Editing Sites Research Articles

Mitogenome Assembly Reveals Gene Migration and RNA Editing Events in Plateau Hongliu (Myricaria elegans Royle.)

The Plateau Hongliu (Myricaria elegans Royle.) is a woody shrub halophyte that thrives in arid areas of western Tibet, in the Himalayan Mountains. It is acclaimed as superior in saline stress acclimation and as a critical pharmaceutical resource of the Tibetan traditional herb. Nevertheless, the mitogenome in the genus Myricaria remains unknown. Here, using the Illumina and PacBio sequencing assays, the first complete mitogenome of the M. elegans revealed a multi-branched skeleton with a total length of 416,354 bp and GC content of 44.33%, comprising two circular molecules (M1 and 2). The complete mitogenome annotates 31 unique protein-encoding genes (PEGs), fifteen tRNAs, and three rRNA genes. The UAA exhibits the most prominent codon usage preference as a termination, followed by UUA codons for leucine. The mitogenome contains 99 simple sequence repeats and 353 pairs of dispersed repeats, displaying the most frequent in palindromic repeats. Gene transfer analyses identified 8438 bp of 18 homologous fragments from the plastome, accounting for 2.03% of the total length. Using the PREP suite, 350 C-U RNA editing sites were predicted, of which nad4 and ccmB were on the top frequency. Syntenic and phylogenetic analyses suggested weakly conserved patterns of M. elegans in Caryophyllales owing to the genome rearrangement. In summary, the deciphered unique features and complexities of the mitogenome in M. elegans provide novel insights into understanding the evolution and biological conservation underlying climate resilience in halophytes.

Development and assessment of an RNA editing-based risk model for the prognosis of cervical cancer patients.

RNA editing, as an epigenetic mechanism, exhibits a strong correlation with the occurrence and development of cancers. Nevertheless, few studies have been conducted to investigate the impact of RNA editing on cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). In order to study the connection between RNA editing and CESC patients' prognoses, we obtained CESC-related information from The Cancer Genome Atlas (TCGA) database and randomly allocated the patients into the training group or testing group. An RNA editing-based risk model for CESC patients was established by Cox regression analysis and least absolute shrinkage and selection operator (LASSO). According to the median score generated by this RNA editing-based risk model, patients were categorized into subgroups with high and low risks. We further constructed the nomogram by risk scores and clinical characteristics and analyzed the impact of RNA editing levels on host gene expression levels and adenosine deaminase acting on RNA. Finally, we also compared the biological functions and pathways of differentially expressed genes (DEGs) between different subgroups by enrichment analysis. In this risk model, we screened out 6 RNA editing sites with significant prognostic value. The constructed nomogram performed well in forecasting patients' prognoses. Furthermore, the level of RNA editing at the prognostic site exhibited a strong correlation with host gene expression. In the high-risk subgroup, we observed multiple biological functions and pathways associated with immune response, cell proliferation, and tumor progression. This study establishes an RNA editing-based risk model that helps forecast patients' prognoses and offers a new understanding of the underlying mechanism of RNA editing in CESC.

Nuclear genomes of dinoflagellates reveal evolutionarily conserved pattern of RNA editing relative to stress response

Dinoflagellates are a group of diverse protists with complex genomes whose gene expression regulation mechanisms remain little known. RNA editing is a post-transcriptional regulatory mechanism of gene expression utilized by diverse species, and has been described primarily in the plastid and mitochondrial genomes of dinoflagellates. Its role in post-transcriptional regulation in the nuclear genomes of dinoflagellates remains largely unexplored. Here, integrating genome and transcriptome data from two dinoflagellate taxa in a comparative analysis, we identified 10,486 and 69,953 putative RNA editing sites in the nuclear genomes of the coral symbiont, Durusdinium trenchii CCMP2556 and the free-living bloom-forming taxon, Prorocentrum cordatum CCMP1329. We recovered all 12 possible types of RNA edits, with more edits representing transitions than transversions. In contrast to other eukaryotes, we found a dominance of A-to-T transversion in non-coding regions, many of which were condition-specific. Overall, the RNA editing sites implicate 7.5% of D. trenchii genes and 13.2% of P. cordatum genes. Some sites (1.5% in D. trenchii and more-substantially 62.3% in P. cordatum) were edited at significantly different frequencies in distinct growth conditions. The distribution of editing types and locations exhibited conserved patterns between the two phylogenetically distant species. Interestingly, A-to-T editing within the untranslated regions appear to be associated with upregulation of the edited genes in response to heat stress. These results lend support to the hypothesis that RNA editing is a key molecular mechanism that underpins regulation of gene expression in dinoflagellates.

