Widespread alternative splicing dysregulation occurs presymptomatically in CAG expansion spinocerebellar ataxias.

The spinocerebellar ataxias (SCAs) are a group of dominantly inherited neurodegenerative diseases, several of which are caused by CAG expansion mutations (SCAs 1, 2, 3, 6, 7 and 12) and more broadly belong to the large family of over 40 microsatellite expansion diseases. While dysregulation of alternative splicing is a well defined driver of disease pathogenesis across several microsatellite diseases, the contribution of alternative splicing in CAG expansion SCAs is poorly understood. Furthermore, despite extensive studies on differential gene expression, there remains a gap in our understanding of presymptomatic transcriptomic drivers of disease. We sought to address these knowledge gaps through a comprehensive study of 29 publicly available RNA-sequencing datasets. We identified that dysregulation of alternative splicing is widespread across CAG expansion mouse models of SCAs 1, 3 and 7. These changes were detected presymptomatically, persisted throughout disease progression, were repeat length-dependent, and were present in brain regions implicated in SCA pathogenesis including the cerebellum, pons and medulla. Across disease progression, changes in alternative splicing occurred in genes that function in pathways and processes known to be impaired in SCAs, such as ion channels, synaptic signalling, transcriptional regulation and the cytoskeleton. We validated several key alternative splicing events with known functional consequences, including Trpc3 exon 9 and Kcnma1 exon 23b, in the Atxn1154Q/2Q mouse model. Finally, we demonstrated that alternative splicing dysregulation is responsive to therapeutic intervention in CAG expansion SCAs with Atxn1 targeting antisense oligonucleotide rescuing key splicing events. Taken together, these data demonstrate that widespread presymptomatic dysregulation of alternative splicing in CAG expansion SCAs may contribute to disease onset, early neuronal dysfunction and may represent novel biomarkers across this devastating group of neurodegenerative disorders.

Disease-associated inosine misincorporation into RNA hinders translation.

Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis.

Repeat length increases disease penetrance and severity in C9orf72 ALS/FTD BAC transgenic mice.

C9orf72 ALS/FTD patients show remarkable clinical heterogeneity, but the complex biology of the repeat expansion mutation has limited our understanding of the disease. BAC transgenic mice were used to better understand the molecular mechanisms and repeat length effects of C9orf72 ALS/FTD. Genetic analyses of these mice demonstrate that the BAC transgene and not integration site effects cause ALS/FTD phenotypes. Transcriptomic changes in cell proliferation, inflammation and neuronal pathways are found late in disease and alternative splicing changes provide early molecular markers that worsen with disease progression. Isogenic sublines of mice with 800, 500 or 50 G4C2 repeats generated from the single-copy C9-500 line show longer repeats result in earlier onset, increased disease penetrance and increased levels of RNA foci and dipeptide RAN protein aggregates. These data demonstrate G4C2 repeat length is an important driver of disease and identify alternative splicing changes as early biomarkers of C9orf72 ALS/FTD.

RNA structure probing to characterize RNA-protein interactions on a low abundance pre-mRNA in living cells.

In vivo RNA structure analysis has become a powerful tool in molecular biology, largely due to the coupling of an increasingly diverse set of chemical approaches with high-throughput sequencing. This has resulted in a transition from single target to transcriptome-wide approaches. However, these methods require sequencing depths that preclude studying low abundance targets, which are not sufficiently captured in transcriptome-wide approaches. Here we present a ligation-free method to enrich for low abundance RNA sequences, which improves the diversity of molecules analyzed and results in improved analysis. In addition, this method is compatible with any choice of chemical adduct or read-out approach. We utilized this approach to study an autoregulated event in the pre-mRNA of the splicing factor, muscleblind-like splicing regulator 1 (MBNL1).

A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels.

