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.

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.

Muscleblind-like 2 controls the hypoxia response of cancer cells.

Hypoxia is a hallmark of solid cancers, supporting proliferation, angiogenesis, and escape from apoptosis. There is still limited understanding of how cancer cells adapt to hypoxic conditions and survive. We analyzed transcriptome changes of human lung and breast cancer cells under chronic hypoxia. Hypoxia induced highly concordant changes in transcript abundance, but divergent splicing responses, underlining the cell type-specificity of alternative splicing programs. While RNA-binding proteins were predominantly reduced, hypoxia specifically induced muscleblind-like protein 2 (MBNL2). Strikingly, MBNL2 induction was critical for hypoxia adaptation by controlling the transcript abundance of hypoxia response genes, such as vascular endothelial growth factor A (VEGFA) MBNL2 depletion reduced the proliferation and migration of cancer cells, demonstrating an important role of MBNL2 as cancer driver. Hypoxia control is specific for MBNL2 and not shared by its paralog MBNL1. Thus, our study revealed MBNL2 as central mediator of cancer cell responses to hypoxia, regulating the expression and alternative splicing of hypoxia-induced genes.

Loss of MBNL leads to disruption of developmentally regulated alternative polyadenylation in RNA-mediated disease.

Inhibition of muscleblind-like (MBNL) activity due to sequestration by microsatellite expansion RNAs is a major pathogenic event in the RNA-mediated disease myotonic dystrophy (DM). Although MBNL1 and MBNL2 bind to nascent transcripts to regulate alternative splicing during muscle and brain development, another major binding site for the MBNL protein family is the 3' untranslated region of target RNAs. Here, we report that depletion of Mbnl proteins in mouse embryo fibroblasts leads to misregulation of thousands of alternative polyadenylation events. HITS-CLIP and minigene reporter analyses indicate that these polyadenylation switches are a direct consequence of MBNL binding to target RNAs. Misregulated alternative polyadenylation also occurs in skeletal muscle in a mouse polyCUG model and human DM, resulting in the persistence of neonatal polyadenylation patterns. These findings reveal an additional developmental function for MBNL proteins and demonstrate that DM is characterized by misregulation of pre-mRNA processing at multiple levels.

AON-induced splice-switching and DMPK pre-mRNA degradation as potential therapeutic approaches for Myotonic Dystrophy type 1.

Expansion of an unstable CTG repeat in the 3'UTR of the DMPK gene causes Myotonic Dystrophy type 1 (DM1). CUG-expanded DMPK transcripts (CUGexp) sequester Muscleblind-like (MBNL) alternative splicing regulators in ribonuclear inclusions (foci), leading to abnormalities in RNA processing and splicing. To alleviate the burden of CUGexp, we tested therapeutic approach utilizing antisense oligonucleotides (AONs)-mediated DMPK splice-switching and degradation of mutated pre-mRNA. Experimental design involved: (i) skipping of selected constitutive exons to induce frameshifting and decay of toxic mRNAs by an RNA surveillance mechanism, and (ii) exclusion of the alternative exon 15 (e15) carrying CUGexp from DMPK mRNA. While first strategy failed to stimulate DMPK mRNA decay, exclusion of e15 enhanced DMPK nuclear export but triggered accumulation of potentially harmful spliced out pre-mRNA fragment containing CUGexp. Neutralization of this fragment with antisense gapmers complementary to intronic sequences preceding e15 failed to diminish DM1-specific spliceopathy due to AONs' chemistry-related toxicity. However, intronic gapmers alone reduced the level of DMPK mRNA and mitigated DM1-related cellular phenotypes including spliceopathy and nuclear foci. Thus, a combination of the correct chemistry and experimental approach should be carefully considered to design a safe AON-based therapeutic strategy for DM1.

Intron retention induced by microsatellite expansions as a disease biomarker.

