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At least 1,351 records · Page 75Linked to original sources
PubMed2004

A non-consensus branch point plays an important role in determining the stability of the 2-kb LAT intron during acute and latent infections of herpes simplex virus type-1.

Herpes simplex virus type 1 (HSV-1) establishes lifelong latent infection in sensory neurons of the peripheral nervous system. During HSV latency, the latency-associated transcripts (LATs) are the only viral transcripts abundantly expressed. The most abundant form of LATs is a 2-kb stable intron spliced from a primary transcript (mLAT). It has been previously reported that a non-consensus branch point influences the stability of the intron (in vitro) in cells transfected with plasmid constructs (J. Virol. 71 (1997) 5849; J. Virol. 71 (1997) 4199). However, it is unknown whether this branch point is important in determining LAT stability in vivo (in the context of virus). To study the role of this stable intron in HSV-1 infection, we have constructed a mutant virus KOS-CONS in which the branch point has been mutated to consensus branch point nucleotides. The accumulation of the 2-kb intron in KOS-CONS-infected cells was greatly reduced. The LAT intron was not detectable in KOS-CONS-infected mouse trigeminal ganglia (TG) during acute and latent phase infection by Northern blot analysis. Replication of the KOS-CONS and the wild-type KOS viruses on Vero cells was determined to be similar, as was the level of HSV-1 DNA in mouse trigeminal ganglia during acute and latent phase infection. Using the mouse TG explant model, the reactivation pattern of both viruses was shown to be similar. Our data suggest that the unique branch point plays a significant role in determining the stability of LAT intron in vivo, but that the stability of the intron does not appear to affect HSV-1 replication, the establishment of latency, or viral reactivation.

Acute Disease↗
PubMed2005

Inhibition of hepatitis C virus replication by pol III-directed overexpression of RNA decoys corresponding to stem-loop structures in the NS5B coding region.

Increasing evidence has shown that the stem-loop (SL) structures in the NS5B coding region of hepatitis C virus (HCV) function as cis-replicating elements that are indispensable for viral replication. We have investigated whether small RNA molecules analogous to the SL structures could inhibit HCV replication. Reporter assays showed that both in vitro transcribed and pol III-directed transcripts corresponding to 5BSL3.1 and 5BSL3.2 efficiently inhibited HCV replicon-encoded luciferase expression. Mutagenesis studies revealed that mutation in 5BSL3.2 which debilitated its binding to NS5B also abolished the ability of 5BSL3.2 RNA to inhibit HCV replication, suggesting that SL RNA inhibits HCV by sequestering the replication complex. Further, adenoviral-mediated expression of the SL RNAs potently blocked the replication of HCV replicon in Huh-7 cells. Importantly, SL RNAs derived from HCV 2a, an evolutionarily distant genotype, were also shown to suppress the replication of HCV 1b replicon in spite of the genetic heterogeneity between the SL elements of the two viruses, implying the potential of SL RNA-based approach to inhibit a wide range of HCV isolates. These results suggest that SL RNA decoys may prove to be useful in the treatment of hepatitis C, which may be advantageous over other sequence-specific gene therapy modalities (such as antisense RNA and siRNA) in preventing the escape of genetic variants.

Adenoviridae↗
PubMed2006

The stable 2-kb LAT intron of herpes simplex stimulates the expression of heat shock proteins and protects cells from stress.

During latency of herpes simplex virus type 1 (HSV-1), the major viral transcript that is detected is the latency-associated transcript (LAT) 2-kb intron. This intron is excised from a larger (approximately 10 kb) primary transcript. Infection of cells with HSV expressing LATs showed that their presence increased accumulation of Hsp70 protein specifically during cold shock. Transfection of cells with a plasmid, expressing the 2-kb LAT intron, showed increased translation compared to cells transfected with plasmids deleted in regions of the intron. Cold shock of cells expressing the intron resulted in an up-regulation of Hsp70 protein, but not Hsp70 mRNA. Furthermore, these cells showed increased cell viability. Using plasmid deletion mutants in the LAT gene sequence, we have shown that the effect requires full-length LAT intron. These findings show that a function of the stable 2-kb LAT intron is to protect cells from cold-induced stress and that this may be mediated via Hsp70.

