Time-resolved profiling of RNA binding proteins throughout the mRNA life cycle.

mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.

Structural atlas of human primary microRNAs generated by SHAPE-MaP.

MicroRNA (miRNA) maturation is critically dependent on structural features of primary transcripts (pri-miRNAs). However, the scarcity of determined pri-miRNA structures has limited our understanding of miRNA maturation. Here, we employed selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), a high-throughput RNA structure probing method, to unravel the secondary structures of 476 high-confidence human pri-miRNAs. Our SHAPE-based structures diverge substantially from those inferred solely from computation, particularly in the apical loop and basal segments, underlining the need for experimental data in RNA structure prediction. By comparing the structures with high-throughput processing data, we determined the optimal structural features of pri-miRNAs. The sequence determinants are influenced substantially by their structural contexts. Moreover, we identified an element termed the bulged GWG motif (bGWG) with a 3' bulge in the lower stem, which promotes processing. Our structure-function mapping better annotates the determinants of pri-miRNA processing and offers practical implications for designing small hairpin RNAs and predicting the impacts of miRNA mutations.

Deadenylation kinetics of mixed poly(A) tails at single-nucleotide resolution.

Shortening of messenger RNA poly(A) tails, or deadenylation, is a rate-limiting step in mRNA decay and is highly regulated during gene expression. The incorporation of non-adenosines in poly(A) tails, or 'mixed tailing', has been observed in vertebrates and viruses. Here, to quantitate the effect of mixed tails, we mathematically modeled deadenylation reactions at single-nucleotide resolution using an in vitro deadenylation system reconstituted with the complete human CCR4-NOT complex. Applying this model, we assessed the disrupting impact of single guanosine, uridine or cytosine to be equivalent to approximately 6, 8 or 11 adenosines, respectively. CCR4-NOT stalls at the 0, -1 and -2 positions relative to the non-adenosine residue. CAF1 and CCR4 enzyme subunits commonly prefer adenosine but exhibit distinct sequence selectivities and stalling positions. Our study provides an analytical framework to monitor deadenylation and reveals the molecular basis of tail sequence-dependent regulation of mRNA stability.

Asynchronous development of memory integration and differentiation influences temporal memory organization.

Adults remember items with shared contexts as occurring closer in time to one another than those associated with different contexts, even when their objective temporal distance is fixed. Such temporal memory biases are thought to reflect within-event integration and between-event differentiation processes that organize events according to their contextual similarities and differences, respectively. Within-event integration and between-event differentiation are hypothesized to differentially rely on binding and control processes, which may develop at different ages. To test this hypothesis, 5- to 12-year-olds and adults (N = 134) studied quartets of image pairs that contained either the same scene (same-context) or different scenes (different-context). Participants remembered same-context items as occurring closer in time by older childhood (7-9 years), whereas different-context items were remembered as occurring farther apart by early adolescence (10-12 years). The differential emergence of these temporal memory biases suggests within-event integration and between-event differentiation emerge at different ages. RESEARCH HIGHLIGHTS: Children are less likely than adults to use contextual information (e.g., location) to organize their continuous experience in memory, as indicated by temporal memory biases. Biases reflecting within-event integration (i.e., remembering elements with a shared context as occurring closer together in time) emerged in late childhood. Biases reflecting between-event differentiation (i.e., remembering elements from different contexts as occurring farther apart in time) emerged in early adolescence. The differential emergence of biases reflecting within-event integration and between-event differentiation suggests they are distinct, yet complementary, processes that support developmental improvements in event memory organization.

Impaired cholinergic integrity of the colon and pancreas in dementia with Lewy bodies.

