Comprehensive identification, characterization, and expression analysis of the MORF gene family in Brassica napus.

BACKGROUND: RNA editing in chloroplast and mitochondrion transcripts of plants is an important type of post-transcriptional RNA modification in which members of the multiple organellar RNA editing factor gene family (MORF) play a crucial role. However, a systematic identification and characterization of MORF members in Brassica napus is still lacking. RESULTS: In this study, a total of 43 MORF genes were identified from the genome of the Brassica napus cultivar "Zhongshuang 11". The Brassica napus MORF (BnMORF) family members were divided into three groups through phylogenetic analysis. BnMORF genes distributed on 14 chromosomes and expanded due to segmental duplication and whole genome duplication repetitions. The majority of BnMORF proteins were predicted to be localized to mitochondria and chloroplasts. The promoter cis-regulatory element analysis, spatial-temporal expression profiling, and co-expression network of BnMORF genes indicated the involvement of BnMORF genes in stress and phytohormone responses, as well as growth and development. CONCLUSION: This study provides a comprehensive analysis of BnMORF genes and lays a foundation for further exploring their physiological functions in Brassica napus.

Synthesis and pharmacodynamic evaluation of Dihydropteridone derivatives against PDCoV in vivo and in vitro.

Porcine Delta Coronavirus (PDCoV) infection can induce serious dehydration, diarrhea and even death of piglets, which has caused huge losses to the breeding industry. PDCoV has been reported to have the potential for cross species transmission, and even reports of infecting humans have emerged. At present, there are still no effective prevention and control measures for PDCoV. In this study, we have designed and synthesized a series of unreported Dihydropteridone derivatives. All of these compounds were evaluated for the against PDCoV in vivo and in vitro for the first time. In this study, antiviral activity (17.34 ± 7.20 µM) and low cytotoxicity (>800 µM) was found in compound W8. Compound W8 exerts antiviral effect on PDCoV by inhibiting cell apoptosis and inflammatory factors caused by virus infection in vitro. In addition, lung and small intestinal lesions caused by PDCoV infection in mice could be significantly reduced by compound W8. These findings highlight the potential of compound W8 as a valuable therapeutic option against PDCoV infection, and lay a foundation for further research and development in this field.

FOS Inhibits the Differentiation of Intramuscular Adipocytes in Goats.

Goat intramuscular fat (IMF) deposition is precisely regulated by many key genes as well as transcription factors. Nevertheless, the potential of the regulators of goat IMF deposition remains undefined. In this work, we reported that the transcription factor FOS is expressed at a low level at the early differentiation stage and at a high level in late differentiation. The overexpression of FOS inhibited intramuscular adipocyte lipid accumulation and significantly downregulated the expressions of PPARγ, C/EBPß, C/EBPα, AP2, SREBP1, FASN, ACC, HSL, and ATGL. Consistently, the knockdown of FOS, facilitated by two distinct siRNAs, significantly promoted intramuscular adipocyte lipid accumulation. Moreover, our analysis revealed multiple potential binding sites for FOS on the promoters of PPARγ, C/EBPß, and C/EBPα. The expression changes in PPARγ, C/EBPß, and C/EBPα during intramuscular adipogenesis were opposite to that of FOS. In summary, FOS inhibits intramuscular lipogenesis in goats and potentially negatively regulates the expressions of PPARγ, C/EBPß, and C/EBPα genes. Our research will provide valuable data for the underlying molecular mechanism of the FOS regulation network of intramuscular lipogenesis.

The ATP Synthase γ Subunit ATPC1 Regulates RNA Editing in Chloroplasts.

RNA editing is the process of modifying RNA molecules by inserting, deleting, or substituting nucleotides. In flowering plants, RNA editing occurs predominantly in RNAs encoded by the organellar genomes of mitochondria and chloroplasts, and the main type of editing involves the substitution of cytidine with uridine at specific sites. Abnormal RNA editing in plants can affect gene expression, organelle function, plant growth, and reproduction. In this study, we report that ATPC1, the gamma subunit of ATP synthase in Arabidopsis chloroplasts, has an unexpected role in the regulation of editing at multiple sites of plastid RNAs. The loss of function of ATPC1 severely arrests chloroplast development, causing a pale-green phenotype and early seedling lethality. Disruption of ATPC1 increases the editing of matK-640, rps12-i-58, atpH-3'UTR-13210, and ycf2-as-91535 sites while decreasing the editing of rpl23-89, rpoA-200, rpoC1-488, and ndhD-2 sites. We further show that ATPC1 participates in RNA editing by interacting with known multiple-site chloroplast RNA editing factors, including MORFs, ORRM1, and OZ1. The transcriptome in the atpc1 mutant is profoundly affected, with a pattern of defective expression of chloroplast development-related genes. These results reveal that the ATP synthase γ subunit ATPC1 is involved in multiple-site RNA editing in Arabidopsis chloroplasts.