Expansions of simple sequence repeats, or microsatellites, have been linked to ∼30 neurological-neuromuscular diseases. While these expansions occur in coding and noncoding regions, microsatellite sequence and repeat length diversity is more prominent in introns with eight different trinucleotide to hexanucleotide repeats, causing hereditary diseases such as myotonic dystrophy type 2 (DM2), Fuchs endothelial corneal dystrophy (FECD), and C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Here, we test the hypothesis that these GC-rich intronic microsatellite expansions selectively trigger host intron retention (IR). Using DM2, FECD, and C9-ALS/FTD as examples, we demonstrate that retention is readily detectable in affected tissues and peripheral blood lymphocytes and conclude that IR screening constitutes a rapid and inexpensive biomarker for intronic repeat expansion disease.

Unfavorable Medical Diagnosis - Oncological Patients' Experiences and Preferences.

The study aims at analyzing the experiences of Polish patients who received an unfavorable medical diagnosis. In order to reach a random group of patients with an unfavorable diagnosis, we started cooperating with 19 independent patient organizations and associations. A nationwide study, based on a self-constructed e-survey questionnaire, was carried out with the use of the CAWI method. Three hundred and fourteen patients in total participated in the study, all of whom had received an unfavorable diagnosis, including 119 oncological patients and 195 suffering from other chronic diseases. This paper focuses on the analysis of the opinions expressed by oncological patients. It also highlights the differences in oncological and non-oncological patients' reactions to an unfavorable diagnosis. Caution and tactfulness displayed by a physician while conveying an unfavorable diagnosis were assessed negatively by 36% of the respondents, 8 in 10 oncological patients claim that Polish doctors are insufficiently prepared to communicate bad news. As far as the model of the physician-patient relationship is concerned, over half of the surveyed (59%) prefers partnership, 18% opts for an informative model, 16% chooses a model based on dialogue and empathy and 7% is in favor of a paternalistic model. We have demonstrated that the very location where diagnosis is conveyed affects the quality of medical communication: patients visiting a private doctor's office have a better chance to express their doubts than the ones treated at a hospital or a public outpatient clinic. The majority of oncological patients, especially individuals under the age of 40, prefer shared decision-making.

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.

MBNL splicing activity depends on RNA binding site structural context.

Muscleblind-like (MBNL) proteins are conserved RNA-binding factors involved in alternative splicing (AS) regulation during development. While AS is controlled by distribution of MBNL paralogs and isoforms, the affinity of these proteins for specific RNA-binding regions and their location within transcripts, it is currently unclear how RNA structure impacts MBNL-mediated AS regulation. Here, we defined the RNA structural determinants affecting MBNL-dependent AS activity using both cellular and biochemical assays. While enhanced inclusion of MBNL-regulated alternative exons is controlled by the arrangement and number of MBNL binding sites within unstructured RNA, when these sites are embedded in a RNA hairpin MBNL binds preferentially to one side of stem region. Surprisingly, binding of MBNL proteins to RNA targets did not entirely correlate with AS efficiency. Moreover, comparison of MBNL proteins revealed structure-dependent competitive behavior between the paralogs. Our results showed that the structure of targeted RNAs is a prevalent component of the mechanism of alternative splicing regulation by MBNLs.

Intrinsic Regulatory Role of RNA Structural Arrangement in Alternative Splicing Control.

Alternative splicing is a highly sophisticated process, playing a significant role in posttranscriptional gene expression and underlying the diversity and complexity of organisms. Its regulation is multilayered, including an intrinsic role of RNA structural arrangement which undergoes time- and tissue-specific alterations. In this review, we describe the principles of RNA structural arrangement and briefly decipher its cis- and trans-acting cellular modulators which serve as crucial determinants of biological functionality of the RNA structure. Subsequently, we engage in a discussion about the RNA structure-mediated mechanisms of alternative splicing regulation. On one hand, the impairment of formation of optimal RNA structures may have critical consequences for the splicing outcome and further contribute to understanding the pathomechanism of severe disorders. On the other hand, the structural aspects of RNA became significant features taken into consideration in the endeavor of finding potential therapeutic treatments. Both aspects have been addressed by us emphasizing the importance of ongoing studies in both fields.

