a-nonsequencing-approach-for-the-rapid-detection-of-rna-editing

A Nonsequencing Approach for the Rapid Detection of RNA Editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...

Japan Advanced Institute of Science and Technology

During pre-mRNA splicing, exons in the primary transcript are precisely connected to generate an mRNA. Intron lariat RNAs are formed as by-products of this process. In addition, some exonic circular RNAs (circRNAs) may also result from exon skipping as by-products. Lariat RNAs and circRNAs are both RNase R resistant RNAs. RNase R is a strong 3' to 5' exoribonuclease, which efficiently degrades linear RNAs, such as mRNAs and rRNAs; therefore, the circular parts of lariat RNAs and the circRNAs can be segregated from eukaryotic total RNAs by their RNase R resistance. Thus, RNase R resistant RNAs could provide unexplored splicing information not available from mRNAs. Analyses of these RNAs identified repeating splicing phenomena, such as re-splicing of mature mRNAs and nested splicing. Moreover, circRNA might function as microRNA sponges. There is an enormous variety of endogenous circRNAs, which are generally synthesized in cells and tissues. 

A view of pre-mRNA splicing from RNase R resistant RNAs.

RBFox proteins are well-known alternative splicing regulators. We have shown previously that during neuronal differentiation of P19 cells induced by all-trans retinoic acid and cell aggregation, RBFox1 shows markedly increased temporal expression. To find its key splicing regulation, we examined the effect of RBFox1 on 33 previously reported and validated neuronal splicing events of P19 cells. We observed that alternative splicing of three genes, specifically, amyloid precursor protein (APP), disks large homolog 3 (DLG3), and G protein, alpha activating activity polypeptide O (GNAO1), was altered by transient RBFox1 expression in HEK293 and HeLa cells. Moreover, an RBFox1 mutant (RBFox1FA) that was unable to bind the target RNA sequence ((U)GCAUG) did not induce these splicing events. APP generates amyloid beta peptides that are involved in the pathology of Alzheimer's disease, and therefore we examined APP alternative splicing regulation by RBFox1 and other splicing regulators. Our results indicated that RBFox proteins promote the skipping of APP exon 7, but not the inclusion of exon 8. We made APP6789 minigenes and observed that two (U)GCAUG sequences, located upstream of exon 7 and in exon 7, functioned to induce skipping of exon 7 by RBFox proteins. Overall, RBFox proteins may shift APP from exon 7 containing isoforms, APP770 and APP751, toward the exon 7 lacking isoform, APP695, which is predominant in neural tissues. 

Alternative splicing regulation of APP exon 7 by RBFox proteins.

Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45-55 of the  gene, might improve patients' symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45-55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44-56 connected mRNA transcript of the  using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5' splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate  circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.

Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot.

Many genetic diseases are caused by T-to-C point mutations. Hence, editing of mutated genes represents a promising strategy for treating these disorders. We engineered an artificial RNA editase by combining the deaminase domain of APOBEC1 (apolipoprotein B mRNA editing catalytic polypeptide 1) with a guideRNA (gRNA) which is complementary to target mRNA. In this artificial enzyme system, gRNA is bound to MS2 stem-loop, and deaminase domain, which has the ability to convert mutated target nucleotide C-to-U, is fused to MS2 coat protein. As a target RNA, we used RNA encoding blue fluorescent protein (BFP) which was derived from the gene encoding GFP by 199 T > C mutation. Upon transient expression of both components (deaminase and gRNA), we observed GFP by confocal microscopy, indicating that mutated 199C in BFP had been converted to U, restoring original sequence of GFP. This result was confirmed by PCR-RFLP and Sanger's sequencing using cDNA from transfected cells, revealing an editing efficiency of approximately 21%. Although deep RNA sequencing result showed some off-target editing events in this system, we successfully developed an artificial RNA editing system using artificial deaminase (APOBEC1) in combination with MS2 system could lead to therapies that treat genetic disease by restoring wild-type sequence at the mRNA level.

RNA editing of BFP, a point mutant of GFP, using artificial APOBEC1 deaminase to restore the genetic code.

Tabebuia pallida (Lindl.) Miers (T. pallida) is a well-known native Caribbean medicinal plant. The leaves and barks of T. pallida are used as traditional medicine in the form of herbal or medicinal tea to manage cancer, fever, and pain. Moreover, extracts from the leaves of T. pallida showed anticancer activity. However, the chemical profile and mechanism of anticancer activity of T. pallida leaves (TPL), stem bark (TPSB), root bark (TPRB) and flowers (TPF) remain unexplored.

