Modulatory role of RNA helicases in MBNL-dependent alternative splicing regulation.

Muscleblind-like splicing regulators (MBNLs) activate or repress the inclusion of alternative splicing (AS) events, enabling the developmental transition of fetal mRNA splicing isoforms to their adult forms. Herein, we sought to elaborate the mechanism by which MBNLs mediate AS related to biological processes. We evaluated the functional role of DEAD-box (DDX) RNA helicases, DDX5 and DDX17 in MBNL-dependent AS regulation. Whole-transcriptome analysis and validation approaches revealed a handful of MBNLs-dependent AS events to be affected by DDX5 and DDX17 in mostly an opposite manner. The opposite expression patterns of these two groups of factors during muscle development and coordination of fetal-to-adult splicing transition indicate the importance of these proteins at early stages of development. The identified pathways of how the helicases modulate MBNL splicing activity include DDX5 and DDX17-dependent changes in the ratio of MBNL splicing isoforms and most likely changes in accessibility of MBNL-binding sites. Another pathway involves the mode of action of the helicases independent of MBNL activity. These findings lead to a deeper understanding of the network of interdependencies between RNA-binding proteins and constitute a valuable element in the discussion on developmental homeostasis and pathological states in which the studied protein factors play a significant role.

Autistic traits in myotonic dystrophy type 1 due to MBNL inhibition and RNA mis-splicing.

Tandem repeat expansions are enriched in autism spectrum disorder, including CTG expansion in the DMPK gene that underlines myotonic muscular dystrophy type 1. Although the clinical connection of autism to myotonic dystrophy is corroborated, the molecular links remained unknown. Here, we show a mechanistic path of autism via repeat expansion in myotonic dystrophy. We found that inhibition of muscleblind-like (MBNL) splicing factors by expanded CUG RNAs alerts the splicing of autism-risk genes during brain development especially a class of autism-relevant microexons. To provide in vivo evidence that the CTG expansion and MBNL inhibition axis leads to the presentation of autistic traits, we demonstrate that CTG expansion and MBNL-null mouse models recapitulate autism-relevant mis-splicing profiles and demonstrate social deficits. Our findings indicate that DMPK CTG expansion-associated autism arises from developmental mis-splicing. Understanding this pathomechanistic connection provides an opportunity for greater in-depth investigations of mechanistic threads in autism.

Doctors' attitudes in the situation of delivering bad news: patients' experience and expectations.

Introduction: The purpose of our research was to find out patients' preferences concerning their doctors' attitudes and behaviour as they deliver bad news to them. Material and methods: In national research conducted from February to October 2017 using the computer-assisted web interview (CAWI) technique, we studied the statements of 314 adult patients who had received bad medical news from their doctors. Seventy-nine per cent of them were women and 21% were men. Fifty-nine per cent had higher education and 33% had secondary education. A specially designed closed question survey was used as a tool to collect the data. Results: Most of the patients (59.6%) expected a doctor-patient relationship based on partnership and collective decisions concerning further treatment. Patients wanted their doctors to be honest with them, to provide them with solid information and an opportunity to ask questions and discuss the suggested solutions. Less than 2 out of 10 patients expected "an empathy specialist". The patients who evaluated their doctors' behaviour and the way bad news was delivered to them negatively were more likely to change doctors or terminate their treatment. Conclusions: The doctor-patient relationship when an unfavourable diagnosis is being communicated is an important aspect, which defines the way people who participate in this difficult situation behave and communicate. Doctors' behaviour during DBN should meet the patients' expectations. Such an attitude guarantees trust towards doctors and results in more positive evaluations on them. Most importantly, it translates directly into the patients' therapeutic behaviours and treatment effects.

Delivering Bad News: Emotional Perspective and Coping Strategies of Medical Students.

Purpose: The aim of our study was to analyze students' emotional experiences and coping mechanisms regarding the situation of delivering bad news (DBN). Patients and Methods: 291 fifth- and sixth-year students from 14 medical universities took part in our study. Their responses were analyzed from the perspective of their experience - whether the individuals received bad news, delivered bad news themselves, witnessed bad news being delivered, or had no experience at all. We used content analysis (CA) to examine the responses and analyzed classified variables using χ2 tests, logistic regression, and predictive modeling with multiple correspondence analysis (MCA). Results: Students who had experience as a deliverer were more likely to experience failure and guilt (p=0.005). Predictions of anxiety (59.3%), stress (41.9%), and sadness (33.7%) were the highest in the students with no DBN experience. These students were most likely to make statements suggesting lack of methods and strategies for coping with difficult emotions. Students, who were immersed in DBN experiences, more often expressed the need to talk about their emotions with an experienced doctor, a family member or a friend, and used those conversations as a coping strategy. Conclusion: The most important aspects that influence the quality of delivering bad news are both communication skills and emotional context. Training regarding DBN should not only contain those two elements, but also consider the development of coping mechanisms.

