Loss of MBNL1 induces RNA misprocessing in the thymus and peripheral blood

ARTICLE https://doi.org/10.1038/s41467-020-15962-x OPEN Loss of MBNL1 induces RNA misprocessing in the thymus and peripheral blood 1234567890():,; Łukasz J. Sznajder 1,5,6 ✉, Marina M. Scotti1,5, Jihae Shin1,3, Katarzyna Taylor1,4, Franjo Ivankovic Curtis A. Nutter1, Faaiq N. Aslam1, S. H. Subramony2, Laura P. W. Ranum1 & Maurice S. Swanson 1, 1,6 ✉ The thymus is a primary lymphoid organ that plays an essential role in T lymphocyte maturation and selection during development of one arm of the mammalian adaptive immune response. Although transcriptional mechanisms have been well documented in thymocyte development, co-/post-transcriptional modifications are also important but have received less attention. Here we demonstrate that the RNA alternative splicing factor MBNL1, which is sequestered in nuclear RNA foci by C(C)UG microsatellite expansions in myotonic dystrophy (DM), is essential for normal thymus development and function. Mbnl1 129S1 knockout mice develop postnatal thymic hyperplasia with thymocyte accumulation. Transcriptome analysis indicates numerous gene expression and RNA mis-splicing events, including transcription factors from the TCF/LEF family. CNBP, the gene containing an intronic CCTG microsatellite expansion in DM type 2 (DM2), is coordinately expressed with MBNL1 in the developing thymus and DM2 CCTG expansions induce similar transcriptome alterations in DM2 blood, which thus serve as disease-specific biomarkers. 1 Department of Molecular Genetics and Microbiology, Center for NeuroGenetics and the Genetics Institute, University of Florida, College of Medicine, Gainesville, FL 32610, USA. 2 Department of Neurology, Center for NeuroGenetics, University of Florida, College of Medicine, Gainesville, FL 32610, USA. Present address: Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School and Rutgers Cancer Institute of New Jersey, Newark, NJ 07103, USA. 4Present address: Laboratory of Gene Therapy, Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89 61-614 Poznań, Poland. 5These authors contributed equally: Łukasz J. Sznajder, Marina M. Scotti. 6These authors jointly supervised this work: Łukasz J. Sznajder, Maurice S. Swanson. ✉email: ; mswanson@ufl.edu 3 NATURE COMMUNICATIONS | (2020)11:2022 | https://doi.org/10.1038/s41467-020-15962-x | www.nature.com/naturecommunications 1 ARTICLE T NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15962-x he thymus plays a crucial role in adaptive immunity by providing a conducive environment for T lymphocyte (T cell) differentiation1,2. As thymocytes mature, approximately 90–95% undergo apoptosis, either from lack of effective T-Cell Receptor (TCR) recombination and signaling, or alternatively, in response to negative selection to ablate high-affinity TCRs capable of recognizing self-antigens3. Defects in T cell selection and/or the loss of mature T cell populations, can result in a compromised immune state where cytotoxic CD8+ T cells are unable to exert cytotoxic functions against intracellular pathogens, or helper CD4+ T cells are unable to effectively provide B cell help to drive protective humoral immunity. While transcriptional regulation has been shown to play a vital role in both thymic organogenesis and T cell development4, co-/post-transcriptional events have also been implicated in thymocyte differentiation5,6. Indeed, alternative splicing (AS) adds another layer of complexity and diversity for developmental and tissue-restricted expression and splicing patterns for many gene transcripts that shift during development and aging7–9. Due to the complexity of interactions between the RNA sequence elements and trans-acting splicing factors that control splicing and 3′-end cleavage/polyadenylation, RNA processing is particularly susceptible to mutations implicated in disease10,11. For example, the Muscleblind-like (MBNL) family of alternative splicing factors triggers the switch from fetal to adult splicing programs for its RNA targets and loss of MBNL1 activity is a major pathogenic factor in the multisystemic disease myotonic dystrophy (DM) type 1 (DM1) and type 2 (DM2)12,13. DM1 and DM2 are autosomal dominant disorders caused by microsatellite, or short tandem repeat (STR), CTG and CCTG expansions (exp) encoded in either the 3′ untranslated region (UTR) of DMPK (DM1) or the first intron of CNBP (DM2), respectively. In both DM types, transcription of these mutant STRs results in the expression of C(C)UGexp RNAs that are retained in the nucleus as RNA foci together with MBNL proteins14. This MBNL sequestration process results in downstream pre-mRNA misprocessing, including errors in AS and alternative 3′-end cleavage/polyadenylation (APA)15,16 that result in pathological manifestations17. Although DM1 and DM2 are classified as a muscular dystrophy, the immune system is also affected and both DM types are characterized by a number of cellular and humoral abnormalities in peripheral blood. For example, although hypogammaglobulinemia and low lymphocyte counts occur in both DM types, they are especially prevalent in DM2 and are associated with an increased risk of autoimmune disease in DM218,19. In addition, thymic hyperplasia and thymoma, as well as increased risk for other cancer types, have been reported in DM20–23. Although the immune phenotype contributes to DM1 and DM2 complexity, the consequence of MBNL depletion on adaptive immunity has not been investigated. The thymus is active in developing mice and highly active in the pre-pubescent period in humans, but subsequently undergoes progressive involution with reduced thymic output. In this study, we report that loss of MBNL1 expression in 129S1-Mbnl1ΔE3/ΔE3 knockout (KO) mice results in postnatal thymic hyperplasia and thymocyte accumulation and we identify misprocessing of developmental splicing events critical for T lymphocyte maturation. Importantly, these splicing changes are also detectable in DM2, but not DM1, peripheral blood. Additionally, we provide evidence that the degree of splicing dysregulation is proportional to DM2 CCTG STR length and CNBP intron 1 retention level. Based on this analysis, we propose a set of AS events that are readily detectable in whole blood and serve as biomarkers for DM2 disease. 2 Results MBNL1 loss leads to dysregulation of thymic gene expression. Our previous in situ hybridization study of Mbnl gene expression during mouse embryogenesis revealed that Mbnl1 is highly expressed in the thymus suggesting that the MBNL1 protein regulates RNA processing during thymic development24. To confirm this observation and extend our understanding of Mbnl1 developmental expression, we retrieved publicly available RNA sequencing (RNA-seq) data of embryonic (E12.5-E18.5) and newborn (P0) mouse thymus25. Differential gene expression analysis confirmed that Mbnl1 expression increased du (...truncated)