LncRNA HCP5-Encoded Protein Regulates Ferroptosis to Promote the Progression of Triple-Negative Breast Cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) are a class of RNA molecules that are longer than 200 nucleotides and were initially believed to lack encoding capability. However, recent research has found open reading frames (ORFs) within lncRNAs, suggesting that they may have coding capacity. Despite this discovery, the mechanisms by which lncRNA-encoded products are involved in cancer are not well understood. The current study aims to investigate whether lncRNA HCP5-encoded products promote triple-negative breast cancer (TNBC) by regulating ferroptosis. METHODS: We used bioinformatics to predict the coding capacity of lncRNA HCP5 and conducted molecular biology experiments and a xenograft assay in nude mice to investigate the mechanism of its encoded products. We also evaluated the expression of the HCP5-encoded products in a breast cancer tissue microarray. RESULTS: Our analysis revealed that the ORF in lncRNA HCP5 can encode a protein with 132-amino acid (aa), which we named HCP5-132aa. Further experiments showed that HCP5-132aa promotes TNBC growth by regulating GPX4 expression and lipid ROS level through the ferroptosis pathway. Additionally, we found that the breast cancer patients with high levels of HCP5-132aa have poorer prognosis. CONCLUSIONS: Our study suggests that overexpression of lncRNA HCP5-encoded protein is a critical oncogenic event in TNBC, as it regulates ferroptosis. These findings could provide new therapeutic targets for the treatment of TNBC.

ncFO: A Comprehensive Resource of Curated and Predicted ncRNAs Associated with Ferroptosis.

Ferroptosis is a form of regulated cell death driven by the accumulation of lipid hydroperoxides. Regulation of ferroptosis might be beneficial to cancer treatment. Non-coding RNAs (ncRNAs) are a class of RNA transcripts that generally cannot encode proteins and have been demonstrated to play critical roles in regulating ferroptosis. Herein, we developed ncFO, the ncRNA-ferroptosis association database, to document the manually curated and predicted ncRNAs that are associated with ferroptosis. Collectively, ncFO contains 90 experimentally verified entries, including 46 microRNAs (miRNAs), 21 long non-coding RNAs (lncRNAs), and 17 circular RNAs (circRNAs). In addition, ncFO also incorporates two online prediction tools based on the regulation and co-expression of ncRNA and ferroptosis genes. Using default parameters, we obtained 3260 predicted entries, including 598 miRNAs and 178 lncRNAs, by regulation, as well as 2,592,661 predicted entries, including 967 miRNAs and 9632 lncRNAs, by ncRNA-ferroptosis gene co-expression in more than 8000 samples across 20 cancer types. The detailed information of each entry includes ncRNA name, disease, species, tissue, target, regulation, publication time, and PubMed identifier. ncFO also provides survival analysis and differential expression analysis for ncRNAs. In summary, ncFO offers a user-friendly platform to search and predict ferroptosis-associated ncRNAs, which might facilitate research on ferroptosis and discover potential targets for cancer treatment. ncFO can be accessed at http://www.jianglab.cn/ncFO/.

Development of a universal antibiotic resistance screening reporter for improving efficiency of cytosine and adenine base editing.

Base editing has emerged as a revolutionary technology for single nucleotide modifications. The cytosine and adenine base editors (CBEs and ABEs) have demonstrated great potential in clinical and fundamental research. However, screening and isolating target-edited cells remains challenging. In the current study, we developed a universal Adenine and Cytosine Base-Editing Antibiotic Resistance Screening Reporter (ACBE-ARSR) for improving the editing efficiency. To develop the reporter, the CBE-ARSR was first constructed and shown to be capable of enriching cells for those that had undergone CBE editing activity. Then, the ACBE-ARSR was constructed and was further validated in the editing assays by four different CBEs and two versions of ABE at several different genomic loci. Our results demonstrated that ACBE-ARSR, compared to the reporter of transfection (RoT) screening strategy, improved the editing efficiency of CBE and ABE by 4.6- and 1.9-fold on average, respectively. We found the highest CBE and ABE editing efficiencies as enriched by ACBE-ARSR reached 90% and 88.7%. Moreover, we also demonstrated ACBE-ARSR could be employed for enhancing simultaneous multiplexed genome editing. In conclusion, both CBE and ABE activity can be improved significantly using our novel ACBE-ARSR screening strategy, which we believe will facilitate the development of base editors and their application in biomedical and fundamental research studies.

Prokaryotic Argonaute Protein from Natronobacterium gregoryi Requires RNAs To Activate for DNA Interference In Vivo.