A cohort study reveals different dynamics of SARS-CoV-2-specific antibody formation after Comirnaty and Vaxzevria vaccination.

The Coronavirus (COVID-19) Disease Pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected millions of people worldwide, prompting a collective effort from the global scientific community to develop a vaccine against it. This study purports to investigate the influence of factors such as sex, age, type of vaccination (Comirnaty, BNT162b2, Pfizer Inc. or Vaxzevria, ChAdOx1-S, Oxford/AstraZeneca), and time since vaccine administration on the process of antibody production. Both of them are based on the introduction of SARS-CoV-2 spike protein (S protein) to the body using different mechanisms (mRNA and recombinant adenovirus, respectively). S protein is responsible for host cell attachment and penetration via its receptor-binding domain (RBD domain). The level of anti-RBD IgG antibodies was tested with an ELISA-based immunodiagnostic assay in serum samples from a total of 1395 patients at 3 time points: before vaccination, after the first dose, and after the second dose. Our novel statistical model, the Generalized Additive Model, revealed variability in antibody production dynamics for both vaccines. Interestingly, no discernible variation in antibody levels between men and women was found. A nonlinear relationship between age and antibody production was observed, characterized by decreased antibody levels for people up to 30 and over 60 years of age, with a lack of correlation in the middle age range. Collectively, our findings further the understanding of the mechanism driving vaccine-induced immunity. Additionally, we propose the Generalized Additive Model as a standardized way of presenting data in similar research.

Applying Machine Learning to Construct a Model of Risk of Depression in Patients Following Cardiac Surgery with the Use of the SF-12 Survey.

BACKGROUND: Depression is a common problem in patients with cardiovascular diseases. Identifying a risk factor model of depression has been postulated. A model of the risk of depression would provide a better understanding of this disorder in this population. We sought to construct a model of the risk factors of depression in patients following cardiac surgery, with the use of machine learning. METHODS AND MEASURES: Two hundred and seventeen patients (65.4% men; mean age 65.14 years) were asked to complete the short form health survey-12 (SF-12v.2), three months after hospital discharge. Those at risk of depression were identified based on the SF-12 mental component summary (MCS). Centroid class principal component analysis (CCPCA) and the classification and regression tree (CART) were used to design a model. RESULTS: A risk of depression was identified in 29.03% of patients. The following variables explained 82.53% of the variance in depression risk: vitality, limitation of activities due to emotional problems (role-emotional, RE), New York Heart Association (NYHA) class, and heart failure. Additionally, CART revealed that decreased vitality increased the risk of depression to 45.44% and an RE score > 68.75 increased it to 63.11%. In the group with an RE score < 68.75, the NYHA class increased the risk to 41.85%, and heart failure further increased it to 44.75%. CONCLUSION: Assessing fatigue and vitality can help health professionals with identifying patients at risk of depression. In addition, assessing functional status and dimensions of fatigue, as well as the impact of emotional state on daily functioning, can help determine effective intervention options.

Medical or Common Knowledge? Knowledge of Medical Professionals on Obesity Diagnosis Criteria and Treatment.

