Post-transcriptional mechanisms controlling neurogenesis and direct neuronal reprogramming

Neurogenesis is a tightly regulated process in time and space both in the developing embryo and in adult neurogenic niches.A drastic change in the transcriptome and proteome of radial glial cells or neural stem cells towards the neuronal state is achieved due to sophisticated mechanisms of epigenetic,transcriptional,and post-transcriptional regulation.Understanding these neurogenic mechanisms is of major importance,not only for shedding light on very complex and crucial developmental processes,but also for the identification of putative reprogramming factors,that harbor hierarchically central regulatory roles in the course of neurogenesis and bare thus the capacity to drive direct reprogramming towards the neuronal fate.The major transcriptional programs that orchestrate the neurogenic process have been the focus of research for many years and key neurogenic transcription factors,as well as repressor complexes,have been identified and employed in direct reprogramming protocols to convert non-neuronal cells,into functional neurons.The post-transcriptional regulation of gene expression during nervous system development has emerged as another important and intricate regulatory layer,strongly contributing to the complexity of the mechanisms controlling neurogenesis and neuronal function.In particular,recent advances are highlighting the importance of specific RNA binding proteins that control major steps of mRNA life cycle during neurogenesis,such as alternative splicing,polyadenylation,stability,and translation.Apart from the RNA binding proteins,microRNAs,a class of small non-coding RNAs that block the translation of their target mRNAs,have also been shown to play crucial roles in all the stages of the neurogenic process,from neural stem/progenitor cell proliferation,neuronal differentiation and migration,to functional maturation.Here,we provide an overview of the most prominent post-transcriptional mechanisms mediated by RNA binding proteins and microRNAs during the neurogenic process,giving particular emphasis on the interplay of specific RNA binding proteins with neurogenic microRNAs.Taking under consideration that the molecular mechanisms of neurogenesis exert high similarity to the ones driving direct neuronal reprogramming,we also discuss the current advances in in vitro and in vivo direct neuronal reprogramming approaches that have employed microRNAs or RNA binding proteins as reprogramming factors,highlighting the so far known mechanisms of their reprogramming action.

Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene(VdThit)confers resistance to Verticillium wilt in cotton

Verticillium wilt(VW),induced by the soil-borne fungus Verticillium dahliae(Vd),poses a substantial threat to a diverse array of plant species.Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures.In this study,we successfully generated two stable transgenic lines of cotton(Gossypium hirsutum L.),VdThitRNAi-1 and VdThit-RNAi-2,using host-induced gene silencing(HIGS)technology to introduce double-stranded RNA(dsRNA)targeting the thiamine transporter protein gene(VdThit).Southern blot analysis confirmed the presence of a single-copy insertion in each line.Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type(WT).The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions.Real-time quantitative PCR(qRT-PCR)analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton.Small RNA sequencing(sRNA-Seq)analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines.Additionally,the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd.Under field conditions,VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT.In summary,our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton,thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.

Features,Mechanisms and Applies of Post-transcriptional Gene Silencing in Transgenic Plants

Since transgene silencing was found in transgenic plants,many scholars have studied it extensively and considered that it has three functional mechanisms:post dependent gene silencing,transcriptional gene silencing,post transcriptional gene silencing.At the moment,people have mainly focused on the study of post transcriptional gene silencing and found its features:extensivity,conduction and peculiarity,also put forward some hypothesis for its mechanisms,for example,RNA threshold model,aberrant RNA model,inter or intra molecular base pairing model and so on.Furthermore,post transcriptional gene silencing is being applied in gene engineering of plants.Recently the people have found that post transcriptional gene silencing has bearing on capacity plants resisting virus.Many researchers have studied post transcriptional gene silencing,but there are some questions which need be solved in the future.This article summarizes progresses in features,mechanisms,applies of post transcriptional gene silencing about transgenic plants.

Host-Induced Gene Silencing of Effector AGLIP1 Enhanced Resistance of Rice to Rhizoctonia solani AG1-IA

Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing(TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1, were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into si RNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection.

Stress response silencing by an E3 ligase mutated in neurodegeneration

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis,yet their prolonged activation induces apoptosis and disrupts organismal health1-3.How stress responses are turned off at the right time and place remains poorly understood.Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress.