Dementia with Lewy bodies is characterized by a high burden of autonomic dysfunction and Lewy pathology in peripheral organs and components of the sympathetic and parasympathetic nervous system. Parasympathetic terminals may be quantified with 18F-fluoroetoxybenzovesamicol, a PET tracer that binds to the vesicular acetylcholine transporter in cholinergic presynaptic terminals. Parasympathetic imaging may be useful for diagnostics, improving our understanding of autonomic dysfunction and for clarifying the spatiotemporal relationship of neuronal degeneration in prodromal disease. Therefore, we aimed to investigate the cholinergic parasympathetic integrity in peripheral organs and central autonomic regions of subjects with dementia with Lewy bodies and its association with subjective and objective measures of autonomic dysfunction. We hypothesized that organs with known parasympathetic innervation, especially the pancreas and colon, would have impaired cholinergic integrity. To achieve these aims, we conducted a cross-sectional comparison study including 23 newly diagnosed non-diabetic subjects with dementia with Lewy bodies (74 ± 6 years, 83% male) and 21 elderly control subjects (74 ± 6 years, 67% male). We obtained whole-body images to quantify PET uptake in peripheral organs and brain images to quantify PET uptake in regions of the brainstem and hypothalamus. Autonomic dysfunction was assessed with questionnaires and measurements of orthostatic blood pressure. Subjects with dementia with Lewy bodies displayed reduced cholinergic tracer uptake in the pancreas (32% reduction, P = 0.0003) and colon (19% reduction, P = 0.0048), but not in organs with little or no parasympathetic innervation. Tracer uptake in a region of the medulla oblongata overlapping the dorsal motor nucleus of the vagus correlated with autonomic symptoms (rs = -0.54, P = 0.0077) and changes in orthostatic blood pressure (rs = 0.76, P < 0.0001). Tracer uptake in the pedunculopontine region correlated with autonomic symptoms (rs = -0.52, P = 0.0104) and a measure of non-motor symptoms (rs = -0.47, P = 0.0230). In conclusion, our findings provide the first imaging-based evidence of impaired cholinergic integrity of the pancreas and colon in dementia with Lewy bodies. The observed changes may reflect parasympathetic denervation, implying that this process is initiated well before the point of diagnosis. The findings also support that cholinergic denervation in the brainstem contributes to dysautonomia.

Studying the Development of Navigation Using Virtual Environments.

Research on spatial navigation is essential to understanding how mobile species adapt to their environments. Such research increasingly uses virtual environments (VEs) because, although VE has drawbacks, it allows for standardization of procedures, precision in measuring behaviors, ease in introducing variation, and cross-investigator comparability. Developmental researchers have used a wide range of VE testing methods, including desktop computers, gaming consoles, virtual reality, and phone applications. We survey the paradigms to guide researchers' choices, organizing them by their characteristics using a framework proposed by Girard (2022) in which navigation is reactive or deliberative, and may be tied to sensory input or not. This organization highlights what representations each paradigm indicates. VE tools have enriched our picture of the development of navigation, but much research remains to be done, e.g., determining retest reliability, comparing performance on different paradigms, validating performance against real-world behavior and open sharing. Reliable and valid assessments available on open-science repositories are essential for work on the development of navigation, its neural bases, and its implications for other cognitive domains.

[The value of lamina cribrosa in the diagnosis and treatment of glaucoma. Remodeling of lamina cribrosa collagen and approaches to its therapeutic treatment]. / Znachenie struktury reshetchatoi membrany sklery v diagnostike i lechenii glaukomy. Remodelirovanie kollagena reshetchatoi membrany i puti terapevticheskogo vozdeistviya na nego.

Among the first structures suffering damage with an increase in intraocular pressure (IOP) and in early stage of glaucoma are the lamina cribrosa (LC) and peripapillary sclera (ppScl). Changes in these structures occur at the molecular and cellular level. Extracellular matrix (ECM) is the basis of connective tissue, provides mechanical support for the cells, facilitates intercellular interactions and transport of chemicals, including in LC and ppScl. Mechanical stress causes remodeling and disorganization of the ECM, which leads to changes in the structure of the tissue itself, an increase in its rigidity and a decrease in elasticity. Taking into account the molecular and cellular mechanisms of damage to LC and ppScl, various researchers have developed strategies and tactics for therapeutic intervention on these structures, contributing to a decrease in ECM secretion and, as a consequence, suspension of their remodeling. These approaches may in the future form the basis for the treatment of glaucomatous optic neuropathy.

[The improvement of cognitive rehabilitation of persons of older and senile age].

The article presents results of study of patients of elderly age within the framework of pilot project on the basis of the geriatric department of the State Budget Institution of the Rostov Oblast "The Hospital for War Veterans". The specially questionnaire was developed with participation of the charitable foundation "Memory of Generations" and the public movement "Medics-Volunteers". The social hygienic characteristics of studied contingent was analyzed. The statistical processing of obtained data was carried out considering subjective status of patients.

Methyl Donor Nutrient Intake and Incidence of Type 2 Diabetes: Results From Three Large U.S. Cohorts.