INTRODUCTION: Proper diagnosis of obesity, its severity, and complications and their effective treatment requires an interdisciplinary healthcare approach. Nevertheless, obesity remains under-identified and undertreated. Academic knowledge concerning obesity pathology, diagnosis, and treatment is advancing. It is not clear whether this translates into clinical practice. The goal of the study was to assess the knowledge of healthcare professionals (HCPs) on obesity and particularly on the criteria for diagnosis as well as for conservative and surgical treatment. METHODS: This cross-sectional study was conducted among active HCPs (N = 184), including physicians, nurses, physiotherapists, and paramedics who had contact with adult patients with obesity. The proprietary research survey, implemented in an online tool, was used to assess knowledge on the diagnosis and treatment of obesity and self-assessment of that knowledge. The analysis was limited to the following: body mass index (BMI) definition, BMI values, visceral obesity definition, bariatric surgery indications, choice of treatment method, role of diet and physical activity, knowledge of obesity pharmacotherapy, length of obesity pharmacotherapy, financing of bariatric procedures, and goals of bariatric treatment. The correct answers were determined according to the Polish guidelines for the diagnosis and treatment of obesity. RESULTS: Half of the respondents (52.2%) were doctors, 20.7% were nurses and midwives, 19.0% were physiotherapists, and 8.2% were other medical professionals. Among questions related to knowledge on obesity, 67.1% of respondents provided correct answers, with respondents answering questions concerning obesity diagnosis correctly more frequently (70.1%) than those concerning methods of treatment (64.6%). The largest number of correct answers was related to the definition of BMI and normal BMI values. The smallest number of correct answers pertained to the diagnostic criteria for visceral obesity and pharmacological treatment of obesity. There was no statistically significant impact of a responder's knowledge levels on the obesity of different HCPs. Workplace and participation in training sessions were found to have the largest impact on the level of knowledge on obesity. HCPs' own assessment of their knowledge on obesity was negatively correlated with their actual level of knowledge. CONCLUSION: The prevalence of overweight and obesity implies that essentially every HCP has daily contact with patients with excessive body weight. Knowledge of BMI values cannot be considered as exclusively medical knowledge: these values were established years ago and are present in widely available sources. Our research showed that 32.9% of HCPs did not have sufficient knowledge about how to diagnose and treat obesity.

Fatigue, Pro-Social Attitude and Quality of Life as Predictors of Empathy in Medical and Social-Oriented Students.

Empathy is significant in professions that require establishing proper contact as a condition for providing help. Identifying factors related to empathy is important for understanding how to teach empathic behavior. The main goal of this study was to find variables related to empathy in a group of students from two universities: medical and social oriented (N = 1701). The study group consisted of female (81%) and male (19%) participants, aged between 18-20 (37%), 21-23 (49%), or 24 years and above (14%). A self-designed questionnaire was used to collect socio-demographical information, with additional questions (social self-esteem, prosocial attitude, subjective quality of life). Empathy was measured with the EQ-40, fatigue with CHFQ-PL, and stress with PSS-10. The results showed a statistically significant regression model for empathy. A high quality of life and having feelings of pleasure when helping other people allows to predict a high level of empathy, especially among females. Higher levels of fatigue and social self-esteem, the latter of which is measured here by the belief that you are more important than others, predicts lower empathy. There were no differences between students from two different kinds of universities when taking into consideration stress levels, subjective quality of life, and prosocial attitude. However, students from the medical university were more exhausted and more convinced that their value was greater than others, as compared to the students studying social sciences. When teaching empathic behavior, it is beneficial to attempt to maintain or restore students' well-being and reduce fatigue and to teach how to achieve such effects in the future. Learning the balance between compassion, willingness to help, and self-compassion also seems to be important.

Sustainable recovery of MBNL activity in autoregulatory feedback loop in myotonic dystrophy.

Muscleblind-like proteins (MBNLs) are RNA-binding proteins essential for the developmental regulation of various processes including alternative splicing. Their activity is misregulated in myotonic dystrophy type 1 (DM1), an incurable genetic, neuro-muscular disorder caused by uncontrolled expansion of CTG repeats. Mutant RNAs containing hundreds or thousands of repeats efficiently sequester MBNL proteins. As a consequence, global alternative splicing abnormalities are induced. Importantly, the size of expansion differs significantly not only between patients but also between different parts of the same muscle as a consequence of somatic expansion. One of the potential therapeutic strategies in DM is overexpression of MBNLs. However, gene therapy tools might induce excessive activity of MBNLs, what in turn might change the metabolism of many RNAs. To overcome these limitations, we designed an autoregulated MBNL1 overexpression system. The genetic construct contains an MBNL1-coding sequence separated by the fragment of ATP2A1 pre-mRNA with an MBNL-sensitive alternative exon containing stop codon in the reading frame of MBNL1. Inclusion of this exon leads to the arrangement of an inactive form of the protein, but exclusion gives rise to fully active MBNL1. This approach enables the autoregulation of the amount of overexpressed MBNL1 with high dynamic range which ensures a homogeneous level of this protein in cells treated with the genetic construct. We demonstrated beneficial effects of an autoregulated construct on alternative splicing patterns in DM1 models and cells derived from patients with DM1.

Influence of Personal Experiences of Medical Students on Their Assessment of Delivering Bad News.

