletal pain research. Meta-analyses indicated small-to-moderate benefits of active treatments over control interventions, across subgroups of manual therapies, exercise, and rehabilitation, and psychological intervention trials. Multiple meta-regression modelling demonstrated that similarity between sham control and tested interventions predicted variability in pain-related outcomes, attrition, and blinding effectiveness. Influential variables were differences relating to the extent of intervention exposure, participant experience, and treatment environments. The results support the supposed link between blinding methods and effect sizes, based on a large and systematically sourced overview of methods. However, challenges to effective blinding are complex and often difficult to discern from trial reports. Nonetheless, these insights have the potential to change trial design, conduct, and reporting and will inform guideline development.

Structural basis for recognition of transcriptional terminator structures by ProQ/FinO domain RNA chaperones.

The ProQ/FinO family of RNA binding proteins mediate sRNA-directed gene regulation throughout gram-negative bacteria. Here, we investigate the structural basis for RNA recognition by ProQ/FinO proteins, through the crystal structure of the ProQ/FinO domain of the Legionella pneumophila DNA uptake regulator, RocC, bound to the transcriptional terminator of its primary partner, the sRNA RocR. The structure reveals specific recognition of the 3' nucleotide of the terminator by a conserved pocket involving a ß-turn-α-helix motif, while the hairpin portion of the terminator is recognized by a conserved α-helical N-cap motif. Structure-guided mutagenesis reveals key RNA contact residues that are critical for RocC/RocR to repress the uptake of environmental DNA in L. pneumophila. Structural analysis and RNA binding studies reveal that other ProQ/FinO domains also recognize related transcriptional terminators with different specificities for the length of the 3' ssRNA tail.

Role of pri-miRNA tertiary structure in miR-17~92 miRNA biogenesis.

MicroRNAs (miRNAs) regulate gene expression in a variety of biological pathways such as development and tumourigenesis. miRNAs are initially expressed as long primary transcripts (pri-miRNAs) that undergo sequential processing by Drosha and then Dicer to yield mature miRNAs. miR-17~92 is a miRNA cluster that encodes 6 miRNAs and while it is essential for development it also has reported oncogenic activity. To date, the role of RNA structure in miRNA biogenesis has only been considered in terms of the secondary structural elements required for processing of pri-miRNAs by Drosha. Here we report that the miR-17~92 cluster has a compact globular tertiary structure where miRNAs internalized within the core of the folded structure are processed less efficiently than miRNAs on the surface of the structure. Increased miR-92 expression resulting from disruption of the compact miR-17~92 structure results in increased repression of integrin α5 mRNA, a known target of miR-92a. In summary, we describe the first example of pri-miRNA structure modulating differential expression of constituent miRNAs.

Spliceosome structure: piece by piece.

Processing of pre-mRNAs by RNA splicing is an essential step in the maturation of protein coding RNAs in eukaryotes. Structural studies of the cellular splicing machinery, the spliceosome, are a major challenge in structural biology due to the size and complexity of the splicing ensemble. Specifically, the structural details of splice site recognition and the architecture of the spliceosome active site are poorly understood. X-ray and NMR techniques have been successfully used to address these questions defining the structure of individual domains, isolated splicing proteins, spliceosomal RNA fragments and recently the U1 snRNP multiprotein.RNA complex. These results combined with extant biochemical and genetic data have yielded important insights as well as posing fresh questions with respect to the regulation and mechanism of this critical gene regulatory process.

Multidisciplinary Approach to Improve Sepsis Outcomes.

Severe sepsis and septic shock cause significant morbidity and mortality with health care costs approximating $17 billion annually. The Surviving Sepsis Campaign 2012 recommended time-sensitive care bundles to improve outcomes for patients with sepsis. At our community teaching hospital, a review of sepsis management for patients admitted to a medical intensive care unit (ICU) between December 2015 and March 2016 found 70.8% compliance with timing of lactate draw, 65.3% compliance for blood cultures, and 51.4% compliance with antibiotic administration recommendations. Thus, a quality improvement initiative to improve detection and time to bundle completion for ICU-level patients was designed. Previous studies suggest that utilization of sepsis alert systems and sepsis response teams in the emergency department setting is associated with improved compliance with recommended sepsis bundles and improved hospital mortality. Therefore, a "sepsis alert" protocol was implemented that used both an electronic alert and an overhead team alert that mobilized nursing, pharmacy, phlebotomy, and a senior internal medicine resident to bedside. In addition, a template to document sepsis diagnosis and bundle adherence was created. After implementation, we noted improvement in appropriately timed serum lactate, 88.6% versus 70.8% (p = .008) with no significant improvements in blood cultures, antibiotic administration, or mortality.

Characterization of a U2AF-independent commitment complex (E') in the mammalian spliceosome assembly pathway.

Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5' splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3' splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E' which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E' complex is characterized by a U1 snRNA-5' splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5' splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.