De novo assembly of the complete mitochondrial genome of pepino (Solanum muricatum) using PacBio HiFi sequencing: insights into structure, phylogenetic implications, and RNA editing

BackgroundSolanum muricatum is an emerging horticultural fruit crop with rich nutritional and antioxidant properties. Although the chromosome-scale genome of this species has been sequenced, its mitochondrial genome sequence has not been reported to date.ResultsPacBio HiFi sequencing was used to assemble the circular mitogenome of S. muricatum, which was 433,466 bp in length. In total, 38 protein-coding, 19 tRNA, and 3 rRNA genes were annotated. The reticulate mitochondrial conformations with multiple junctions were verified by polymerase chain reaction, and codon usage, sequence repeats, and gene migration from chloroplast to mitochondrial genome were determined. A collinearity analysis of eight Solanum mitogenomes revealed high structural variability. Overall, 585 RNA editing sites in protein coding genes were identified based on RNA-seq data. Among them, mttB was the most frequently edited (52 times), followed by ccmB (46 times). A phylogenetic analysis based on the S. muricatum mitogenome and those of 39 other taxa (including 25 Solanaceae species) revealed the evolutionary and taxonomic status of S. muricatum.ConclusionsWe provide the first report of the assembled and annotated S. muricatum mitogenome. This information will help to lay the groundwork for future research on the evolutionary biology of Solanaceae species. Furthermore, the results will assist the development of molecular breeding strategies for S. muricatum based on the most beneficial agronomic traits of this species.

Increasing CRISPR/Cas9-mediated gene editing efficiency in T7 phage by reducing the escape rate based on insight into the survival mechanism.

Bacteriophages have been used across various fields, and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of bacteriophages. However, some bacteriophages can escape from the cleavage of Cas protein, such as Cas9, and decrease the efficiency of genome editing. This study focuses on the bacteriophage T7, which is widely utilized but whose mechanism of evading the cleavage of CRISPR/Cas9 has not been elucidated. First, we test the escape rates of T7 phage at different cleavage sites, ranging from 10 -2 to 10 -5. The sequencing results show that DNA point mutations and microhomology-mediated end joining (MMEJ) at the target sites are the main causes. Next, we indicate the existence of the hotspot DNA region of MMEJ and successfully reduce MMEJ events by designing targeted sites that bypass the hotspot DNA region. Moreover, we also knock out the ATP-dependent DNA ligase 1. 3 gene, which may be involved in the MMEJ event, and the frequency of MMEJ at 4. 3 is reduced from 83% to 18%. Finally, the genome editing efficiency in T7 Δ 1. 3 increases from 20% to 100%. This study reveals the mechanism of T7 phage evasion from the cleavage of CRISPR/Cas9 and demonstrates that the special design of editing sites or the deletion of key gene 1. 3 can reduce MMEJ events and enhance gene editing efficiency. These findings will contribute to advancing CRISPR/Cas-based tools for efficient genome editing in phages and provide a theoretical foundation for the broader application of phages.

Elucidating the multichromosomal structure within the Brasenia schreberi mitochondrial genome through assembly and analysis