BACKGROUND: We aimed to identify which attitudes and emotions accompany latter-year medical students as they experience situations where bad news is communicated. METHODS: A cross-sectional study was conducted using the computer-assisted web interview (CAWI) methodology in a group of 321 fifth- and sixth-year medical students from 14 medical universities in Poland. Correlations were analyzed using Pearson's χ2 test. For the categorical variables, subject profiles were analyzed using K-means clustering. RESULTS: Students' self-assessments of their competence in delivering bad news (DBN) differed depending on the type of experience they had with it. More than half of the students had observed a situation of DBN (63.6%) and as many as 26.5% of the participants had received bad news themselves. These two groups were less likely to declare a lack of DBN-related skills (43.4% and 33.4%, respectively) than others. In this study, 9% of the students had personally delivered bad news. Only 13.4% of these students rated their DBN skills as insufficient. They were also the least likely to express concern regarding high levels of stress (29.6%) and anxiety (48%). CONCLUSIONS: The ability to personally deliver bad medical news to a patient was the most effective form of gaining experience in DBN. Being a bearer of bad news may help students develop their own strategies for coping with difficult emotions and develop their professional competences, leading to improved medical care and patient comfort.

Comparing Students of Medical and Social Sciences in Terms of Self-Assessment of Perceived Stress, Quality of Life, and Personal Characteristics.

The aim of this study was to compare medical and social sciences students' outcomes in terms of self-perceived stress, quality of life, and personality traits. We put particular emphasis on external and internal differences in students of specific fields-medicine, nursing, psychology, and pedagogy. In a survey, 1,783 students from Medical University of Gdansk and University of Gdansk participated in our study, of whom 1,223 were included in the final statistical analysis. All of them were evaluated using valid and reliable questionnaires-TIPI-PL, PSS-10, and a one-item scale of quality of life. Stress turned out to have a negative effect on quality of life, regardless of the type of field of study. Moreover, students from different fields varied in terms of personality factors: conscientiousness, agreeableness, openness to experience, and emotional stability. In conclusion, many students regardless of their field suffer from high stress and report low quality of life, which potentially further affects their academic performance and social life.

The "CONNECT" Protocol: Delivering Bad News by Phone or Video Call.

The COVID-19 pandemic has changed the principles of communication within the health-care setting. Communication skills as developed for use in the context of a direct and personal encounter have become insufficient. As a result, numerous health professionals came to believe they were not sufficiently prepared to deliver bad news in relation to medical care. The CONNECT protocol is a tool designed to help health-care professionals in the delivery of such messages. The name of the protocol is an acronym derived from C - context, O - organization, NN - near and niceties, E - emotions, C - counseling, T - taking care. The objective of the protocol is to improve the effectiveness of the delivery of bad news in relation to medical care by ensuring proper organization of the key elements of the encounter with the patient and/or their family.

Partners in crime: Proteins implicated in RNA repeat expansion diseases.

Short tandem repeats are repetitive nucleotide sequences robustly distributed in the human genome. Their expansion underlies the pathogenesis of multiple neurological disorders, including Huntington's disease, amyotrophic lateral sclerosis, and frontotemporal dementia, fragile X-associated tremor/ataxia syndrome, and myotonic dystrophies, known as repeat expansion disorders (REDs). Several molecular pathomechanisms associated with toxic RNA containing expanded repeats (RNAexp ) are shared among REDs and contribute to disease progression, however, detailed mechanistic insight into those processes is limited. To deepen our understanding of the interplay between toxic RNAexp molecules and multiple protein partners, in this review, we discuss the roles of selected RNA-binding proteins (RBPs) that interact with RNAexp and thus act as "partners in crime" in the progression of REDs. We gather current findings concerning RBPs involved at different stages of the RNAexp life cycle, such as transcription, splicing, transport, and AUG-independent translation of expanded repeats. We argue that the activity of selected RBPs can be unique or common among REDs depending on the expanded repeat type. We also present proteins that are functionally depleted due to sequestration on RNAexp within nuclear foci and those which participate in RNAexp -dependent innate immunity activation. Moreover, we discuss the utility of selected RBPs as targets in the development of therapeutic strategies. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA in Disease and Development > RNA in Disease.

Delivering Bad News: Self-Assessment and Educational Preferences of Medical Students.