Application of ion pair chromatography coupled with mass spectrometry to assess antisense oligonucleotides concentrations in living cells.

Antisense oligonucleotides (ASOs) are synthetic bioactive compounds used as therapeutic agents in clinical trials. They act by binding to complementary sequences of the targeted nucleic acids in cells. Assessing the efficiency of ASO delivery to cells or tissues and the stability of these compounds in different biological systems is important. To answer these questions, we developed a new, quick and reliable method to determine the concentrations of different types of ASOs in treated cells. Ultra-high performance liquid chromatography coupled with mass spectrometry was used for the first time for the separation and determination of the studied compounds in total RNA extracts. To develop a method with the highest possible sensitivity, a central composite design was used to comprehensively optimize the MS parameters. Moreover, the effects of the type and concentration of the ion pair reagent on sensitivity were also examined. Finally, a mobile phase containing methanol, hexafluoroisopropanol and N,N-dimethylbutylamine was selected. The optimized method allowed good linearity, accuracy, precision and sensitivity of ASO detection. Next, these compounds were delivered into cells via transfection at a concentration of 25 nM or 125 nM in 1 mL of cell culture medium. After 48 hours, total RNA was isolated from the treated cells and analyzed with the use of the newly developed method. For the cells treated with a higher concentration of ASO composed of phosphorothioate 2'-O-methyl RNA units, the concentration in solution was 0.96 ± 0.06 µM, while in the case of shorter ASO composed of locked nucleic acid units, it was 0.72 ± 0.06 µM in the total RNA extract.

Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript.

Muscleblind-like proteins (MBNLs) are regulators of RNA metabolism. During tissue differentiation the level of MBNLs increases, while their functional insufficiency plays a crucial role in myotonic dystrophy (DM). Deep sequencing of RNA molecules cross-linked to immunoprecipitated protein particles (CLIP-seq) revealed that MBNL1 binds to MBNL1 exon 1 (e1) encoding both the major part of 5΄UTR and an amino-terminal region of MBNL1 protein. We tested several hypotheses regarding the possible autoregulatory function of MBNL1 binding to its own transcript. Our data indicate that MBNLs induce skipping of e1 from precursor MBNL1 mRNA and that e1 exclusion may impact transcript association with polysomes and translation. Furthermore, e1-deficient protein isoform lacking the first two zinc fingers is highly unstable and its EGFP fusion protein has severely compromised splicing activity. We also show that MBNL1 can be transcribed from three different promoters and that the transcription initiation site determines the mode of e1 regulation. Taken together, we demonstrate that MBNL proteins control steady-state levels of MBNL1 through an interaction with e1 in its precursor mRNA. Insights from our study open a new avenue in therapies against DM based on manipulation of the transcription initiation site and e1 splicing of MBNL1 mRNA.

Design of a "Mini" Nucleic Acid Probe for Cooperative Binding of an RNA-Repeated Transcript Associated with Myotonic Dystrophy Type 1.

Toxic RNAs containing expanded trinucleotide repeats are the cause of many neuromuscular disorders, one being myotonic dystrophy type 1 (DM1). DM1 is triggered by CTG-repeat expansion in the 3'-untranslated region of the DMPK gene, resulting in a toxic gain of RNA function through sequestration of MBNL1 protein, among others. Herein, we report the development of a relatively short miniPEG-γ peptide nucleic acid probe, two triplet repeats in length, containing terminal pyrene moieties, that is capable of binding rCUG repeats in a sequence-specific and selective manner. The newly designed probe can discriminate the pathogenic rCUGexp from the wild-type transcript and disrupt the rCUGexp-MBNL1 complex. The work provides a proof of concept for the development of relatively short nucleic acid probes for targeting RNA-repeat expansions associated with DM1 and other related neuromuscular disorders.