Brasenia schreberi, a plant species traditionally utilized in Chinese medicine and cuisine, represents an early evolutionary stage among flowering plants (angiosperms). While the plastid genome of this species has been published, its mitochondrial genome (mitogenome) has not been extensively explored, with a notable absence of thorough comparative analyses of its organellar genomes. In our study, we had assembled the entire mitogenome of B. schreberi utilizing the sequencing data derived from both Illumina platform and Oxford Nanopore. The B. schreberi mitogenome mostly exists as six circular DNA molecules, with the largest being 628,257 base pairs (bp) and the smallest 110,220 bp, amounting to 1.49 megabases (Mb). Then we annotated the mitogenome of B. schreberi. The mitogenome encompasses a total of 71 genes: 40 of these are coding proteins genes (PCGs), 28 are genes for transfer RNA (tRNA), and the remaining 3 are genes for ribosomal RNA (rRNA). In the analysis of codon usage, we noted a unique codon preference specific to each amino acid. The most commonly used codons exhibited an average RSCU of 1.36, indicating a noticeable bias in codon selection. In the repeat sequence analysis, a total of 553 simple sequence repeats (SSRs) were identified, 1,822 dispersed repeats (comprising 1,015 forward and 807 palindromic repeats), and 608 long terminal repeats (LTRs). Additionally, in the analysis of homologous sequences between organelle genomes, we detected 38 homologous sequences derived from the plastid genome, each exceeding 500 bp, within the B. schreberi mitochondrial genome. Notably, ten tRNA genes (trnC-GCA, trnM-CAU, trnI-CAU, trnQ-UUG, trnN-GUU, trnT-GGU, trnW-CCA, trnA-UGC, trnI-GAU, and trnV-GAC) appear to have been completely transferred from the chloroplast to the mitogenome. Utilizing the Deepred-mt to predict the RNA editing sites in the mitogenome, we have identified 675 high-quality RNA editing sites in the 40 mitochondrial PCGs. In the final stage of our study, we performed an analysis of colinearity and inferred the phylogenetic relationship of B. schreberi with other angiosperms, utilizing the mitochondrial PCGs as a basis. The results showed that the non-coding regions of the B. schreberi mitogenome are characterized by an abundance of repetitive sequences and exogenous sequences, and B. schreberi is more closely related with Euryale ferox.

FokI-RYdCas9 Mediates Nearly PAM-Less and High-Precise Gene Editing in Human Cells.

The demand for high-precision CRISPR/Cas9 systems in biomedicine is experiencing a notable upsurge. The editing system fdCas9 employs a dual-sgRNA strategy to enhance editing accuracy. However, the application of fdCas9 is constrained by the stringent requirement for two protospacer adjacent motifs (PAMs) of Cas9. Here, we devised an optimized editor, fRYdCas9, by merging FokI with the nearly PAM-less RYdCas9 variant, and two fRYdCas9 systems formed a dimer in a proper spacer length to accomplish DNA cleavage. In comparison to fdCas9, fRYdCas9 demonstrates a substantial increase in the number of editable genomic sites, approximately 330-fold, while maintaining a comparable level of editing efficiency. Through meticulous experimental validation, we determined that the optimal spacer length between two FokI guided by RYdCas9 is 16 base pairs. Moreover, fRYdCas9 exhibits a near PAM-less feature, along with no on-target motif preference via the library screening. Meanwhile, fRYdCas9 effectively addresses the potential risks of off-targets, as analyzed through whole genome sequencing (WGS). Mouse embryonic editing shows fRYdCas9 has robust editing capabilities. This study introduces a potentially beneficial alternative for accurate gene editing in therapeutic applications and fundamental research.

RNA editing factor SlORRM2 regulates the formation of fruit pointed tips in tomato.

Domestication of tomato (Solanum lycopersicum) has led to large variation in fruit size and morphology. The development of the distal end of the fruit is a critical factor in determining its overall shape. However, the intricate mechanisms underlying distal fruit development require further exploration. This study aimed to investigate the regulatory role of an organelle RNA recognition motif (RRM)-containing protein SlORRM2 in tomato fruit morphology development. Mutant plants lacking SlORRM2 exhibited fruits with pointed tips at the distal end. However, this phenotype could be successfully restored through the implementation of a "functional complementation" strategy. Our findings suggest that the formation of pointed tips in the fruits of the CR-slorrm2 mutants is linked to alterations in the development of the ovary and style. We observed a substantial decrease in the levels of indole-3-acetic acid (IAA) and altered expression of IAA-related response genes in the ovary and style tissues of CR-slorrm2. Moreover, our data demonstrated that SlORRM2 plays a role in regulating mitochondrial RNA editing sites, particularly within genes encoding various respiratory chain subunits. Additionally, the CR-slorrm2 mutants exhibited modified organellar morphology and increased levels of reactive oxygen species. These findings provide valuable insights into the mechanisms underlying the formation of fruit pointed tips in tomato and offer genetic resources for tomato breeding.

Insights into U-to-C RNA editing from the lycophyte Phylloglossum drummondii.