BACKGROUND: Numerous reports indicate the educational deficiencies of medical students in delivering bad-news-related skills. Evaluation of the performance of training programs in this area should be one of the key components of the educational process. The purpose of this study was to analyze medical students' preferences and educational needs regarding DBN (delivering bad news). The effect of clinical experience on the self-assessment of skills was analyzed. METHODS: The quantitative survey was conducted using the CAWI technique. The study involved 321 fifth- and sixth-year medical students from 14 medical universities in Poland. Pearson's χ2 test was used for statistical analysis. The profile of respondents for categorical variables was determined by KMeans analysis. RESULTS: As many as 75.1% of students revealed that they did not feel sufficiently prepared for DBN. Only 18.7% reported having adequate competence in this area. More than half of the inquired students (63.6%) witnessed a situation during their clinical practice in which a physician provided a patient with information about an unfavorable diagnosis. These students were less likely to declare that they could not deliver BN (43.4%) than students who had no such experience (58.2%). As many as 86.3% of the respondents reported the need for more time in DBN skills training. Students mostly preferred active teaching methods. CONCLUSIONS: Understanding students' learning needs and preferences can help medical schools optimize their education programs to develop DBN-related competencies.

Distribution of alternative untranslated regions within the mRNA of the CELF1 splicing factor affects its expression.

CUG-binding protein, ELAV-like Family Member 1 (CELF1) plays an important role during the development of different tissues, such as striated muscle and brain tissue. CELF1 is an RNA-binding protein that regulates RNA metabolism processes, e.g., alternative splicing, and antagonizes other RNA-binding proteins, such as Muscleblind-like proteins (MBNLs). Abnormal activity of both classes of proteins plays a crucial role in the pathogenesis of myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults. In this work, we show that alternative splicing of exons forming both the 5' and 3' untranslated regions (UTRs) of CELF1 mRNA is efficiently regulated during development and tissue differentiation and is disrupted in skeletal muscles in the context of DM1. Alternative splicing of the CELF1 5'UTR leads to translation of two potential protein isoforms that differ in the lengths of their N-terminal domains. We also show that the MBNL and CELF proteins regulate the distribution of mRNA splicing isoforms with different 5'UTRs and 3'UTRs and affect the CELF1 expression by changing its sensitivity to specific microRNAs or RNA-binding proteins. Together, our findings show the existence of different mechanisms of regulation of CELF1 expression through the distribution of various 5' and 3' UTR isoforms within CELF1 mRNA.

Attitudes of Medical Professionals Towards Discrimination of Patients with Obesity.

PURPOSE: Many studies show that discriminatory practices are common in healthcare institutions, also in the form of medical staff's inappropriate behaviour. Weight stigma may not only become a source of unpleasant experience for the patients but also cause them to withdraw from the treatment, which may further exacerbate their condition. PATIENTS AND METHODS: In a nationwide study, we asked 184 medical professionals, about their experiences and opinions on the discrimination of patients with obesity. The study was conducted in 2020, with the use of CAWI method, based on an original survey created for this study. RESULTS: Most of the medical professionals (68.5%) estimated that the problem of worse attitudes towards patients suffering from obesity is a common phenomenon. About 48.4% witnessed medical staff's discriminatory behaviours. The most frequent forms of inappropriate behaviours pertained to interpersonal relations - mocking the appearance (96.6%), looks of disgust and repulsion (96.2%), lack of reaction to offensive remarks (92.0%) or scaring a patient with the necessity to lose weight (57.7%). The participants of the study pointed to limited access to dedicated medical equipment (62.4%) as a discriminatory systemic limitation. CONCLUSION: Discrimination of patients with obesity is a social issue, which also occurs within the health care system. Limited access to medical resources, gaps in knowledge of obesity and an insufficient level of soft skills in health care professionals are the key deficiencies, which hinder effective treatment.

Intensity, dynamics and deficiencies of empathy in medical and non-medical students.