OBJECTIVE: We examined whether intake of methyl donor nutrients, including vitamins B2, B6, and B12 and folate, from foods and/or supplements is associated with type 2 diabetes risk. RESEARCH DESIGN AND METHODS: We included 203,644 women and men from the Nurses' Health Study (1984-2016), Nurses' Health Study 2 (1991-2017), and Health Professionals Follow-Up Study (1986-2016). Dietary data were collected every 2-4 years with use of semiquantitative food-frequency questionnaires. Cox proportional hazards models with time-varying covariates were used to evaluate associations between each nutrient and type 2 diabetes risk. We combined cohort-specific hazard ratios (HRs) using inverse variance-weighted fixed-effects meta-analyses. RESULTS: During 4,900,181 person-years of follow-up, we documented 19,475 incident type 2 diabetes cases. In multivariable-adjusted meta-analyses, participants in the highest quintiles of total vitamin B2 and B6 intakes had lower risk of diabetes compared with those in the lowest quintiles (HR 0.93 [95% CI 0.89, 0.98] for B2 and 0.93 [0.89, 0.97] for B6). With stratification by source, significant associations remained for B2 from food but not from supplements. Neither association for B6 from food nor association for B6 from supplements attained significance. No association was observed between total B12 intake and diabetes. However, B12 from food was marginally associated with higher diabetes risk (1.05 [1.00-1.11]) but not after additional adjustment for red meat intake (1.04 [0.99-1.10]). No evidence of association was observed between intakes of folate and diabetes. CONCLUSIONS: The results of our study suggest that higher intake of vitamin B2 and B6, especially B2 from food sources, may be associated with a modestly lower type 2 diabetes risk.

Functional viromic screens uncover regulatory RNA elements.

The number of sequenced viral genomes has surged recently, presenting an opportunity to understand viral diversity and uncover unknown regulatory mechanisms. Here, we conducted a screening of 30,367 viral segments from 143 species representing 96 genera and 37 families. Using a library of viral segments in 3' UTR, we identified hundreds of elements impacting RNA abundance, translation, and nucleocytoplasmic distribution. To illustrate the power of this approach, we investigated K5, an element conserved in kobuviruses, and found its potent ability to enhance mRNA stability and translation in various contexts, including adeno-associated viral vectors and synthetic mRNAs. Moreover, we identified a previously uncharacterized protein, ZCCHC2, as a critical host factor for K5. ZCCHC2 recruits the terminal nucleotidyl transferase TENT4 to elongate poly(A) tails with mixed sequences, delaying deadenylation. This study provides a unique resource for virus and RNA research and highlights the potential of the virosphere for biological discoveries.

Role of the proline-rich disordered domain of DROSHA in intronic microRNA processing.

DROSHA serves as a gatekeeper of the microRNA (miRNA) pathway by processing primary transcripts (pri-miRNAs). While the functions of structured domains of DROSHA have been well documented, the contribution of N-terminal proline-rich disordered domain (PRD) remains elusive. Here we show that the PRD promotes the processing of miRNA hairpins located within introns. We identified a DROSHA isoform (p140) lacking the PRD, which is produced by proteolytic cleavage. Small RNA sequencing revealed that p140 is significantly impaired in the maturation of intronic miRNAs. Consistently, our minigene constructs demonstrated that PRD enhances the processing of intronic hairpins, but not those in exons. Splice site mutations did not affect the PRD's enhancing effect on intronic constructs, suggesting that the PRD acts independently of splicing reaction by interacting with sequences residing within introns. The N-terminal regions from zebrafish and Xenopus DROSHA can replace the human counterpart, indicating functional conservation despite poor sequence alignment. Moreover, we found that rapidly evolving intronic miRNAs are generally more dependent on PRD than conserved ones, suggesting a role of PRD in miRNA evolution. Our study reveals a new layer of miRNA regulation mediated by a low-complexity disordered domain that senses the genomic contexts of miRNA loci.

Improving Compliance With Institutional Performance on Train of Four Monitoring.

