Modern genetic engineering technologies, such as base editing and prime editing (PE), have proven to provide the efficient and reliable genome editing tools that obviate the need for donor templates and double-strand breaks (DSBs) introduced in DNA. Relatively new, they quickly gained recognition for their accuracy, simplicity, and multiplexing capabilities. The review summarizes the new literature on the technologies and considers their architecture, methods to create editors, specificity, efficiency, and versatility. Advantages and disadvantages of the editors are discussed along with their prospective use in basic and applied research. The review may be useful for planning genome editing studies and analyzing their results to solve various problems of fundamental biology, biotechnology, medicine, and agriculture.
In previous studies, we found that the zinc finger proteins Su(Hw) and CG9890 interact with the Drosophila SAGA complex and participate in the formation of the active chromatin structure and transcription regulation. In this research, we discovered the interaction of the DUB module of the SAGA complex with another zinc finger protein, CG9609. ChIP-Seq analysis was performed, and CG9609 binding sites in the Drosophila genome were identified. Analysis of binding sites showed that they are localized predominantly at gene promoters. The CG9609 protein has been shown to be involved in the regulation of gene expression.
Eukaryotic translation release factor eRF1 is an important cellular protein that plays a key role in translation termination, nonsense-mediated mRNA decay (NMD), and readthrough of stop codons. The amount of eRF1 in the cell influences all these processes. The mechanism of regulation of eRF1 translation through an autoregulatory NMD-dependent expression circuit has been described for plants and fungi, but the mechanisms of regulation of human eRF1 translation have not yet been studied. Using reporter constructs, we studied the effect of eRF1 mRNA elements on its translation in cell-free translation systems and HEK293 cell culture. Our data indicate the absence of an NMD-dependent autoregulatory circuit for human eRF1 expression. We found that the translation of the eRF1 coding sequence is most strongly influenced by the 5′ untranslated region of eRF1 mRNA and the start codon of the upstream open reading frame. According to the transcription start database, eRF1 mRNA is characterized by high heterogeneity of the transcription start and a variable 5' untranslated region in length. In addition, the start codon of the CDS in eRF1 mRNA is located within the known translational regulator of short 5' untranslated regions (TISU), which also stimulates mRNA transcription of genes with high transcription start heterogeneity. We hypothesize that regulation of human eRF1 synthesis occurs at both the transcriptional and translational levels. At the transcription level, the length of the eRF1 5' untranslated region and the number of the upstream open reading frames in it are regulated. This regulation in turn, regulates the production of eRF1 at the translation level.
The sensitivity of human glioblastoma cells to virus-mediated oncolysis was investigated on five patient-derived cell lines. Primary glioblastoma cells (Gbl13n, Gbl16n, Gbl17n, Gbl25n, and Gbl27n) were infected with tenfold serial dilutions of the Leningrad-3 strain of the mumps virus, and virus reproduction and cytotoxicity were monitored for 96 −120 h. Immortalized human non-tumor NKE cells were used as controls to determine the virus specificity. Four out of the five glioblastoma cell lines examined were susceptible to mumps virus infection, whereas no virus reproduction was observed in the non-tumor cell line. Moreover, the level of proapoptotic caspase-3 activity was increased in all infected cells 48 h after infection. The kinetics of viral RNA accumulation in the studied glioblastoma cell lines was comparable with the rate of cell death. The data suggest that glioblastoma cell lines were permissive for the mumps virus. Glioblastoma cell lines differed in type I IFN production in response to the mumps virus infection. In addition, it was shown that MV infection was able to induce immunogenic death of glioblastoma cells.
Small non-coding RNAs (sRNAs, also called sncRNAs) known as gene expression regulatory factors are capable of modulating mRNA functions through complementary base pairing. A number of studies has shown that when exposed to radiation, the expression of drug resistance genes increases in some cells. Here, in Escherichia coli subjected to 12C6+ heavy ion beams or X-ray exposure, five sRNAs (sRNA120, sRNA127, sRNA109, sRNA72, sRNA53) with elevated expression were identified by deep sequencing and sRNAscanner prediction. To investigate whether they have a potential role in drug resistance, we engineered strains overexpressing these sRNAs, and assessed their survival rate under sub-lethal antibiotic concentrations. It was noteworthy that under Gentamicin, Norfloxacin, Chloramphenicol and Cfotaxime, the survival rate of ::sRNA53 strain was 1.0667, 1.0251, 1.3797 and 3.9327 times higher, respectively, than for the control and strains overexpressing other sncRNAs. TargetRNA2 software identified lsrA as a likely target gene regulated by sRNA53 based on binding free energy calculations. We analyzed the interaction sites between sRNA53 and lsrA and measured the expression of these molecules in various mutants using RT-qPCR. We also investigated the regulation of Luxs/AI-2 system by the lsr operon and the biofilm formation of mutants. In the ::sRNA53 strain, the log2FC for sRNA53 and lsrA concurrently escalated by 1.8533 and 1.7367-fold. Additionally, the biofilm formation ability of ::sRNA53, ::lsrA, and ::sRNA53::lsrA (co-expression) strains was increased 5.4542, 3.946, and 7.1758-fold, respectively, compared to wild-type MG1655. Based on these data, we can conclude that sRNA53 plays a critical role in the development of antibiotic resistance in E. coli. Apparently, the action of sRNA53 targets the lsrA gene, which, by modulating the Luxs/AI-2 system, affects the ability to biofilm formation and drug resistance of the bacterium. The study shows that a new sRNA, named sRNA53, is involved in the formation of tolerance to sub-lethal doses of various antibiotics.
