Search Results (6)

Adenosine deaminases acting on RNA (ADARs) have double-stranded RNA binding domains and a deaminase domain (DD). We used the MS2 system and specific guide RNAs to direct ADAR1-DD to target adenosines in the mRNA encoding-enhanced green fluorescence protein. Using this system in transfected HEK-293 cells, we evaluated the effects of changing the length and position of the guide RNA on the efficiency of conversion of amber (TAG) and ochre (TAA) stop codons to tryptophan (TGG) in the target. Guide RNAs of 19, 21 and 23 nt were positioned upstream and downstream of the MS2-RNA, providing a total of six guide RNAs. The upstream guide RNAs were more functionally effective than the downstream guide RNAs, with the following hierarchy of efficiency: 21 nt > 23 nt > 19 nt. The highest editing efficiency was 16.6%. Off-target editing was not detected in the guide RNA complementary region but was detected 50 nt downstream of the target. The editing efficiency was proportional to the amount of transfected deaminase but inversely proportional to the amount of the transfected guide RNA. Our results suggest that specific RNA editing requires precise optimization of the ratio of enzyme, guide RNA, and target RNA. Full article

Site-directed RNA editing (SDRE) technologies have great potential in gene therapy. Our group has developed a strategy to redirect exogenous adenosine deaminases acting on RNA (ADARs) to specific sites by making editable structures using antisense RNA oligonucleotides. Improving the editing efficiency of the MS2-ADAR system is important in treating undesirable G-to-A point mutations. This work demonstrates an effective strategy to enhance the editing efficiency of this SDRE system. The strategy involves changing the number of MS2 stem-loops on both sides of the antisense RNA and the mismatch base on the antisense part. The enhanced green fluorescent protein (EGFP) with W58X mutation is used as the reporter gene. Subsequently, we adjusted the amount of plasmids for transfection to tune the expression level of the guide RNA, and finally, we observed the fluorescence signal after transfection. After equalizing number of MS2 stem-loops at both sides of the antisense RNA, high editing efficiency was achieved. In the same level of guide RNA expression, when the paired base position was the target uridine, the editing efficiency was higher than cytidine, adenosine, and guanosine. Full article

RNA editing is an epitranscriptomic modification, leading to targeted changes in RNA transcripts. It is mediated by the action of ADAR (adenosine deaminases acting on double-stranded (ds) RNA and APOBEC (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like) deaminases and appears to play a major role in the pathogenesis of many diseases. Here, we assessed its role in experimental autoimmune encephalomyelitis (EAE), a widely used non-clinical model of autoimmune inflammatory diseases of the central nervous system (CNS), which resembles many aspects of human multiple sclerosis (MS). We have analyzed in silico data from microglia isolated at different timepoints through disease progression to identify the global editing events and validated the selected targets in murine tissue samples. To further evaluate the functional role of RNA editing, we induced EAE in transgenic animals lacking expression of APOBEC-1. We found that RNA-editing events, mediated by the APOBEC and ADAR deaminases, are significantly reduced throughout the course of disease, possibly affecting the protein expression necessary for normal neurological function. Moreover, the severity of the EAE model was significantly higher in APOBEC-1 knock-out mice, compared to wild-type controls. Our results implicate regulatory epitranscriptomic mechanisms in EAE pathogenesis that could be extrapolated to MS and other neurodegenerative disorders (NDs) with common clinical and molecular features. Full article

Intrinsically disordered myelin basic protein (MBP) is one of the key autoantigens in autoimmune neurodegeneration and multiple sclerosis particularly. MBP is highly positively charged and lacks distinct structure in solution and therefore its intracellular partners are still mostly enigmatic. Here we used combination of formaldehyde-induced cross-linking followed by immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate the interaction network of MBP in mammalian cells and provide the list of potential MBP interacting proteins. Our data suggest that the largest group of MBP-interacting proteins belongs to cellular proteins involved in the protein translation machinery, as well as in the spatial and temporal regulation of translation. MBP interacts with core ribosomal proteins, RNA helicase Ddx28 and RNA-binding proteins STAU1, TDP-43, ADAR-1 and hnRNP A0, which are involved in various stages of RNA biogenesis and processing, including specific maintaining MBP-coding mRNA. Among MBP partners we identified CTNND1, which has previously been shown to be necessary for myelinating Schwann cells for cell-cell interactions and the formation of a normal myelin sheath. MBP binds proteins MAGEB2/D2 associated with neurotrophin receptor p75NTR, involved in pathways that promote neuronal survival and neuronal death. Finally, we observed that MBP interacts with RNF40–a component of heterotetrameric Rnf40/Rnf20 E3 ligase complex, recruited by Egr2, which is the central transcriptional regulator of peripheral myelination. Concluding, our data suggest that MBP may be more actively involved in myelination not only as a main building block but also as a self-regulating element. Full article

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in μs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZα_ADAR1)–DNA complex. The global search results indicated that a α-helical segment of hZα_ADAR1 provided the main contribution to the three-state conformational changes of a hZα_ADAR1—DNA complex with a slow B–Z exchange process. The two global exchange rate constants, k_ex and k_ZB, were found to be 844 and 9.8 s⁻¹, respectively, in agreement with two regimes of residue-dependent chemical shift differences—the “dominant oscillatory regime” and “semi-oscillatory regime”. We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes. Full article

Site-directed RNA editing (SDRE) technologies have great potential for treating genetic diseases caused by point mutations. Our group and other researchers have developed SDRE methods utilizing adenosine deaminases acting on RNA (ADARs) and guide RNAs recruiting ADARs to target RNAs bearing point mutations. In general, efficient SDRE relies on introducing numerous guide RNAs relative to target genes. However, achieving a large ratio is not possible for gene therapy applications. In order to achieve a realistic ratio, we herein developed a system that can introduce an equal number of genes and guide RNAs into cultured cells using a fusion protein comprising an ADAR fragment and a plasmid vector containing one copy of each gene on a single construct. We transfected the single construct into HEK293T cells and achieved relatively high efficiency (up to 42%). The results demonstrate that efficient SDRE is possible when the copy number is similar for all three factors (target gene, guide RNA, and ADAR enzyme). This method is expected to be capable of highly efficient gene repair in vivo, making it applicable for gene therapy. Full article