A CTG repeat expansion in the DMPK gene is the causative mutation of myotonic dystrophy type 1 (DM1). Transcription of the expanded CTG repeat produces toxic gain-of-function CUG RNA, leading to disease symptoms. A screening platform that targets production or stability of the toxic CUG RNA in a selective manner has the potential to provide new biological and therapeutic insights. A DM1 HeLa cell model was generated that stably expresses a toxic r(CUG)480 and an analogous r(CUG)0 control from DMPK and was used to measure the ratio-metric level of r(CUG)480 versus r(CUG)0. This DM1 HeLa model recapitulates pathogenic hallmarks of DM1, including CUG ribonuclear foci and missplicing of pre-mRNA targets of the muscleblind (MBNL) alternative splicing factors. Repeat-selective screening using this cell line led to the unexpected identification of multiple microtubule inhibitors as hits that selectively reduce r(CUG)480 levels and partially rescue MBNL-dependent missplicing. These results were validated by using the Food and Drug Administration-approved clinical microtubule inhibitor colchicine in DM1 mouse and primary patient cell models. The mechanism of action was found to involve selective reduced transcription of the CTG expansion that we hypothesize to involve the LINC (linker of nucleoskeleton and cytoskeleton) complex. The unanticipated identification of microtubule inhibitors as selective modulators of toxic CUG RNA opens research directions for this form of muscular dystrophy and may shed light on the biology of CTG repeat expansion and inform therapeutic avenues. This approach has the potential to identify modulators of expanded repeat-containing gene expression for over 30 microsatellite expansion disorders.

Transcriptome alterations in myotonic dystrophy skeletal muscle and heart.

Myotonic dystrophy (dystrophia myotonica, DM) is a multi-systemic disease caused by expanded CTG or CCTG microsatellite repeats. Characterized by symptoms in muscle, heart and central nervous system, among others, it is one of the most variable diseases known. A major pathogenic event in DM is the sequestration of muscleblind-like proteins by CUG or CCUG repeat-containing RNAs transcribed from expanded repeats, and differences in the extent of MBNL sequestration dependent on repeat length and expression level may account for some portion of the variability. However, many other cellular pathways are reported to be perturbed in DM, and the severity of specific disease symptoms varies among individuals. To help understand this variability and facilitate research into DM, we generated 120 RNASeq transcriptomes from skeletal and heart muscle derived from healthy and DM1 biopsies and autopsies. A limited number of DM2 and Duchenne muscular dystrophy samples were also sequenced. We analyzed splicing and gene expression, identified tissue-specific changes in RNA processing and uncovered transcriptome changes strongly correlating with muscle strength. We created a web resource at http://DMseq.org that hosts raw and processed transcriptome data and provides a lightweight, responsive interface that enables browsing of processed data across the genome.

Intronic sequence elements impede exon ligation and trigger a discard pathway that yields functional telomerase RNA in fission yeast.

The fission yeast telomerase RNA (TER1) precursor harbors an intron immediately downstream from its mature 3' end. Unlike most introns, which are removed from precursor RNAs by the spliceosome in two sequential but tightly coupled transesterification reactions, TER1 only undergoes the first cleavage reaction during telomerase RNA maturation. The mechanism underlying spliceosome-mediated 3' end processing has remained unclear. We now demonstrate that a strong branch site (BS), a long distance to the 3' splice site (3' SS), and a weak polypyrimidine (Py) tract act synergistically to attenuate the transition from the first to the second step of splicing. The observation that a strong BS antagonizes the second step of splicing in the context of TER1 suggests that the BS-U2 snRNA interaction is disrupted after the first step and thus much earlier than previously thought. The slow transition from first to second step triggers the Prp22 DExD/H-box helicase-dependent rejection of the cleaved products and Prp43-dependent "discard" of the splicing intermediates. Our findings explain how the spliceosome can function in 3' end processing and provide new insights into the mechanism of splicing.

An engineered RNA binding protein with improved splicing regulation.

The muscleblind-like (MBNL) family of proteins are key developmental regulators of alternative splicing. Sequestration of MBNL proteins by expanded CUG/CCUG repeat RNA transcripts is a major pathogenic mechanism in the neuromuscular disorder myotonic dystrophy (DM). MBNL1 contains four zinc finger (ZF) motifs that form two tandem RNA binding domains (ZF1-2 and ZF3-4) which each bind YGCY RNA motifs. In an effort to determine the differences in function between these domains, we designed and characterized synthetic MBNL proteins with duplicate ZF1-2 or ZF3-4 domains, referred to as MBNL-AA and MBNL-BB, respectively. Analysis of splicing regulation revealed that MBNL-AA had up to 5-fold increased splicing activity while MBNL-BB had 4-fold decreased activity compared to a MBNL protein with the canonical arrangement of zinc finger domains. RNA binding analysis revealed that the variations in splicing activity are due to differences in RNA binding specificities between the two ZF domains rather than binding affinity. Our findings indicate that ZF1-2 drives splicing regulation via recognition of YGCY RNA motifs while ZF3-4 acts as a general RNA binding domain. Our studies suggest that synthetic MBNL proteins with improved or altered splicing activity have the potential to be used as both tools for investigating splicing regulation and protein therapeutics for DM and other microsatellite diseases.