The lycophyte Phylloglossum drummondii is the sole inhabitant of its genus in the Huperzioideae group and one of a small minority of plants which perform uridine to cytidine RNA editing. We assembled the P.drummondii chloroplast and mitochondrial genomes and used RNA sequence data to build a comprehensive profile of organellar RNA editing events. In addition to many C-to-U editing events in both organelles, we found just four U-to-C editing events in the mitochondrial transcripts cob, nad1, nad5 and rpl2. These events are conserved in related lycophytes in the genera Huperzia and Phlegmariurus. De novo transcriptomes for three of these lycophytes were assembled to search for putative U-to-C RNA editing enzymes. Four putative U-to-C editing factors could be matched to the four mitochondrial U-to-C editing sites. Due to the unusually few numbers of U-to-C RNA editing sites, P. drummondii and related lycophytes are useful models for studying this poorly understood mechanism.

Characterization and Function Analysis of miRNA Editing during Fat Deposition in Chinese Indigenous Ningxiang Pigs.

This study aimed to identify active miRNA editing sites during adipose development in Ningxiang pigs and analyze their characteristics and functions. Based on small RNA-seq data from the subcutaneous adipose tissues of Ningxiang pigs at four stages-30 days (piglet), 90 days (nursery), 150 days (early fattening), and 210 days (late fattening)-we constructed a developmental map of miRNA editing in the adipose tissues of Ningxiang pigs. A total of 505 miRNA editing sites were identified using the revised pipeline, with C-to-U editing types being the most prevalent, followed by U-to-C, A-to-G, and G-to-U. Importantly, these four types of miRNA editing exhibited base preferences. The number of editing sites showed obvious differences among age groups, with the highest occurrence of miRNA editing events observed at 90 days of age and the lowest at 150 days of age. A total of nine miRNA editing sites were identified in the miRNA seed region, with significant differences in editing levels (p < 0.05) located in ssc-miR-23a, ssc-miR-27a, ssc-miR-30b-5p, ssc-miR-15a, ssc-miR-497, ssc-miR-15b, and ssc-miR-425-5p, respectively. Target gene prediction and KEGG enrichment analyses indicated that the editing of miR-497 might potentially regulate fat deposition by inhibiting adipose synthesis via influencing target binding. These results provide new insights into the regulatory mechanism of pig fat deposition.

New comparative genomic evidence supporting the proteomic diversification role of A-to-I RNA editing in insects.

Adenosine-to-inosine (A-to-I) RNA editing, resembling A-to-G mutation, confers adaptiveness by increasing proteomic diversity in a temporal-spatial manner. This evolutionary theory named "proteomic diversifying hypothesis" has only partially been tested in very few organisms like Drosophila melanogaster, mainly by observing the positive selection on nonsynonymous editing events. To find additional genome-wide evidences supporting this interesting assumption, we retrieved the genomes of four Drosophila species and collected 20 deep-sequenced transcriptomes of different developmental stages and neuron populations of D. melanogaster. We systematically profiled the RNA editomes in these samples and performed meticulous comparative genomic analyses. Further evidences were found to support the diversifying hypothesis. (1) None of the nonsynonymous editing sites in D. melanogaster had ancestral G-alleles, while the silent editing sites had an unignorable fraction of ancestral G-alleles; (2) Only very few nonsynonymous editing sites in D. melanogaster had corresponding G-alleles derived in the genomes of sibling species, and the fraction of such situation was significantly lower than that of silent editing sites; (3) The few nonsynonymous editing with corresponding G-alleles had significantly more variable editing levels (across samples) than other nonsynonymous editing sites in D. melanogaster. The proteomic diversifying nature of RNA editing in Drosophila excludes the restorative role which favors an ancestral G-allele. The few fixed G-alleles in sibling species might facilitate the adaptation to particular environment and the corresponding nonsynonymous editing in D. melanogaster would introduce stronger advantage of flexible proteomic diversification. With multi-Omics data, our study consolidates the nature of evolutionary significance of A-to-I RNA editing sites in model insects.