A Dimeric 2,9-Diamino-1,10-phenanthroline Derivative Improves Alternative Splicing in Myotonic Dystrophy Type†1 Cell and Mouse Models.

Expanded r(CUG) repeats are the cause of the neurological disorder myotonic dystrophy type 1 (DM1). The pathological features of DM1 include the formation of ribonuclear foci containing expanded r(CUG) repeats, which sequester the MBNL1 protein and lead to the misregulation of alternative pre-mRNA splicing. Small molecules that bind to the r(CUG) repeats and improve alternative splicing have therapeutic potential in the treatment of DM1. Herein, the synthesis of DDAP (a dimeric form of the CUG-binding molecule DAP reported previously), its binding properties to r(CUG) repeats, and its effect on the misregulation of splicing are reported. The surface plasmon resonance assay, circular dichroism spectra, and ESI-TOF mass spectrometry results confirmed the binding of DDAP to r(CUG)9 repeats. Studies on a DM1 cell model and a DM1 mouse model revealed that DDAP was partially effective in the recovery of the pre-mRNA splicing defects. The mechanism underlying this recovery was studied in vitro through a competitive binding assay, and suggested that DDAP could interfere with the binding of MBNL1 to r(CUG) repeats in a concentration-dependent manner.

MBNL expression in autoregulatory feedback loops.

Muscleblind-like (MBNL) proteins bind to hundreds of pre- and mature mRNAs to regulate their alternative splicing, alternative polyadenylation, stability and subcellular localization. Once MBNLs are withheld from transcript regulation, cellular machineries generate products inapt for precise embryonal/adult developmental tasks and myotonic dystrophy, a devastating multi-systemic genetic disorder, develops. We have recently demonstrated that all three MBNL paralogs are capable of fine-tuning cellular content of one of the three MBNL paralogs, MBNL1, by binding to the first coding exon (e1) of its pre-mRNA. Intriguingly, this autoregulatory feedback loop grounded on alternative splicing of e1 appears to play a crucial role in delaying the onset of myotonic dystrophy. Here, we describe this process in the context of other autoregulatory and regulatory loops that maintain the content and diverse functions of MBNL proteins at optimal level in health and disease, thus supporting the overall cellular homeostasis.

Mechanistic determinants of MBNL activity.

Muscleblind-like (MBNL) proteins are critical RNA processing factors in development. MBNL activity is disrupted in the neuromuscular disease myotonic dystrophy type 1 (DM1), due to the instability of a non-coding microsatellite in the DMPK gene and the expression of CUG expansion (CUGexp) RNAs. Pathogenic interactions between MBNL and CUGexp RNA lead to the formation of nuclear complexes termed foci and prevent MBNL function in pre-mRNA processing. The existence of multiple MBNL genes, as well as multiple protein isoforms, raises the question of whether different MBNL proteins possess unique or redundant functions. To address this question, we coexpressed three MBNL paralogs in cells at equivalent levels and characterized both specific and redundant roles of these proteins in alternative splicing and RNA foci dynamics. When coexpressed in the same cells, MBNL1, MBNL2 and MBNL3 bind the same RNA motifs with different affinities. While MBNL1 demonstrated the highest splicing activity, MBNL3 showed the lowest. When forming RNA foci, MBNL1 is the most mobile paralog, while MBNL3 is rather static and the most densely packed on CUGexp RNA. Therefore, our results demonstrate that MBNL paralogs and gene-specific isoforms possess inherent functional differences, an outcome that could be enlisted to improve therapeutic strategies for DM1.