Soft-tissue fat tumours: differentiating malignant from benign using proton density fat fraction quantification MRI.

AIM: To evaluate if quantifying proton density fat fraction (PDFF) would be useful in separating lipoma, atypical lipomatous tumour (ALT) and liposarcoma in the extremities and trunk. In addition, differentiating ALT versus non-classical lipomas using magnetic resonance imaging (MRI)-based fatty acid composition (FAC) and three-dimensional (3D) texture analysis was tested. MATERIAL AND METHODS: This prospective study (undertaken between 2014-2017; comprising 20 women, 21 men) was approved by the Regional Ethical Review Board and informed consent was obtained from all participants. For PDFF and FAC 3D spoiled gradient multi-echo images were acquired. PDFF was analysed in 16 lipomas (25-76 years), 14 ALTs (42-78 years) and 11 myxoid liposarcomas (31-68 years). The difference of mean PDFF was tested with one-way analysis of variance. A support vector machine algorithm was used to find the separating mean PDFF values. RESULTS: Mean PDFF for lipomas was 90% (range 76-98%), for ALT 83% (range 62-91%), and for liposarcoma 4% (range 0-21%). The difference of mean PDFF for liposarcomas versus ALT and lipoma was significant (p=0.0001, for both), and for ALT versus lipoma (p=0.021). The optimal threshold for separating liposarcoma from ALT and lipoma was 41.5%, and for ALT and lipoma 85%. Texture analysis could not separate ALT and non-classical lipomas, while the difference for FAC unsaturation degree was significant (p=0.013). CONCLUSION: Measuring PDFF is a promising complement to standard MRI, to separate liposarcomas from ALT and lipomas. Lipomas that are not solely composed of fat cannot confidently be separated from ALT using PDFF, FAC, or texture analysis.

Activated platelet aggregation is transiently impaired also by a reduced dose of protamine.

Objectives: Protamine reduces platelet aggregation after cardiopulmonary bypass (CPB). We studied the inhibitory effect of a reduced protamine dose, the duration of impaired platelet function and the possible correlation to postoperative bleeding. Design: Platelet function was assessed by impedance aggregometry in 30 patients undergoing cardiac surgery with CPB at baseline, before protamine administration, after 70% and 100% of the calculated protamine dose, after 20 minutes and at arrival to the intensive care unit. Adenosine diphosphate (ADP), thrombin receptor activating peptide-6 (TRAP), arachidonic acid (AA) and collagen (COL) were used as activators. Blood loss was measured during operation and three hours after surgery. Results are presented as median (25th-75th percentile). Results: Platelet aggregation decreased markedly after the initial dose of protamine (70%) with all activators; ADP 89 (71-110) to 54 (35-78), TRAP 143 (116-167) to 109 (77-136), both p < .01; AA 25 (16-49) to 17 (12-24) and COL 92 (47-103) to 60 (38-81) U, both p < .05. No further decrease was seen after 100% protamine. The effect was transient and after twenty minutes platelet aggregation had started to recover; ADP 76 (54-106), TRAP 138 (95-158), AA 20 (10-35), COL 70 (51-93) U. Blood loss during operation correlated to aggregometry measured at baseline and after protaminization. Conclusions: Protamine after CPB induces a marked decrease in platelet aggregation already at a protamine-heparin ratio of 0.7:1. The impairment seems to be transient and recovery had started after 20 minutes.

Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy.

Alternative splicing is a regulated process that results in expression of specific mRNA and protein isoforms. Alternative splicing factors determine the relative abundance of each isoform. Here we focus on MBNL1, a splicing factor misregulated in the disease myotonic dystrophy. By altering the concentration of MBNL1 in cells across a broad dynamic range, we show that different splicing events require different amounts of MBNL1 for half-maximal response, and respond more or less steeply to MBNL1. Motifs around MBNL1 exon 5 were studied to assess how cis-elements mediate the MBNL1 dose-dependent splicing response. A framework was developed to estimate MBNL concentration using splicing responses alone, validated in the cell-based model, and applied to myotonic dystrophy patient muscle. Using this framework, we evaluated the ability of individual and combinations of splicing events to predict functional MBNL concentration in human biopsies, as well as their performance as biomarkers to assay mild, moderate, and severe cases of DM.

Mitigating RNA Toxicity in Myotonic Dystrophy using Small Molecules.