Dissecting the molecular puzzle of the editosome core in Arabidopsis organelles

Over the last decade, the composition of the C-to-U RNA editing complex in embryophyte organelles has turned out to be much more complex than first expected. While PPR proteins were initially thought to act alone, significant evidences have clearly depicted a sophisticated mechanism with numerous protein-protein interaction involving PPR and non-PPR proteins. Moreover, the identification of specific functional partnership between PPRs also suggests that, in addition to the highly specific PPRs directly involved in the RNA target recognition, non-RNA-specific ones are required. Although some of them, such as DYW1 and DYW2, were shown to be the catalytic domains of the editing complex, the molecular function of others, such as NUWA, remains elusive. It was suggested that they might stabilize the complex by acting as a scaffold. We here performed functional complementation of the crr28–2 mutant with truncated CRR28 proteins mimicking PPR without the catalytic domain and show that they exhibit a specific dependency to one of the catalytic proteins DYW1 or DYW2. Moreover, we also characterized the role of the PPR NUWA in the editing reaction and show that it likely acts as a scaffolding factor. NUWA is no longer required for efficient editing of the CLB19 editing sites once this RNA specific PPR is fused to the DYW catalytic domain of its partner DYW2. Altogether, our results strongly support a flexible, evolutive and resilient editing complex in which RNA binding activity, editing activity and stabilization/scaffolding function can be provided by one or more PPRs.

Assembly and comparative analysis of the complete multichromosomal mitochondrial genome of Cymbidium ensifolium, an orchid of high economic and ornamental value

BackgroundOrchidaceae is one of the largest groups of angiosperms, and most species have high economic value and scientific research value due to their ornamental and medicinal properties. In China, Chinese Cymbidium is a popular ornamental orchid with high economic value and a long history. However, to date, no detailed information on the mitochondrial genome of any species of Chinese Cymbidium has been published.ResultsHere, we present the complete assembly and annotation of the mitochondrial genome of Cymbidium ensifolium (L.) Sw. The mitogenome of C. ensifolium was 560,647 bp in length and consisted of 19 circular subgenomes ranging in size from 21,995 bp to 48,212 bp. The genome encoded 35 protein-coding genes, 36 tRNAs, 3 rRNAs, and 3405 ORFs. Repeat sequence analysis and prediction of RNA editing sites revealed a total of 915 dispersed repeats, 162 simple repeats, 45 tandem repeats, and 530 RNA editing sites. Analysis of codon usage showed a preference for codons ending in A/T. Interorganellar DNA transfer was identified in 13 of the 19 chromosomes, with plastid-derived DNA fragments representing 6.81% of the C. ensifolium mitochondrial genome. The homologous fragments of the mitochondrial genome and nuclear genome were also analysed. Comparative analysis showed that the GC content was conserved, but the size, structure, and gene content of the mitogenomes varied greatly among plants with multichromosomal mitogenome structure. Phylogenetic analysis based on the mitogenomes reflected the evolutionary and taxonomic statuses of C. ensifolium. Interestingly, compared with the mitogenomes of Cymbidium lancifolium Hook. and Cymbidium macrorhizon Lindl., the mitogenome of C. ensifolium lost 8 ribosomal protein-coding genes.ConclusionIn this study, we assembled and annotated the mitogenome of C. ensifolium and compared it with the mitogenomes of other Liliidae and plants with multichromosomal mitogenome structures. Our findings enrich the mitochondrial genome database of orchid plants and reveal the rapid structural evolution of Cymbidium mitochondrial genomes, highlighting the potential for mitochondrial genes to help decipher plant evolutionary history.

Is RNA editing truly absent in the complex thalloid liverworts (Marchantiopsida)? Evidence of extensive RNA editing from Cyathodium cavernarum.

RNA editing is a crucial modification in plants' organellar transcripts that converts cytidine to uridine (C-to-U; and sometimes uridine to cytidine) in RNA molecules. This post-transcriptional process is controlled by the PLS-class protein with a DYW domain, which belongs to the pentatricopeptide repeat (PPR) protein family. RNA editing is widespread in land plants; however, complex thalloid liverworts (Marchantiopsida) are the only group reported to lack both RNA editing and DYW-PPR protein. The liverwort Cyathodium cavernarum (Marchantiopsida, Cyathodiaceae), typically found in cave habitats, was newly found to have 129 C-to-U RNA editing sites in its chloroplast and 172 sites in its mitochondria. The Cyathodium genus, specifically C. cavernarum, has a large number of PPR editing factor genes, including 251 DYW-type PPR proteins. These DYW-type PPR proteins may be responsible for C-to-U RNA editing in C. cavernarum. Cyathodium cavernarum possesses both PPR DYW proteins and RNA editing. Our analysis suggests that the remarkable RNA editing capability of C. cavernarum may have been acquired alongside the emergence of DYW-type PPR editing factors. These findings provide insight into the evolutionary pattern of RNA editing in land plants.