Background: We performed a multistep quality improvement project related to neuromuscular blockade and monitoring to evaluate the effectiveness of a comprehensive quality improvement program based upon the Multi-institutional Perioperative Outcomes Group (MPOG) Anesthesiology Performance Improvement and Reporting Exchange (ASPIRE) metrics targeted specifically at improving train of four (TOF) monitoring rates. Methods: We adapted the plan-do-study-act (PDSA) framework and implemented 2 PDSA cycles between January 2021 and December 2021. PDSA Cycle 1 (Phase I) and PDSA Cycle 2 (Phase II) included a multipart program consisting of (1) a departmental survey assessing attitudes toward intended results, outcomes, and barriers for TOF monitoring, (2) personalized MPOG ASPIRE quality performance reports displaying provider performance, (3) a dashboard access to help providers complete a case-by-case review, and (4) a web-based app spaced education module concerning TOF monitoring and residual neuromuscular blockade. Our primary outcome was to identify the facilitators and barriers to implementation of our intervention aimed at increasing TOF monitoring. Results: In Phase I, 25 anesthesia providers participated in the preintervention and postintervention needs assessment survey and received personalized quality metric reports. In Phase II, 222 providers participated in the preintervention needs assessment survey and 201 participated in the postintervention survey. Thematic analysis of Phase I survey data aimed at identifying the facilitators and barriers to implementation of a program aimed at increasing TOF monitoring revealed the following: intended results were centered on quality of patient care, barriers to implementation largely encompassed issues with technology/equipment and the increased burden placed on providers, and important outcomes were focused on patient outcomes and improving provider knowledge. Results of Phase II survey data was similar to that of Phase I. Notably in Phase II a few additional barriers to implementation were mentioned including a fear of loss of individualization due to standardization of patient care plan, differences between the attending overseeing the case and the in-room provider who is making decisions/completing documentation, and the frequency of intraoperative handovers. Compared to preintervention, postintervention compliance with TOF monitoring increased from 42% to 70% (28% absolute difference across N = 10 169 cases; P < .001). Conclusions: Implementation of a structured quality improvement program using a novel educational intervention showed improvements in process metrics regarding neuromuscular monitoring, while giving us a better understanding of how best to implement improvements in this metric at this magnitude.

An app a day: Results of pre- and post-surveys of knowledge, attitudes, and practices (KAP) regarding antimicrobial stewardship principles among nurses who utilized a novel learning platform.

Background: Nurses perform several functions that are integral for antimicrobial stewardship (AMS). However, nurses are underrepresented in research and underutilized in implementation of AMS interventions. The objective of this pilot study was to assess the effect of asynchronous microlearning on inpatient nursing staff knowledge, attitudes, and practices (KAP) regarding AMS principles. Methods: A team of pharmacists, physicians, and nurses developed 9 case-based, multiple-choice questions with accompanying educational explanations on associated AMS principles. One case was delivered to participants daily via an institutional web-based application (QuizTime). A KAP survey with 20 questions on a 5-point Likert scale was administered before and after the intervention. Survey results were compared using a Wilcoxon signed-rank test. Results: Participants' mean survey score after the intervention demonstrated statistically significant improvement for 18 (90%) of 20 items compared to before the intervention. Participants' confidence improved in key AMS activities: (1) differentiating between colonization and infection (mean difference, 0.63; P < .001), (2) identifying unnecessary urine cultures and inappropriate treatment of urinary tract infections (mean difference, 0.94; P < .001), (3) recognizing opportunities for intravenous to oral therapy conversion (mean difference, 1.07; P < .001), and (4) assessing for antibiotic-associated adverse effects (mean difference, 0.54; P < .001). Conclusions: Nursing education provided through an asynchronous, microlearning format via a mobile platform resulted in statistically significant improvement in most KAP topics. Nurses are integral members of a multidisciplinary AMS team, and novel education methods can help equip them with the necessary AMS tools. This pilot study forms the basis for expanded AMS educational efforts in all healthcare professionals.

C-reactive protein partially mediates the inverse association between coffee consumption and risk of type 2 diabetes: The UK Biobank and the Rotterdam study cohorts.