Cold Temperature↗
PubMed2006

Characterization of a spliced exon product of herpes simplex type-1 latency-associated transcript in productively infected cells.

The latency-associated transcripts (LATs) of herpes simplex virus type-1 (HSV-1) are the only viral RNAs accumulating during latent infections in the sensory ganglia of the peripheral nervous system. The major form of LAT that accumulates in latently infected neurons is a 2 kb intron, spliced from a much less abundant 8.3 primary transcript. The spliced exon mRNA has been hard to detect. However, in this study, we have examined the spliced exon RNA in productively infected cells using ribonuclease protection (RPA), and quantitative RT-PCR (q-PCR) assays. We were able to detect the LAT exon RNA in productively infected SY5Y cells (a human neuronal cell line). The level of the LAT exon RNA was found to be approximately 5% that of the 2 kb intron RNA and thus is likely to be relatively unstable. Quantitative RT-PCR (q-PCR) assays were used to examine the LAT exon RNA and its properties. They confirmed that the LAT exon mRNA is present at a very low level in productively infected cells, compared to the levels of other viral transcripts. Furthermore, experiments showed that the LAT exon mRNA is expressed as a true late gene, and appears to be polyadenylated. In SY5Y cells, in contrast to most late viral transcripts, the LAT exon RNA was found to be mainly nuclear localized during the late stage of a productive infection. Interestingly, more LAT exon RNA was found in the cytoplasm in differentiated compared to undifferentiated SY5Y cells, suggesting the nucleocytoplasmic distribution of the LAT exon RNA and its related function may be influenced by the differentiation state of cells.

Cell Differentiation↗
PubMed2004

RNA interference as a new biotechnological tool for the control of virus diseases in plants.

RNA silencing occurs in a wide variety of organisms, including protozoa, fungi, plants and animals and involves recognition of a target RNA and initiation of a sequence-specific RNA degradation pathway in the cytoplasm. In the last few years, there have been considerable advances in our understanding of post-transcriptional gene silencing (PTGS). This mechanism is conceived as a natural antiviral defense system in plants that is activated as a response to double-stranded RNA (dsRNA) formed during virus replication. To develop new approaches for plant protection against virus diseases based on PTGS we have expanded previous findings on RNA interference (RNAi) in animals by using dsRNA to specifically interfere with virus infection in plants. This approach differs from strategies based on transgenic expression of RNAs but still relies on PTGS as a means to achieve pathogen-derived resistance (PDR). Our findings suggest that exogenously supplied dsRNA could form the basis for the development of an environmentally safe, new biotechnological tool aimed at protecting crops against virus diseases, provided that some limitations of the current status of the approach could be overcome.

Biotechnology↗
PubMed2005

Identification of a protein encoded in the herpes simplex virus type 1 latency associated transcript promoter region.

Herpes simplex virus type 1 (HSV-1) establishes a lifelong latency in the neurons of its host. Sporadically, the latent virus reactivates and spreads back to the original site of infection and causes recrudescent diseases. The only gene actively transcribed during neuronal latency is the latency associated transcript (LAT) gene. Several transcripts have been detected in the important LAT promoter region. However, no polypeptides coded by these transcripts are known. In this communication, we reported the cloning, sequencing, and characterization of a transcript immediately upstream of LAT. We designated this gene UOL (Upstream of LAT). The UOL RNA is polyadenylated, expressed as a late gene in infected cells, transcribed in the same direction as LAT, and contains an open reading frame (ORF) capable of encoding a protein of 96 amino acids with a predicted molecular mass of 11 kDa. The UOL transcript contains 466 nucleotides in length. The 5' end of the UOL transcript starts at nucleotide 118,266 and the 3' end of the UOL transcript ends at nucleotide 118,731 based on the published 17syn+ genomic sequence. The UOL protein was detected in infected cell lysates by immunoprecipitation using an antibody raised against UOL ORF synthetic peptide. More importantly, sera from mice infected with wild-type HSV-1 but not sera from mice infected with a mutant with the UOL region deleted recognized the UOL ORF, expressed in Escherichia coli, on Western blots. These results suggest that a UOL protein is in HSV-1 infected tissue culture cells and in mice infected with HSV-1.