Abstract LB360: Establishment of predictive biomarkers for carcinogenesis in ulcerative colitis using RNA editing analyses

Abstract Background: Ulcerative colitis (UC) is a chronic inflammatory disease continuous from the rectum. We previously reported that chronic inflammation may induce the RNA-editing enzyme ADAR1 and promote carcinogenesis (Journal of Crohn's and Colitis, 2023). However, the relationship between RNA editing and carcinogenesis in UC remains unresolved because we could not identify specific RNA editing sites. Therefore, in this study, we performed a new comprehensive RNA analysis using RNA sequencing to clarify the role of RNA editing in the carcinogenesis process in UC and to construct a prediction model of carcinogenic risk. Methods: We performed immunostaining of ADAR1, an RNA-editing enzyme, in 139 cases of ulcerative colitis. In addition, normal colorectal tissue, UC inflammatory phase, normal rectal mucosa of UC carcinoma cases, and UC carcinoma sites were submitted to RNA-Seq testing in 6 cases each (24 cases total). Bioinformatics analysis was performed to compare RNA editing sites in each group. Results: RNA-seq analysis showed a significant increase in global RNA editing sites in the inflammatory phase compared to normal colon tissue (p = 0.02). This phenomenon was due to increased ADAR1 expression associated with type 1 interferon. We hypothesized that global RNA editing was decreased in normal colonic mucosa during the remission phase but that some sites of prolonged RNA editing might promote carcinogenesis. We attempted to identify carcinogenesis-specific RNA editing sites. We identified 72 cancer-specific RNA editing sites, mainly in long noncoding RNAs such as NEAT1 and in the 3'UTR region of mRNAs such as CTSS. Assuming that the accumulation of these RNA editing leads to carcinogenesis, pathway analysis revealed that pathways promoting cancer invasion and stemness are activated. When ROC curves were generated using these editing sites as markers, the AUC was 0.935, suggesting that analysis of RNA editing in the colonic mucosa of UC patients may be able to predict the risk of carcinogenesis. Conclusions: We identified potential editing sites for carcinogenesis in UC, allowing the identification of carcinogenic mechanisms in UC and the establishment of predictive markers for carcinogenic risk. Citation Format: Toshiaki Takahashi, Kunitoshi Shigeyasu, Yoshitaka Kondo, Hibiki Umeda, Kazuya Moriwake, Masashi Kayano, Kaori Nitta, Fuminori Teraishi, Yuzo Umeda, Hiroshi Tazawa, Toshiyoshi Fujiwara. Establishment of predictive biomarkers for carcinogenesis in ulcerative colitis using RNA editing analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB360.

Complete chloroplast genomes of pallavicinia longispina stephani and pleurozia subinflata (austin) austin

Recent progress in sequencing technology and the increasing number of chloroplast genomes available in liverworts have greatly improved our understanding of not only genome structure and evolution, but also its phylogeny and evolution. However, the plastome resource of this group is still ineffective, as the plastomes have been sequenced from a small number of species. In the present study, two newly sequenced chloroplast genomes from liverworts, one from Pallavicinia longispina Stephani and the other from Pleurozia subinflata (Austin) Austin were reported. The former genome has a length of 122,410 bp, with GC content 36.90%, and the latter has a length of 118,441 bp, with GC content 32.22%. A total of 132 and 127 genes were determined in the plastomes of P. longispina and P. subinflata, respectively. Furthermore, 41 short sequence repeats (SSRs), 419 editing sites and 15 non-adjacent repeats in the plastome of P. longispina, and 65 SSRs, 288 editing sites, and nine non-adjacent repeats in the plastome of P. subinflata were detected. Valuable genomic resources for further research on liverwort phylogeny, evolution, and population genetics are provided from this study. Bangladesh J. Bot. 53(1): 109-113, 2024 (March)

High-Throughput Screening of PAM-Flexible Cas9 Variants for Expanded Genome Editing in the Silkworm (Bombyx mori).

