The possibility of using stem cell-derived cardiomyocytes opens a new platform for modeling cardiac cell differentiation and disease or the development of new drugs. Progress in this field can be accelerated by high-throughput screening (HTS) technology combined with promoter reporter system. The goal of the study was to create and evaluate a responsive promoter reporter system that allows monitoring of iPSC differentiation towards cardiomyocytes. The lentiviral promoter reporter system was based on troponin 2 (TNNT2) and alpha cardiac actin (ACTC) with firefly luciferase and mCherry, respectively. The system was evaluated in two in vitro models. vitro cell culture (confirmed by I-FISH, ddPCR, qPCR). Second, differentiated and contracting control cardiomyocytes (obtained from control non-reporter transduced iPSCs) were subsequently transduced with TNNT2-luc-T2A-Puro-CMV-GFP and hACTC-mcherry-WPRE-EF1-Neo lentiviruses to observe the functionality of obtained cardiomyocytes. Our results indicated that the reporter modified cell lines can be used for HTS applications, but it is essential to monitor the stability of the reporter sequence during extended cell in vitro culture. First, system followed the differentiation of TNNT2-luc-T2A-Puro-mCMV-GFP and hACTC-mcherry-WPRE-EF1-Neo from transduced iPSC line towards cardiomyocytes and revealed the significant decrease in both inserts copy number during the prolonged in
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 (RNA exp ) 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 RNA exp molecules and multiple protein partners, in this review, we discuss the roles of selected RNA-binding proteins (RBPs) that interact with RNA exp and thus act as "partners in crime" in the progression of REDs. We gather current findings concerning RBPs involved at different stages of the RNA exp 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 RNA exp within nuclear foci and those which participate in RNA exp -dependent innate immunity activation.Abbreviations: 3 0 UTR/5 0 UTR, 3 0 /5 0 untranslated regions; AS, alternative splicing; ASO, antisense oligonucleotide; C9-ALS/FTD, C9orf72-linked amyotrophic lateral sclerosis and frontotemporal dementia; CAG exp , expanded CAG repeats; CCUG exp , expanded CCUG repeats; CGG exp , expanded CGG repeats; CTG exp , expanded CTG repeats; CUG exp , expanded CUG repeats;
RNA helicases are known from their ability to bind and unwind double-stranded RNA initiating RNA processing events. These evolutionary conserved RNA binding proteins are broadly expressed in a variety of tissues; however, we can distinguish those, which represent tissue-specific expression pattern and play unique roles in certain cell lineages. For instance, some RNA helicases mediate transcriptomic changes triggering cell differentiation which results in specification and establishment of germline in a developing embryo. Others act as safeguards responsible for maintenance of DNA integrity in germ cell. In this article, we focus on selected DEAD/DEAH-box RNA helicases involved in germline development and spermatogenesis presenting their diverse functions and implications for male fertility.
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