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

While human immunodeficiency virus 1(HIV-1) infectionis controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from the body. Despite decades of research there is still no effective vaccine to prevent HIV-1 infection. Therefore, the possibility of an RNA interference(RNAi)-based cure has become an increasingly explored approach. Endogenous gene expression is controlled at both, transcriptional and post-transcriptional levels by noncoding RNAs, which act through diverse molecular mechanisms including RNAi. RNAi has the potential to control the turning on/off of specific genes through transcriptional gene silencing(TGS), as well as finetuning their expression through post-transcriptional gene silencing(PTGS). In this review we will describe in detail the canonical RNAi pathways for PTGS and TGS, the relationship of TGS with other silencing mechanisms and will discuss a variety of approaches developed to suppress HIV-1 via manipulation of RNAi. We will briefly compare RNAi strategies against other approaches developed to target the virus, highlighting their potential to overcome the major obstacle to finding a cure, which is the specific targeting of the HIV-1 reservoir within latently infected cells.

Writing the Silenced Female Body, Scrutinizing the Grand Narrative of Colonization and Decolonization: The Body in Zoë Wicomb’s David’s Story

David’s Story by Zoë Wicomb addresses the complexities of representing female suffering and the limitations of traditional historical narratives in capturing the experiences of marginalized bodies.It challenges the grand narratives of national history by emphasizing the indispensable role of women’s experiences.Through characters like Dulcie and Rachael,Wicomb portrays the female body as a site of resistance and resilience,highlighting the need for more nuanced and inclusive ways of documenting history.Underscoring the inexpressibility of trauma and the limitations of language and representation,the novel self-reflexively acknowledges its own aporia of completing the narrative,embodying the ongoing struggle to capture the full breadth of human experience.

Differences of the Silencing Effect at Cellular Level and Individual Level

[Objective]The aim was to compare the silencing effect of three vectors containing shRNA quartet of different lengths at cellular level and individual level.[Method]To silence EGFP gene,three shRNA quartets of 21,27 and 29 bp composed of hU6,mU6,h7SK and hH1 promoters were constructed and then cloned into silencing vectors.After the vectors were transfected into Vero cells or injected into mouse muscle,real-time fluorescence quantitative PCR was conducted to detect the mRNA level of EGFP gene.[Result]All the three vectors showed better silencing effect;in which,the silencing effect at the cellular level was far higher than that at individual level.[Conclusion]Multi-site tandem expression shRNA may show better silencing effect;intramuscular injection maybe a transgenic method for baby mouse.

Using Gene Silencing Technology to Create Blast Resistant Rice Resources

Rice blast disease is one of the most devastating diseases in rice production,which severely affects the high and stable yield of rice.The formation of appressorium plays a key role in the pathogenesis of Magnaporthe grisea in rice.It has been confirmed that a P-type ATPase （P-ATPase） is involved in the formation of appressorium.A number of small molecular substances are able to enter the pathogen from the host during the interactions between pathogens and hosts,thus resisting the infection of pathogens.In this study,a 232 bp DNA sequence with good specificity from the first exon of P-ATPase gene MgAPT2 was used as an interference fragment and was inserted into interference vector forward and reversely.The interfering vector was then transformed into rice blast-susceptible rice variety Nipponbare via Agrobacterium-mediated transformation.Identification of rice plants inoculated with M.grisea at the seedling stage and detection of the expression level of P-ATPase gene MgAPT2 showed that the expression level of MgAPT2 gene in transgenic plants was reduced and the rice blast resistance was improved.This study provided a new way for the innovation of rice germplasm resources resistant to rice blast disease.

Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injury

We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was signiifcantly enhanced in the model group. Af-ter 8 weeks, the number of horseradish peroxidase-labeled nerve ifbers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and signiifcantly higher than in the model group. The newly formed nerve ifbers and myelinated ner ve ifbers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.

Targeted Silencing of Heparanase Gene by Small Interfering RNA Inhibits Invasiveness and Metastasis of Osteosarcoma Cells

The effects of targeted silencing of heparanase gene by small interfering RNA（siRNA） on invasiveness and metastasis of osteosarcoma cells（MG63 cells） were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA（pGenesil-shRNA） was constructed successfully.MG63 cells were randomly allocated into 3 groups：blank group,empty vector（pGenesil） transfected group and expression vector（pGenesil-shRNA） transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63 cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell prolifera-tion was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels of heparanase were down-regulated by 76.1%（P0.01） and 75.3%（P0.01） respectively in the pGenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.

Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber,Apostichopus japonicus(Selenka),during aestivation

The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling （DNA （cytosine-5）-methyltransferase l, Methyl-CpG-binding domain protein 2）, and Chromodomain-helicase-DNA-binding protein 5） was significantly higher during aestivation （Days 20 and 40）. Similarly, we observed an increase in the expression of genes involved in histone acetylation （Histone deacetylase 3） and Histone-binding protein RBBP4） during the early （Days 5 and 10） and late phases （Days 20 and 40） of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers （Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5） was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.

Effects of gene silencing of CypB on gastric cancer cells

Objective:To determine the effect of gene silencing of cyclophilin B(CypB)on growth and proliferation of gastric cancer cells.Methods:CypB siRNA lentivirus(LV-CypB-si)and control lentivirus(LV-si-con)were produced.CypB expression in gastric cancer cell lines was detected by Western blot.BGC823 and SGC7901 cells were chosen to be infected with LV-sicon and LV-CypB-si,and stable transfectants were isolated.The cell groups transfected with LV-CypB-siRNA,LV-siRNA-con and transfected no carrier were served as the experimental group,the implicit control group and the blank control group respectively.MTT and colony formation assays were used to examine the effect of CypB on the cell growth and proliferation in vitro.Cell cycle was analyzed with flow cytometry.The expression of VEGFR of BGC823-si and SGC7901-si was detected by Western blot.Results:Gene silencing of CypB can inhibit gastric cancer cell growth,proliferation,cell cycle progress and tumorigenesis.CypB expression level was obviously higher in SGC7901 and BGC823 than MKN28 and GES.These two cell lines were infected with LV-si-con and LV-CypB-si respectively.MTT and cloney formation assays showed a significantly decreased rate of cell proliferation from the forth day or the fifth day in cells transfected with LV-CypB-si(P<0.05).Down-regulation of CypB resulted in slightly decreased percentage of S phase and increased percentage of G_1(P<0.05).These findings indicated that CypB could promote the G_1-S transition of gastric cancer cell.In addition,the expression of VEGF of BGC823 and SGC7901 transfected with CypB siRNA was reduced in comparison with the implicit control group and the blank control group.Conclusions:Gene silencing of CypB decreases gastric cancer cells proliferation and in vivo tumorigenesis.These findings indiccate CypB could be a potential biomarker and therapeutic target for gastric cancer.

Establishment and Verification of An Efficient Virus-induced Gene Silencing System in Forsythia

To understand the functional identification of large-scale genomic sequences in Forsythia,tobacco rattle virus(TRV)-mediated virus-induced gene silencing(VIGS),suitable for the plant,was explored in this study.The results showed that the TRV-mediated VIGS system could be successfully used in Forsythia for silencing the reporter gene FsPDS(Forsythia phytoene desaturase)using stem infiltration and leaf infiltrationmethods.All the treated plants were pruned below the injection site after 7–15 d infection;the FsPDS was silenced and typical photobleaching symptoms were observed in newly sprouted leaves at the whole-plant level.Meanwhile,this system has been successfully tested and verified through virus detection and qRT-PCR analysis.After the optimization,Forsythia magnesium chelatase subunit H(FsChlH)was silenced successfully in Forsythia using this system,resulting in yellow leaveswith decreased chlorophyll content.The system was stable,highly efficient and had greater rapidity and convenience,which made it suitable to study the function of genes related to physiological pathways such as growth and development,and metabolic regulation in Forsythia.

Studies on Trans-Generational Transcriptional Silencing of <i>cry</i>1<i>Ac</i>Gene in Tobacco Transgenics

Developing transgenics that express high levels of Cry1Ac protein, and at the same time, are phenotypically normal, has not been an easy task to achieve. It has been routinely observed that most of the transgenic plants that survive, show no or extremely low levels of Cry1Ac protein. However, all of these plants do express the selectable marker, nptII gene. In the present study, we record an interesting observation of how one of the genes (cry1Ac) on a single T-DNA fragment is selectively silenced, keeping the expression of the other gene (nptII) intact. Further, this silenced state is inherited.

Post-transcriptional regulation of vascular endothelial growth factor: Implications for tumor angiogenesis

Vascular endothelial growth factor （VEGF） is a potent secreted mitogen critical for physiologic and tumor angiogenesis. Regulation of VEGF occurs at several levels, including transcription, mRNA stabilization, translation, and differential cellular localization of various isoforms. Recent advances in our understanding of post-transcriptional regulation of VEGF include identification of the stabilizing mRNA binding protein, HuR, and the discovery of internal ribosomal entry sites in the 5＇UTR of the VEGF mRNA. Monoclonal anti-VEGF antibody was recently approved for use in humans, but suffers from the need for high systemic doses. RNA interference （RNAi） technology is being used in vitro and in animal models with promising results. Here, we review the literature on post-transcriptional regulation of VEGF and describe recent progress in targeting these mechanisms for therapeutic benefit.