This review, one in a series on myotonic dystrophy (DM), is focused on the development and potential use of small molecules as therapeutics for DM. The complex mechanisms and pathogenesis of DM are covered in the associated reviews. Here, we examine the various small molecule approaches taken to target the DNA, RNA, and proteins that contribute to disease onset and progression in myotonic dystrophy type 1 (DM1) and 2 (DM2).

Pseudouridine Modification Inhibits Muscleblind-like 1 (MBNL1) Binding to CCUG Repeats and Minimally Structured RNA through Reduced RNA Flexibility.

Myotonic dystrophy type 2 is a genetic neuromuscular disease caused by the expression of expanded CCUG repeat RNAs from the non-coding region of the CCHC-type zinc finger nucleic acid-binding protein (CNBP) gene. These CCUG repeats bind and sequester a family of RNA-binding proteins known as Muscleblind-like 1, 2, and 3 (MBNL1, MBNL2, and MBNL3), and sequestration plays a significant role in pathogenicity. MBNL proteins are alternative splicing regulators that bind to the consensus RNA sequence YGCY (Y = pyrimidine). This consensus sequence is found in the toxic RNAs (CCUG repeats) and in cellular RNA substrates that MBNL proteins have been shown to bind. Replacing the uridine in CCUG repeats with pseudouridine (Ψ) resulted in a modest reduction of MBNL1 binding. Interestingly, Ψ modification of a minimally structured RNA containing YGCY motifs resulted in more robust inhibition of MBNL1 binding. The different levels of inhibition between CCUG repeat and minimally structured RNA binding appear to be due to the ability to modify both pyrimidines in the YGCY motif, which is not possible in the CCUG repeats. Molecular dynamic studies of unmodified and pseudouridylated minimally structured RNAs suggest that reducing the flexibility of the minimally structured RNA leads to reduced binding by MBNL1.

Tumor-induced osteomalacia in association with PTEN-negative Cowden syndrome.

Tumor-induced osteomalacia (TIO) is a rare paraneoplastic condition in which phosphaturic mesenchymal tumors (PMTs) secrete high levels of fibroblast growth factor 23 (FGF23) into the circulation. This results in renal phosphate wasting, hypophosphatemia, muscle weakness, bone pain, and pathological fractures. Recent studies suggest that fibronectin-fibroblast growth factor receptor 1 (FN1-FGFR1) translocations may be a driver of tumorigenesis. We present a patient with TIO who also exhibited clinical findings suggestive of Cowden syndrome (CS), a rare autosomal dominant disorder characterized by numerous benign hamartomas, as well as an increased risk for multiple malignancies, such as thyroid cancer. While CS is a clinical diagnosis, most, but not all, harbor a mutation in the tumor suppressor gene PTEN. Genetic testing revealed a somatic FN1-FGFR1 translocation in the FGF23-producing tumor causing TIO; however, a germline PTEN mutation was not identified. To our knowledge, this is the first reported case of concurrent TIO and CS.

Conservation of context-dependent splicing activity in distant Muscleblind homologs.

The Muscleblind (MBL) protein family is a deeply conserved family of RNA binding proteins that regulate alternative splicing, alternative polyadenylation, RNA stability and RNA localization. Their inactivation due to sequestration by expanded CUG repeats causes symptoms in the neuromuscular disease myotonic dystrophy. MBL zinc fingers are the most highly conserved portion of these proteins, and directly interact with RNA. We identified putative MBL homologs in Ciona intestinalis and Trichoplax adhaerens, and investigated their ability, as well as that of MBL homologs from human/mouse, fly and worm, to regulate alternative splicing. We found that all homologs can regulate alternative splicing in mouse cells, with some regulating over 100 events. The cis-elements through which each homolog exerts its splicing activities are likely to be highly similar to mammalian Muscleblind-like proteins (MBNLs), as suggested by motif analyses and the ability of expanded CUG repeats to inactivate homolog-mediated splicing. While regulation of specific target exons by MBL/MBNL has not been broadly conserved across these species, genes enriched for MBL/MBNL binding sites in their introns may play roles in cell adhesion, ion transport and axon guidance, among other biological pathways, suggesting a specific, conserved role for these proteins across a broad range of metazoan species.

Modifications to toxic CUG RNAs induce structural stability, rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model.