Genome editing provides novel opportunities for the precise genome engineering of diverse organisms. Significant progress has been made in the development of genome-editing tools for Bombyx mori (B. mori) in recent years. Among these, CRISPR/Cas9, which is currently the most commonly used system in lepidopteran insects, recognizes NGG protospacer adjacent motif (PAM) sequences within the target locus. However, Cas9 lacks the ability to target all gene loci in B. mori, indicating the need for Cas9 variants with a larger editing range. In this study, we developed a high-throughput screening platform to validate Cas9 variants at all possible recognizable and editable PAM sites for target sequences in B. mori. This platform enabled us to identify PAM sites that can be recognized by both xCas9 3.7 and SpCas9-NG variants in B. mori and to assess their editing efficiency. Cas9 shows PAM sites every 13 base pairs in the genome, whereas xCas9 3.7 and SpCas9-NG have an average distance of 3.4 and 3.6 base pairs, respectively, between two specific targeting sites. Combining the two Cas9 variants could significantly expand the targeting range of the genome, accelerate research on the B. mori genome, and extend the high-throughput rapid screening platform to other insects, particularly those lacking suitable NGG PAM sequences.

RNA sequences that direct selective ADAR editing from a SELEX library bearing 8-azanebularine

Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs’ ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.

Assembly and comparative analysis of the first complete mitochondrial genome of the invasive water hyacinth, Eichhornia crassipes

Eichhornia crassipes is an aquatic plant in tropical and subtropical regions, renowned for its notorious invasive tendencies. In this study, we assembled the complete mitogenome of E. crassipes into a single circle molecule of 397,361 bp. The mitogenome has 58 unique genes, including 37 protein-coding genes (PCGs), 18 tRNA genes, three rRNA genes, and 47 % GC content. Sixteen (6.93 %) homologous fragments, ranging from 31 bp to 8548 bp, were identified, indicating the transfer of genetic material from chloroplasts to mitochondria. In addition, we detected positive selection in six PCGs (ccmB, ccmC, ccmFC, nad3, nad4 and sdh4), along with the identification of 782 RNA editing sites across 37 mt-PCGs. These findings suggest a potential contribution to the robust adaptation of this invasive plant to the stressful environment. Lastly, we inferred that phylogenetic conflicts of E. crassipes between the plastome and mitogenome may be attributed to the difference in nucleotide substitution rates between the two organelle genomes. In conclusion, our study provided vital genomic resources for further understanding the invasive mechanism of this species and exploring the dynamic evolution of mitogenomes within the monocot clade.

Abstract 6250: The RNA editing landscape of lung cancer in never smokers

Abstract Adenosine-to-Inosine (A-to-I) editing represents a crucial RNA modification mechanism, holding the potential to drive carcinogenesis. This sophisticated post-transcriptional process is catalyzed by a series of ADAR (adenosine deaminase acting on RNA) family members, which can profoundly reshape the cellular epitranscriptomic landscape. While previous explorations have illuminated the important role of A-to-I editing in lung cancer tumorigenesis and prognosis, most studies have focused on lung cancer from smokers. To investigate the contribution on RNA-editing in lung cancers in never smokers (LCINS) , we embarked on a rigorous dissection of A-to-I RNA editing events in 603 LCINS. Harnessing transcriptome and whole-genome sequencing technologies, we identified 15,880 A-to-I sites, of which 75% were positively linked to the enzymatic activity of ADAR1. 92.80% of detected editing sites were located at Alu repetitive regions, with the remainder in non_Alu repeat (4.1%) and non-repeat regions (3.1%). The global RNA editing levels, measured as Alu Editing Index (AEI), were found to be significantly associated with ADAR1 expression, Tumor Mutational Burden, and Neoantigens Burden. A subtle correlation between global RNA editing levels and tumor stage was also noted. Delving into cancer-associated editing events, we identified 5,539 differentially edited sites in tumors compared with normal tissues, which were located at 823 genes, including several key tumor suppressors and oncogenes, like MDM2, BRAF, TP53, BRCA2, ATM, and VHL. Subsequent pathway analysis identified TGF-beta, Notch Signaling, and DNA Damage Response as the key pathways for cancer progression. Using the characterized A-to-I editing profiles, we built a prognostic risk stratification model, with high risk scores significantly linked to poor survival outcomes, even after adjusting for other clinical factors. Our study provides a comprehensive A-to-I editing landscape of lung cancer in never smokers, highlighting the potential roles of RNA modifications in the pathogenesis of this lethal disease. Citation Format: Jian Sang, Wei Zhao, Tongwu Zhang, Maria Teresa Landi. The RNA editing landscape of lung cancer in never smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6250.