Amino Acid Sequence↗
PubMed2006

Conservation patterns in different functional sequence categories of divergent Drosophila species.

We have explored the distributions of fully conserved ungapped blocks in genome-wide pair-wise alignments of recently completed species of Drosophila: D. melanogaster, D. yakuba, D. ananassae, D. pseudoobscura, D. virilis, and D. mojavensis. Based on these distributions we have found that nearly every functional sequence category possesses its own distinctive conservation pattern, sometimes independent of the overall sequence conservation level. In the coding and regulatory regions, the ungapped blocks were longer than in introns, UTRs, and nonfunctional sequences. At the same time, the blocks in the coding regions carried a 3N + 2 signature characteristic of synonymous substitutions in the third-codon position. Larger block sizes in transcription regulatory regions can be explained by the presence of conserved arrays of binding sites for transcription factors. We also have shown that the longest ungapped blocks, or "ultraconserved" sequences, are associated with specific gene groups, including those encoding ion channels and components of the cytoskeleton. We discuss how restraining conservation patterns may help in mapping functional sequence categories and improve genome annotation.

Animals↗
PubMed2026

Decoding the molecular basis of blue grain color codominance in Qingke: Integrative analysis of RNA-seq, DNA methylation, and miRNA-seq.

The grains on single spike of the F1 generation from the cross between blue- and white-grained Qingke (Hordeum vulgare L. var. nudum Hook. f.) are randomly distributed in blue and white colors. This study integrated data from RNA-seq, DNA methylation, and miRNA-seq to analyze this trait. The results showed that the HvF3'5'H gene is likely central to the development of this codominant phenotype. Through cross-validation of three omics approaches, it was found that the HvMYB gene targeted by miR858-z, as well as the WRKY24 and At3g44326 genes targeted by novel-m0152-5p, novel-m0153-5p, and novel-m0154-5p, are correlated with DNA methylation. qRT-PCR analysis confirmed that the four aforementioned genes exhibited variety-specific and developmental stage-specific expression patterns. This study dissects the regulatory network underlying the codominant blue and white grain color divergence on a single Qingke spike from a multi-omics perspective.

DNA Methylation↗
PubMed2026

Dynamic transcriptomic landscape from bulk RNA-seq reveals critical mmu-miR-181a-5p/hif1a and mmu-miR-101a-3p/col1a1 modules for deep second-degree burn wound healing.

Burn injuries constitute a significant global health challenge, with deep partial-thickness burns (deep second-degree) posing particular clinical concerns due to prolonged healing and high scarring risks stemming from reticular dermis damage. Current therapeutic strategies remain largely empirical, reflecting limited understanding of stage-specific regulatory mechanisms. This study systematically investigated the molecular basis of deep partial-thickness burn repair by establishing murine models and performing RNA-seq analysis across healing phases (0, 3, 7, 14 days post-burn, dpb). Integrated bioinformatics revealed pivotal ceRNA and PPI networks, identifying hif1a (hypoxia-responsive immunomodulator) and col1a1 (ECM remodeling hub) as nodal regulators. Mechanistically, mmu-miR-101a-3p and mmu-miR-181a-5p were validated as post-transcriptional repressors of col1a1 and hif1a, respectively. Our work pioneers the discovery of the mmu-miR-181a-5p/hif1a and mmu-miR-101a-3p/col1a1 axes as master regulators of burn repair, offering novel therapeutic targets. The multi-omics dataset and molecular networks established herein provide a foundational resource for wound healing research.

MicroRNAs↗
PubMed2026

Comprehensive analysis of differentially expressed mRNAs, lncRNAs, and miRNAs involved in ovarian differentiation and development in Qihe gibel carp (Carassius gibelio var. Qihe).