BACKGROUND: Empathy is an important competence in the professional development of medical students. The purpose of our study was to compare the levels and scales of empathy in people studying in different educational strategies. METHODS: The study was conducted between April 2019 and March 2020. Medicine, nursing, midwifery, physiotherapy, psychology, pedagogy and sociology students were the participants of this study. University students preparing for medical professions (n = 1001) and students of programs unrelated to medicine (n = 700) underwent the Empathy Quotient test (EQ-40). We have compared results in both study groups with the use of the distribution of density, analysis of variance and student's t-test. RESULTS: The average results received by students of the university preparing for medical professions were lower (M = 42.6) than those of the non-medical university students (M = 45.3) and the differences between the universities turned out to be statistically important (t = - 5.15, df = 1699, p < 0.001). As many as 14.6% of the students in the 1st EQ class were preparing for various medical professions while 9% studied social sciences. 18.2% of all medical programme students (n = 412) manifested the lowest empathy class. Our research has revealed that the students with Asperger profile (AP) and high-functioning autism (HFA) studied at universities preparing for medical professions (n = 18) more frequently than at non-medical universities (n = 5). CONCLUSIONS: We have noticed a serious indicator of erosion in the levels of empathy in medical students and an increase in the number of people with AP and HFA. Empathy decreases in students after the third year of their studies, regardless of the kind of university. We recommend an introduction of career counselling when specialization is being chosen.

Cyclic mismatch binding ligands interact with disease-associated CGG trinucleotide repeats in RNA and suppress their translation.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by a limited expansion of CGG repeats in the FMR1 gene. Degeneration of neurons in FXTAS cell models can be triggered by accumulation of polyglycine protein (FMRpolyG), a by-product of translation initiated upstream to the repeats. Specific aims of our work included testing if naphthyridine-based molecules could (i) block FMRpolyG synthesis by binding to CGG repeats in RNA, (ii) reverse pathological alterations in affected cells and (iii) preserve the content of FMRP, translated from the same FMR1 mRNA. We demonstrate that cyclic mismatch binding ligand CMBL4c binds to RNA structure formed by CGG repeats and attenuates translation of FMRpolyG and formation of nuclear inclusions in cells transfected with vectors expressing RNA with expanded CGG repeats. Moreover, our results indicate that CMBL4c delivery can reduce FMRpolyG-mediated cytotoxicity and apoptosis. Importantly, its therapeutic potential is also observed once the inclusions are already formed. We also show that CMBL4c-driven FMRpolyG loss is accompanied by partial FMRP reduction. As complete loss of FMRP induces FXS in children, future experiments should aim at evaluation of CMBL4c therapeutic intervention in differentiated tissues, in which FMRpolyG translation inhibition might outweigh adverse effects related to FMRP depletion.

Short antisense oligonucleotides alleviate the pleiotropic toxicity of RNA harboring expanded CGG repeats.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an incurable neurodegenerative disorder caused by expansion of CGG repeats in the FMR1 5'UTR. The RNA containing expanded CGG repeats (rCGGexp) causes cell damage by interaction with complementary DNA, forming R-loop structures, sequestration of nuclear proteins involved in RNA metabolism and initiation of translation of polyglycine-containing protein (FMRpolyG), which forms nuclear insoluble inclusions. Here we show the therapeutic potential of short antisense oligonucleotide steric blockers (ASOs) targeting directly the rCGGexp. In nuclei of FXTAS cells ASOs affect R-loop formation and correct miRNA biogenesis and alternative splicing, indicating that nuclear proteins are released from toxic sequestration. In cytoplasm, ASOs significantly decrease the biosynthesis and accumulation of FMRpolyG. Delivery of ASO into a brain of FXTAS mouse model reduces formation of inclusions, improves motor behavior and corrects gene expression profile with marginal signs of toxicity after a few weeks from a treatment.

MEF2C shapes the microtranscriptome during differentiation of skeletal muscles.

Myocyte enhancer factor 2C (MEF2C) is a transcription factor that regulates heart and skeletal muscle differentiation and growth. Several protein-encoding genes were identified as targets of this factor; however, little is known about its contribution to the microtranscriptome composition and dynamics in myogenic programs. In this report, we aimed to address this question. Deep sequencing of small RNAs of human muscle cells revealed a set of microRNAs (miRNAs), including several muscle-specific miRNAs, that are sensitive to MEF2C depletion. As expected, in cells with knockdown of MEF2C, we found mostly downregulated miRNAs; nevertheless, as much as one-third of altered miRNAs were upregulated. The majority of these changes are driven by transcription efficiency. Moreover, we found that MEF2C affects nontemplated 3'-end nucleotide addition of miRNAs, mainly oligouridylation. The rate of these modifications is associated with the level of TUT4 which mediates RNA 3'-uridylation. Finally, we found that a quarter of miRNAs which significantly changed upon differentiation of human skeletal myoblasts is inversely altered in MEF2C deficient cells. We concluded that MEF2C is an essential factor regulating both the quantity and quality of the microtranscriptome, leaving an imprint on the stability and perhaps specificity of many miRNAs during the differentiation of muscle cells.