CUG repeat expansions in the 3' UTR of dystrophia myotonica protein kinase (DMPK) cause myotonic dystrophy type 1 (DM1). As RNA, these repeats elicit toxicity by sequestering splicing proteins, such as MBNL1, into protein-RNA aggregates. Structural studies demonstrate that CUG repeats can form A-form helices, suggesting that repeat secondary structure could be important in pathogenicity. To evaluate this hypothesis, we utilized structure-stabilizing RNA modifications pseudouridine (Ψ) and 2'-O-methylation to determine if stabilization of CUG helical conformations affected toxicity. CUG repeats modified with Ψ or 2'-O-methyl groups exhibited enhanced structural stability and reduced affinity for MBNL1. Molecular dynamics and X-ray crystallography suggest a potential water-bridging mechanism for Ψ-mediated CUG repeat stabilization. Ψ modification of CUG repeats rescued mis-splicing in a DM1 cell model and prevented CUG repeat toxicity in zebrafish embryos. This study indicates that the structure of toxic RNAs has a significant role in controlling the onset of neuromuscular diseases.

Increased ferritin levels in patients with anorexia nervosa: impact of weight gain.

PURPOSE: A few recent studies have found elevated ferritin levels in patients with anorexia nervosa (AN), indicating ferritin as a potential biomarker of disease severity. The purpose of this study was to study how body mass index (BMI) and changes in BMI affect plasma ferritin concentrations in Swedish patients with eating disorders. MATERIALS AND METHODS: In a retrospective computer search from 2009 to 2014, 662 patients with an eating disorder were identified from more than 200,000 individuals with electronic medical records. Three hundred and eighty-nine patients (374 females and 15 males) were found to have at least one p-ferritin value with a corresponding BMI value. Patients with AN were compared to a combined group consisting of patients with bulimia nervosa (BN) and patients with an eating disorder not otherwise specified (EDNOS). RESULTS: Patients with AN had lower BMI compared to the combined group of patients with other eating disorders (BMI = 16.5 ± 1.5, n = 77 vs. 21.0 ± 4.7, n = 312, p < 0.001). Patients with AN also had higher plasma ferritin levels (median 42 µg/L (range 3.3-310) vs. 31 µg/L (range 2.8-280); p < 0.001). As BMI increased in patients with AN, ferritin levels decreased (from a median of 40 µg/L (7-400) to 26 (4-170), n = 47; p < 0.001). DISCUSSION: Measuring ferritin in patients with AN could be valuable in monitoring improvements of nutritional status, but the full clinical value of following ferritin in individual patients has yet to be determined. The study also shows how research can benefit from electronically captured clinical data using electronic health records.

Synthesis of N-substituted aryl amidines by strong base activation of amines.

We describe an efficient method for the direct preparation of N-substituted aryl amidines from nitriles and primary amines. The protocol employs activation of amines by a strong base and provides greater access to a pharmaceutically relevant functional group. This synthetic approach tolerates deactivated nitriles, nitriles with competing substitution sites, and aryl amines.

Role of RNA structure in regulating pre-mRNA splicing.

Pre-mRNA splicing involves removing non-coding introns from RNA transcripts. It is carried out by the spliceosome, along with other auxiliary factors. In general, research in splicing has focused on the sequences within the pre-mRNA, without considering the structures that these sequences might form. We propose that the role of RNA structure deserves more consideration when thinking about splicing mechanisms. RNA structures can inhibit or aid binding of spliceosomal components to the pre-mRNA, or can increase splicing efficiency by bringing important sequences into close proximity. Recent reports have identified proteins and small molecules that can regulate splicing by modulating RNA structures, thereby expanding our knowledge of the mechanisms used to regulate splicing.

Decreased cognitive functions at the age of 66, as measured by the MMSE, associated with having left working life before the age of 60: results from the SNAC study.

AIMS: The age of retirement has financial implications as we tend to live longer, with the result that an increasing number of older inhabitants have to share limited financial resources. However, this is not only a financial issue. It is also of interest to investigate factors related to health and quality of life associated with the age of retirement. The aim of this study was to investigate changes in mood, activity level, and cognition at the age of 66 associated with leaving working life before 60. METHODS: Baseline and follow-up data on 840 participants of the Swedish National Study on Aging and Care - Blekinge was used. Mood was measured by the Montgomery-Åsberg Depression Scale and activity level by 27 survey items. Cognition was measured by the Mini Mental State Examination. RESULTS: Retirement before 60 years of age was not associated with lower cognitive functions and a higher score on depression at baseline, but retirees were less active. Six years later, at the age of 66, a decline in their cognition was found. Retirees were still not more depressed but less active. In a logistic regression analysis, being retired increased the odds ratio for cognitive decline by 1.36-times (OR 2.36) when gender, activity level, education level, and depression were adjusted for. CONCLUSIONS: Participants who retired before the age of 60 declined in cognitive ability over the 6-year study period.