The bony fish Danio rerio (zebrafish) has become one of the important vertebrate model organisms in biomedical cancer research and is used, among other things, for the development of anticancer drugs using xenotransplantation approaches. The ex utero development of zebrafish, optically transparent tissues in the first month of growth, and the immature adaptive immune system during this period greatly facilitate the manipulation of embryos. For highly aggressive cancers where patient survival may be expected to be only a few months, a zebrafish xenograft assay may be the only appropriate method as it requires only four to seven days. Thousands of embryos can be implanted with biopsy tissue from a patient to produce zebrafish xenografts and to use them to screen a large number of drugs and compounds automatically to develop an effective treatment regimen for a specific patient. This review examines the advantages and disadvantages of the zebrafish model in oncology research. The main focus is on the use of zebrafish xenografts to study metastasis and to create avatars in personalized medicine.
As a result of molecular domestication of the gag gene of errantiviruses, the Gagr gene was formed in the genome of Drosophila melanogaster. It has previously been shown that the Gagr gene is transcribed at the highest level in gut tissues relative to other tissues, and its transcription is most effectively induced in females in response to ammonium persulfate added to the nutrient medium. In the present work, the gut transcriptome of females with knockdown of the Gagr gene was studied in all tissues under standard conditions and under stress conditions caused by ammonium persulfate. It was revealed that in females with knockdown of the Gagr gene, the genes of antimicrobial peptides controlled by the Toll and Imd signaling pathways are activated in the gut. Induction of a stress response by ammonium persulfate revealed disruption of the JAK/STAT and JNK/MAPK signaling pathways and an almost complete absence of activation of the ER-stress and UPR-stress pathways in flies with the Gagr gene knockdown. The data obtained confirm the important role of the Gagr gene in maintaining homeostasis and the immune response.
ENY2 is an evolutionarily conserved multifunctional protein and is a member of several complexes that regulate various stages of gene expression. ENY2 is a subunit of the TREX-2 complex, which is necessary for the export of bulk mRNA from the nucleus to the cytoplasm through the nuclear pores in many eukaryotes. The wide range of ENY2 functions suggests that it can also associate with other protein factors or complexes. In a search for proteins that interact with ENY2 of Drosophila melanogaster, a cDNA library was screened in a yeast two-hybrid system. ENY2 was thus found to interact with the RNA-binding protein Paip2. Paip2 directly bound ENY2 in vitro and interacted with ENY2 in vivo at the molecular and genetic levels. Paip2 was capable of association with the ENY2-containing TREX-2 complex. Paip2 was present at the locus of the histone gene cluster. Both Paip2 and ENY2 were detected at histone locus body (HLBs), nuclear structure where coordinated histone mRNA transcription and processing take place. Paip2 and subunits of the TREX-2 complex were shown to associate with histone mRNP particles. A Paip2 knockdown via RNA interference resulted in decreased binding of TREX-2 subunits to histone mRNPs. Thus, Paip2 was identified as a new partner protein of ENY2 within the TREX-2 complex and suggested to participate in TREX-2 binding to histone mRNPs.
SINEs are mobile genetic elements of multicellular eukaryotes that arose during evolution from various tRNAs, as well as from 5S rRNA and 7SL RNA. Like the genes of these RNAs, SINEs are transcribed by RNA polymerase III. The transcripts of some mammalian SINEs have the capability of AAUAAA-dependent polyadenylation, which is unique for transcript generated by RNA polymerase III. Despite a certain similarity with canonical polyadenylation of mRNAs (transcripts of RNA polymerase II), these processes apparently differ significantly. The purpose of this work is to evaluate how important for polyadenylation of SINE transcripts are proteins of the CPSF complex formed by mPSF and mCF subcomplexes which direct mRNA polyadenylation. In HeLa cells, siRNA knockdowns of the CPSF components were carried out, after which the cells were transfected with plasmid constructs containing SINEs. A decrease in polyadenylation of the SINE transcripts as a result of the knockdown of the proteins was evaluated by Northern-hybridization. It turned out that the CPSF components, such as Wdr33 and CPSF30, contributed to the polyadenylation of SINE transcriptions, while the knockdown of CPSF100, CPSF73, and symplekin did not reduce the polyadenylation of these transcripts. Wdr33 and CPSF30, along with the CPSF160 and Fip1 previously studied, are components of the subcomplex mPSF responsible for mRNA polyadenylation. Thus, the available data suggest the importance of all mPSF proteins for polyadenylation of SINE transcripts. At the same time, CPSF100, CPSF73, and symplekin, forming the subcomplex mCF, are responsible for the cleavage of pre-mRNA; therefore, their non-participation in the polyadenylation of SINE transcriptions seems quite natural.
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：