Unfolding the apoptotic mechanism of antioxidant enriched-leaves of Tabebuia pallida (lindl.) miers in EAC cells and mouse model.

The popularity and promise of gene therapy for common genetic diseases are currently increasing. Although effective treatments for genetic disorders are rare, editing of the mutated gene is a possible therapeutic approach for conditions caused by stop codon mutations, including either amber (TAG), opal (TGA) or ochre (TAA) stop codons. Restoration of point-mutated RNAs using artificial RNA editing can be used to modify gene-encoded information and generate functionally distinct proteins from a single gene. By linking the catalytic domain of the RNA editing enzyme, adenosine deaminase acting on RNA (ADAR), to an antisense guide RNA, speciﬁc adenosines (A) can be converted to inosine (I), which is recognized as guanosine (G) during translation. In this study, we engineered the deaminase domain of ADAR1 and the MS2 system to target a specific adenosine and restore the G to A mutations. To this end, the ADAR1 deaminase domain was fused with the RNA binding protein, MS2, which binds to MS2 RNA. Guide RNAs of 19 bp were designed to be complementary to target mRNAs, with either 6X stem-loops downstream of the guide RNA and a CMV promoter, or a 1X MS2 stem-loop on either side of the guide RNA and a U6 promoter. The engineered ADAR1 deaminase domain could convert adenosine to inosine at the desired editing site in EGFP, which was edited to contain an amber (TAG), opal (TGA) or ochre (TAA) stop codon. The system could convert the stop codons to a read-through tryptophan codon (TGG) in a cellular system, leading to fluorescence emission, observed using JuLi microscopy. PCR-RFLP and Sanger sequencing of the target transcript were also conducted, revealing an editing efficiency of 20.97 % for the opal stop codon, and 26 % and 17 % for the 5' and 3' A residues, respectively, in the ochre stop codon, using the double MS2. This was a higher editing rate than that achieved using the MS2-6X guide RNA. Observation of restoration of the read-through codon from the three different stop codons over time demonstrated a relatively low percentage of edited codons after 24 h, which increased after 48 h, but decreased again after 72 h. Successful establishment of this system has the potential to represent a new era in the field of gene therapy.

Double MS2 guided restoration of genetic code in amber (TAG), opal (TGA) and ochre (TAA) stop codon.

Cytidine-to-uridine (C-to-U) RNA editing has been generally observed in land plants; however, reverse (U-to-C) RNA editing is a rare phenomenon. In this study, we investigated the U-to-C RNA editing-related genes in Arabidopsis tissues and the effects on mRNA stability, with a special focus on PPR proteins. A previous study showed the extensive occurrence of U-to-C RNA editing in 12-day and 20-dayold Arabidopsis seedlings. Here, we have demonstrated the effects of this "reverse" RNA editing on the mRNA stability for all seven edited genes. We also identified U-to-C RNA editing in the nuclear PPR gene (AT2G19280) in 12-day-old seedlings of Arabidopsis thaliana. The U-to-C RNA editing sites were found in the untranslated region (3' UTR) of the mature mRNA and may affect its secondary structure. We also examined the correlation between U-to-C RNA editing-related genes and their mRNA abundance. Furthermore, we investigated the effects of U-to-C RNA editing in Arabidopsis using the transcription inhibitor actinomycin D (Act D). The addition of Act D to the seedlings of transgenic Arabidopsis generated by Agrobacterium-mediated transformation showed that single nucleotide base conversion adversely affected the mRNA secondary structure and stability.

Article	Year
A view of pre-mRNA splicing from RNase R resistant RNAs. International journal of molecular sciences\| PubMed ID: 24865493	2014
Alternative splicing regulation of APP exon 7 by RBFox proteins. Neurochemistry international\| PubMed ID: 25125370	2014
Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot. International journal of molecular sciences\| PubMed ID: 27754374	2016
RNA editing of BFP, a point mutant of GFP, using artificial APOBEC1 deaminase to restore the genetic code. Scientific reports\| PubMed ID: 33057101	2020
Unfolding the apoptotic mechanism of antioxidant enriched-leaves of Tabebuia pallida (lindl.) miers in EAC cells and mouse model. Journal of ethnopharmacology\| PubMed ID: 34118341	2021
Double MS2 guided restoration of genetic code in amber (TAG), opal (TGA) and ochre (TAA) stop codon. Enzyme and microbial technology\| PubMed ID: 34311888	2021
The U-to-C RNA editing affects the mRNA stability of nuclear genes in Arabidopsis thaliana. Biochemical and biophysical research communications\| PubMed ID: 34325125	2021

Toshifumi Tshukahara

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