BACKGROUND: Coffee is among the most consumed beverages worldwide. Coffee consumption has been associated with lower risk of type 2 diabetes mellitus (T2D), but underlying mechanisms are not well understood. We aimed to study the role of classic and novel-T2D biomarkers with anti- or pro-inflammatory activity in the association between habitual coffee intake and T2D risk. Furthermore, we studied differences by coffee types and smoking status in this association. METHODS: Using two large population-based cohorts, the UK-Biobank (UKB; n = 145,368) and the Rotterdam Study (RS; n = 7111), we investigated associations of habitual coffee consumption with incident T2D and repeated measures of insulin resistance (HOMA-IR), using Cox proportional hazards and mixed effect models, respectively. Additionally, we studied associations between coffee and subclinical inflammation biomarkers including C-reactive protein (CRP) and IL-13, and adipokines, such as adiponectin and leptin, using linear regression models. Next, we performed formal causal mediation analyses to investigate the role of coffee-associated biomarkers in the association of coffee with T2D. Finally, we evaluated effect modification by coffee type and smoking. All models were adjusted for sociodemographic, lifestyle and health-related factors. RESULTS: During a median follow-up of 13.9 (RS) and 7.4 (UKB) years, 843 and 2290 incident T2D cases occurred, respectively. A 1 cup/day increase in coffee consumption was associated with 4% lower T2D risk (RS, HR = 0.96 [95%CI 0.92; 0.99], p = 0.045; UKB, HR = 0.96 [0.94; 0.98], p < 0.001), with lower HOMA-IR (RS, log-transformed ß = -0.017 [-0.024;-0.010], p < 0.001), and with lower CRP (RS, log-transformed ß = -0.014 [-0.022;-0.005], p = 0.002; UKB, ß = -0.011 [-0.012;-0.009], p < 0.001). We also observed associations of higher coffee consumption with higher serum adiponectin and IL-13 concentrations, and with lower leptin concentrations. Coffee-related CRP levels partially mediated the inverse association of coffee intake with T2D incidence (average mediation effect RS ß = 0.105 (0.014; 0.240), p = 0.016; UKB ß = 6.484 (4.265; 9.339), p < 0.001), with a proportion mediated by CRP from 3.7% [-0.012%; 24.4%] (RS) to 9.8% [5,7%; 25.8%] (UKB). No mediation effect was observed for the other biomarkers. Coffee-T2D and coffee-CRP associations were generally stronger among consumers of ground (filtered or espresso) coffee and among never and former smokers. CONCLUSIONS: Lower subclinical inflammation may partially mediate the beneficial association between coffee consumption and lower T2D risk. Consumers of ground coffee and non-smokers may benefit the most. KEYWORDS (MESH TERMS): coffee consumptions; diabetes mellitus, type 2; inflammation; adipokines; biomarkers; mediation analysis; follow-up studies.

Short poly(A) tails are protected from deadenylation by the LARP1-PABP complex.

Deadenylation generally constitutes the first and pivotal step in eukaryotic messenger RNA decay. Despite its importance in posttranscriptional regulations, the kinetics of deadenylation and its regulation remain largely unexplored. Here we identify La ribonucleoprotein 1, translational regulator (LARP1) as a general decelerator of deadenylation, which acts mainly in the 30-60-nucleotide (nt) poly(A) length window. We measured the steady-state and pulse-chased distribution of poly(A)-tail length, and found that deadenylation slows down in the 30-60-nt range. LARP1 associates preferentially with short tails and its depletion results in accelerated deadenylation specifically in the 30-60-nt range. Consistently, LARP1 knockdown leads to a global reduction of messenger RNA abundance. LARP1 interferes with the CCR4-NOT-mediated deadenylation in vitro by forming a ternary complex with poly(A)-binding protein (PABP) and poly(A). Together, our work reveals a dynamic nature of deadenylation kinetics and a role of LARP1 as a poly(A) length-specific barricade that creates a threshold for deadenylation.

Competencies for the Use of Artificial Intelligence-Based Tools by Health Care Professionals.

PURPOSE: The expanded use of clinical tools that incorporate artificial intelligence (AI) methods has generated calls for specific competencies for effective and ethical use. This qualitative study used expert interviews to define AI-related clinical competencies for health care professionals. METHOD: In 2021, a multidisciplinary team interviewed 15 experts in the use of AI-based tools in health care settings about the clinical competencies health care professionals need to work effectively with such tools. Transcripts of the semistructured interviews were coded and thematically analyzed. Draft competency statements were developed and provided to the experts for feedback. The competencies were finalized using a consensus process across the research team. RESULTS: Six competency domain statements and 25 subcompetencies were formulated from the thematic analysis. The competency domain statements are: (1) basic knowledge of AI: explain what AI is and describe its health care applications; (2) social and ethical implications of AI: explain how social, economic, and political systems influence AI-based tools and how these relationships impact justice, equity, and ethics; (3) AI-enhanced clinical encounters: carry out AI-enhanced clinical encounters that integrate diverse sources of information in creating patient-centered care plans; (4) evidence-based evaluation of AI-based tools: evaluate the quality, accuracy, safety, contextual appropriateness, and biases of AI-based tools and their underlying data sets in providing care to patients and populations; (5) workflow analysis for AI-based tools: analyze and adapt to changes in teams, roles, responsibilities, and workflows resulting from implementation of AI-based tools; and (6) practice-based learning and improvement regarding AI-based tools: participate in continuing professional development and practice-based improvement activities related to use of AI tools in health care. CONCLUSIONS: The 6 clinical competencies identified can be used to guide future teaching and learning programs to maximize the potential benefits of AI-based tools and diminish potential harms.