The Argonaute proteins are present in all three domains of life, which are archaea, bacteria, and eukarya. Unlike the eukaryotic Argonaute proteins, which use small RNA guides to target mRNAs, some prokaryotic Argonaute proteins (pAgos) use a small DNA guide to interfere with DNA and/or RNA targets. However, the mechanisms of pAgo natural function remain unknown. Here, we investigate the mechanism by which pAgo from Natronobacterium gregoryi (NgAgo) targets plasmid and bacteriophage T7 DNA using a heterologous Escherichia coli-based model system. We show that NgAgo expressed from a plasmid linearizes its expression vector. Cotransformation assays demonstrate that NgAgo requires an RNA in trans that is transcribed from the bacteriophage T7 promoter to activate cleavage of a cotransformed plasmid, reminiscent of the trans-RNA function in CRISPR/Cas9. We propose a mechanism to explain how NgAgo eliminates invading foreign DNA and bacteriophage. By leveraging this discovery, we show that NgAgo can be programmed to target a plasmid or a chromosome locus. IMPORTANCE We revealed the mechanism that explains how the NgAgo eliminates the invading foreign DNA and bacteriophage in bacterial cells at 37°C, and by leveraging this discovery, NgAgo can be programmed to target a plasmid or a chromosome locus.

Expression and Functional Analysis of the Argonaute Protein of Thermus thermophilus (TtAgo) in E. coli BL21(DE3).

The prokaryotic Argonaute proteins (pAgos) have been reported to cleave or interfere with DNA targets in a guide-dependent or independent manner. It is often difficult to characterize pAgos in vivo due to the extreme environments favored by their hosts. In the present study, we expressed functional Thermus thermophilus pAgo (TtAgo) in E. coli BL21 (DE3) cells at 37 °C. Initial attempts to express TtAgo in BL21(DE3) cells at 37 °C failed. This was not because of TtAgo mediated general toxicity to the host cells, but instead because of TtAgo-induced loss of its expression plasmid. We employed this discovery to establish a screening system for isolating loss-of-function mutants of TtAgo. The E. colifabI gene was used to help select for full-length TtAgo loss of function mutants, as overexpression of fabI renders the cell to be resistant to the triclosan. We isolated and characterized eight mutations in TtAgo that abrogated function. The ability of TtAgo to induce loss of its expression vector in vivo at 37 °C is an unreported function that is mechanistically different from its reported in vitro activity. These results shed light on the mechanisms by which TtAgo functions as a defense against foreign DNA invasion.

LncRNA-Encoded Peptide: Functions and Predicting Methods.

Long non-coding RNA (lncRNA) was originally defined as the representative of the non-coding RNAs and unable to encode. However, recent reports suggest that some lncRNAs actually contain open reading frames that encode peptides. These coding products play important roles in the pathogenesis of many diseases. Here, we summarize the regulatory pathways of mammalian lncRNA-encoded peptides in influencing muscle function, mRNA stability, gene expression, and so on. We also address the promoting and inhibiting functions of the peptides in different cancers and other diseases. Then we introduce the computational predicting methods and data resources to predict the coding ability of lncRNA. The intention of this review is to provide references for further coding research and contribute to reveal the potential prospects for targeted tumor therapy.

An Improved Genome Engineering Method Using Surrogate Reporter-Coupled Suicidal ZFNs.

Using engineered nucleases such as zinc-finger nucleases (ZFNs) and TALE nuclease (TALEN) to accomplish genome editing often causes high cellular toxicity because of the consistent expression of artificial nucleases and off-targeting effect. And lacking selection marker in modified cells makes it hard to enrich these positive cells. Here we introduce a method by incorporating a surrogate reporter enrichment into a suicidal ZFN system, which is designed by a pair of ZFN expression cassettes flanked with its target sites. Our data demonstrated that this modified system achieved almost the same ZFN activity as the original method but reduced ~40% toxicity. This new suicidal ZFN expression system coupled with a surrogate reporter not only enables decreased cellular toxicity but also makes the genetic modified cells to be enriched by EGFP analysis.

Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure.

The clustered regularly interspaced short palindromic repeats (CRISPR) system has recently been developed into a powerful genome-editing technology, as it requires only two key components (Cas9 protein and sgRNA) to function and further enables multiplex genome targeting and homology-directed repair (HDR) based precise genome editing in a wide variety of organisms. Here, we report a novel and interesting strategy by using the Drosha-mediated sgRNA-shRNA structure to direct Cas9 for multiplex genome targeting and precise genome editing. For multiplex genome targeting assay, we achieved more than 9% simultaneous mutant efficiency for 3 genomic loci among the puromycin-selected cell clones. By introducing the shRNA against DNA ligase IV gene (LIG4) into the sgRNA-shRNA construct, the HDR-based precise genome editing efficiency was improved as more than 2-fold. Our works provide a useful tool for multiplex and precise genome modifying in mammalian cells.