Short antisense-locked nucleic acids (all-LNAs) correct alternative splicing abnormalities in myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by expansion of CTG triplet repeats in 3'-untranslated region of DMPK gene. The pathomechanism of DM1 is driven by accumulation of toxic transcripts containing expanded CUG repeats (CUG(exp)) in nuclear foci which sequester several factors regulating RNA metabolism, such as Muscleblind-like proteins (MBNLs). In this work, we utilized very short chemically modified antisense oligonucleotides composed exclusively of locked nucleic acids (all-LNAs) complementary to CUG repeats, as potential therapeutic agents against DM1. Our in vitro data demonstrated that very short, 8- or 10-unit all-LNAs effectively bound the CUG repeat RNA and prevented the formation of CUG(exp)/MBNL complexes. In proliferating DM1 cells as well as in skeletal muscles of DM1 mouse model the all-LNAs induced the reduction of the number and size of CUG(exp) foci and corrected MBNL-sensitive alternative splicing defects with high efficacy and specificity. The all-LNAs had low impact on the cellular level of CUG(exp)-containing transcripts and did not affect the expression of other transcripts with short CUG repeats. Our data strongly indicate that short all-LNAs complementary to CUG repeats are a promising therapeutic tool against DM1.

Patients' Expectations as to Doctors' Behaviors During Appointed Visits.

Numerous guidelines for students and medical professionals provide the instructions of proper behavior during encounters with patients in a doctor's office. However, they quite often do not consider cultural differences that may affect the doctor-patient relationship. In our study we analyzed Polish patients' expectations (N = 976) for their physicians' actual behavior. We compared our results with analogue studies performed in the United States. We determined that patient expectations concerning a desirable form of verbal and nonverbal communication with a physician vary to a considerable degree. Relatively universal, however, is the wish that the doctors introduce themselves and apply personalized forms of contact.

MBNL proteins and their target RNAs, interaction and splicing regulation.

Muscleblind-like (MBNL) proteins are key regulators of precursor and mature mRNA metabolism in mammals. Based on published and novel data, we explore models of tissue-specific MBNL interaction with RNA. We portray MBNL domains critical for RNA binding and splicing regulation, and the structure of MBNL's normal and pathogenic RNA targets, particularly in the context of myotonic dystrophy (DM), in which expanded CUG or CCUG repeat transcripts sequester several nuclear proteins including MBNLs. We also review the properties of MBNL/RNA complex, including recent data obtained from UV cross-linking and immunoprecipitation (CLIP-Seq), and discuss how this interaction shapes normal MBNL-dependent alternative splicing regulation. Finally, we review how this acquired knowledge about the pathogenic RNA structure and nature of MBNL sequestration can be translated into the design of therapeutic strategies against DM.

Lomofungin and dilomofungin: inhibitors of MBNL1-CUG RNA binding with distinct cellular effects.

Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disorder resulting from expression of RNA containing an expanded CUG repeat (CUG(exp)). The pathogenic RNA is retained in nuclear foci. Poly-(CUG) binding proteins in the Muscleblind-like (MBNL) family are sequestered in foci, causing misregulated alternative splicing of specific pre-mRNAs. Inhibitors of MBNL1-CUG(exp) binding have been shown to restore splicing regulation and correct phenotypes in DM1 models. We therefore conducted a high-throughput screen to identify novel inhibitors of MBNL1-(CUG)12 binding. The most active compound was lomofungin, a natural antimicrobial agent. We found that lomofungin undergoes spontaneous dimerization in DMSO, producing dilomofungin, whose inhibition of MBNL1-(CUG)12 binding was 17-fold more potent than lomofungin itself. However, while dilomofungin displayed the desired binding characteristics in vitro, when applied to cells it produced a large increase of CUG(exp) RNA in nuclear foci, owing to reduced turnover of the CUG(exp) transcript. By comparison, the monomer did not induce CUG(exp) accumulation in cells and was more effective at rescuing a CUG(exp)-induced splicing defect. These results support the feasibility of high-throughput screens to identify compounds targeting toxic RNA, but also demonstrate that ligands for repetitive sequences may have unexpected effects on RNA decay.