A multi-centre polyp detection and segmentation dataset for generalisability assessment.

Polyps in the colon are widely known cancer precursors identified by colonoscopy. Whilst most polyps are benign, the polyp's number, size and surface structure are linked to the risk of colon cancer. Several methods have been developed to automate polyp detection and segmentation. However, the main issue is that they are not tested rigorously on a large multicentre purpose-built dataset, one reason being the lack of a comprehensive public dataset. As a result, the developed methods may not generalise to different population datasets. To this extent, we have curated a dataset from six unique centres incorporating more than 300 patients. The dataset includes both single frame and sequence data with 3762 annotated polyp labels with precise delineation of polyp boundaries verified by six senior gastroenterologists. To our knowledge, this is the most comprehensive detection and pixel-level segmentation dataset (referred to as PolypGen) curated by a team of computational scientists and expert gastroenterologists. The paper provides insight into data construction and annotation strategies, quality assurance, and technical validation.

Sequence determinant of small RNA production by DICER.

RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs)1,2. However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3' overhang and a terminal loop3-11. Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis-acting element, termed the 'GYM motif' (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified 'ruler'-like counting mechanisms from the 5' and 3' ends of pre-miRNA3-6. Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics.

Structure of the human DICER-pre-miRNA complex in a dicing state.

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.

Epigenome-Wide Association Study Reveals CpG Sites Associated with Thyroid Function and Regulatory Effects on KLF9.

Background: Thyroid hormones play a key role in differentiation and metabolism and are known regulators of gene expression through both genomic and epigenetic processes including DNA methylation. The aim of this study was to examine associations between thyroid hormones and DNA methylation. Methods: We carried out a fixed-effect meta-analysis of epigenome-wide association study (EWAS) of blood DNA methylation sites from 8 cohorts from the ThyroidOmics Consortium, incorporating up to 7073 participants of both European and African ancestry, implementing a discovery and replication stage. Statistical analyses were conducted using normalized beta CpG values as dependent and log-transformed thyrotropin (TSH), free thyroxine, and free triiodothyronine levels, respectively, as independent variable in a linear model. The replicated findings were correlated with gene expression levels in whole blood and tested for causal influence of TSH and free thyroxine by two-sample Mendelian randomization (MR). Results: Epigenome-wide significant associations (p-value <1.1E-7) of three CpGs for free thyroxine, five for free triiodothyronine, and two for TSH concentrations were discovered and replicated (combined p-values = 1.5E-9 to 4.3E-28). The associations included CpG sites annotated to KLF9 (cg00049440) and DOT1L (cg04173586) that overlap with all three traits, consistent with hypothalamic-pituitary-thyroid axis physiology. Significant associations were also found for CpGs in FKBP5 for free thyroxine, and at CSNK1D/LINCO1970 and LRRC8D for free triiodothyronine. MR analyses supported a causal effect of thyroid status on DNA methylation of KLF9. DNA methylation of cg00049440 in KLF9 was inversely correlated with KLF9 gene expression in blood. The CpG at CSNK1D/LINC01970 overlapped with thyroid hormone receptor alpha binding peaks in liver cells. The total additive heritability of the methylation levels of the six significant CpG sites was between 25% and 57%. Significant methylation QTLs were identified for CpGs at KLF9, FKBP5, LRRC8D, and CSNK1D/LINC01970. Conclusions: We report novel associations between TSH, thyroid hormones, and blood-based DNA methylation. This study advances our understanding of thyroid hormone action particularly related to KLF9 and serves as a proof-of-concept that integrations of EWAS with other -omics data can provide a valuable tool for unraveling thyroid hormone signaling in humans by complementing and feeding classical in vitro and animal studies.