RNAi-Mediated Silencing of ITPK Gene Reduces Phytic Acid Content,Alters Transcripts of Phytic Acid Biosynthetic Genes,and Modulates Mineral Distribution in Rice Seeds

Phytic acid is the principal storage form of phosphorus in plant seeds and an essential signalling molecule in several regulatory processes of plant development.However,it is known as an anti-nutrient compound owing to its potent chelating property.Thus,reducing the phytic acid content in crops is desirable.Studies involving regulation of MIPS and IPK1 genes to generate low phytate rice have been reported earlier.However,the functional significance of OsITPK and the effect of its down-regulation on phytic acid content and the associated pleiotropic effects on rice have not yet been investigated.In this study,tissue specific RNA interference(RNAi)-mediated down-regulation of a major ITPK homolog(OsITP5/6K-1)resulted in 46.2%decrease in phytic acid content of T2 transgenic seeds with a subsequent 3-fold enhancement in the inorganic phosphorus content.Silencing of OsITP5/6K-1 altered the transcript levels of essential phytic acid pathway genes,without significantly affecting the transcript levels of other OsITPK homologs.Furthermore,the mapping of elements through X-ray microfluorescence analysis revealed significant changes in the spatial distribution pattern and translocation of elements in low phytate seeds.Additionally,low phytate polished seeds exhibited 1.3-fold and 1.6-fold enhancement in iron and zinc content in the grain endosperm,respectively.Silencing of OsITP5/6K-1 also altered the amino acid and myo-inositol content of the transgenic seeds.Our results successfully established that RNAi-mediated silencing of OsITP5/6K-1 gene significantly reduced the phytate levels in seeds without hampering the germination potential of seeds and plant growth.The present study provided an insight into the mechanism of phytic acid biosynthesis pathway.

Silencing Filamin A Inhibits the Invasion and Migration of Breast Cancer Cells by Up-regulating 14-3-3σ

Filamin A and 14-3-3-σ are closely associated with the development of breast cancer. However, the exact relationship between them is still unknown. The present study aimed to examine the interaction of filamin A with 14-3-3-σ in the invasion and migration of breast cancer. RNA interference technology was employed to silence filamin A in MDA-MB-231 cells. Real-time PCR and Westem blotting were used to detect the expression of filamin A and 14-3-3-σ at mRNA and protein levels, respectively. Double immunofluorescence was applied to show their colocalization morphologically. Wound healing assay and Trans-well assay were used to testify the migration and invasion of MDA-MB-231 cells in filamin A-silenced cells. The results showed that silencing filamin A significantly increased the mRNA and protein levels of 14-3-3σ. In addition, double immunofluorescence displayed that filamin A and 14-3-3σ were predominantly colocalized in the cytoplasm of MDA-MB-231 cells. Silencing filamin A led to the enhanced fluorescence of 14-3-3σ. Furthermore, cell functional experiments showed that silencing filamin A inhibited the migration and invasion of MDA-MB-231 cells in vitro. In conclusion, silencing filamin A may inhibit the invasion and migration of breast cancer cells by upregulating 14-3-3σ.

Virtual design and performance prediction of a silencing air cleaner used in an I.C.engine intake system

This paper reports results of the authors’ studies on the virtual design method used in the development of low noise intake system of I.C. engine. The resulting high pass-by noise at level above the legislative target at full throttle when engine speed was around 5200 r/min necessitated a BEM-aided redesign task, following the typical process of design and development of an intake system. During the initial design, based on the acoustic theory and the requirements (1. The air flux of the redesigned should equal to or exceed the value of the original flux; 2. The filtering area must not be degraded), and considering the constraint of space in the engine compartment, total volume and rough internal dimensions were determined. During the detailed design, the exact internal dimensions of the air cleaner were determined, and an effective method was applied to improve the acoustic performance at low frequency. The predicted sound power of the intake system indicated that the objective of reducing the overall engine noise by minimizing intake system noise was achieved.

Gene silencing:Double-stranded RNA mediated mRNA degradation and gene inactivation

The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that double- stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms and are assessing the role of methy- lation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded RNA mediated mRNA degradation and gene inactivation in plants.