Qihe gibel carp (Carassius gibelio var. Qihe) exhibits diverse reproductive modes including gynogenesis and sexual reproduction, yet the molecular mechanisms of ovarian differentiation remain poorly understood. Ovarian tissues at 20, 30, and 60 days after hatching (dah), representing key stages covering early ovarian differentiation and primary oocyte growth, were subjected to whole-transcriptome sequencing. A total of 27,259 mRNAs, 2622 lncRNAs, and 2467 miRNAs were differentially expressed. Cell cycle, transcription, translation, and DNA replication pathways were significantly upregulated from 20 to 60 dah. Oocyte meiosis was enriched from 20 and 30 dah, whereas metabolic pathways (lipid, carbohydrate, and nucleotide metabolism) were enriched from 30 to 60 dah, indicating sequential progression from meiosis initiation to primary oocyte growth with nutrient synthesis. Hub lncRNAs and key ceRNA networks (e.g., MSTRG.28669.5-miR-221-ccnb2) were identified. This study provides the first comprehensive characterization of ncRNA-mediated regulation and ceRNA networks during ovarian development in Qihe gibel carp, establishing a foundation for understanding ovarian differentiation in this species.

Animals↗
PubMed2003

Latent herpesvirus infection in human trigeminal ganglia causes chronic immune response.

The majority of trigeminal ganglia (TGs) are latently infected with alpha-herpesviruses [herpes simplex virus type-1 (HSV-1) and varicella-zoster virus (VZV)]. Whereas HSV-1 periodically reactivates in the TGs, VZV reactivates very rarely. The goal of this study was to determine whether herpesvirus latency is linked to a local immune cell infiltration in human TGs. T cells positive for the CD3 and CD8 markers, and CD68-positive macrophages were found in 30 of 42 examined TGs from 21 healthy individuals. The presence of immune cells correlated constantly with the occurrence of the HSV-1 latency-associated transcript (LAT) and only irregularly with the presence of latent VZV protein. In contrast, uninfected TGs showed no immune cell infiltration. Quantitative RT-PCR revealed that CD8, interferon-gamma, tumor necrosis factor-alpha, IP-10, and RANTES transcripts were significantly induced in TGs latently infected with HSV-1 but not in uninfected TGs. The persisting lymphocytic cell infiltration and the elevated CD8 and cytokine/chemokine expression in the TGs demonstrate for the first time that latent herpesviral infection in humans is accompanied by a chronic inflammatory process at an immunoprivileged site but without any neuronal destruction. The chronic immune response seems to maintain viral latency and influence viral reactivation.

Adolescent↗
PubMed2003

Coordinate regulation of small temporal RNAs at the onset of Drosophila metamorphosis.

The lin-4 and let-7 small temporal RNAs play a central role in controlling the timing of Caenorhabditis elegans cell fate decisions. let-7 has been conserved through evolution, and its expression correlates with adult development in bilateral animals, including Drosophila [Nature 408 (2000), 86]. The best match for lin-4 in Drosophila, miR-125, is also expressed during pupal and adult stages of Drosophila development [Curr. Biol. 12 (2002), 735]. Here, we ask whether the steroid hormone ecdysone induces let-7 or miR-125 expression at the onset of metamorphosis, attempting to link a known temporal regulator in Drosophila with the heterochronic pathway defined in C. elegans. We find that let-7 and miR-125 are coordinately expressed in late larvae and prepupae, in synchrony with the high titer ecdysone pulses that initiate metamorphosis. Unexpectedly, however, their expression is neither dependent on the EcR ecdysone receptor nor inducible by ecdysone in cultured larval organs. Although let-7 and miR-125 can be induced by ecdysone in Kc tissue culture cells, their expression is significantly delayed relative to that seen in the animal. let-7 and miR-125 are encoded adjacent to one another in the genome, and their induction correlates with the transient appearance of an approximately 500-nt RNA transcribed from this region, providing a mechanism to explain their precise coordinate regulation. We conclude that a common precursor RNA containing both let-7 and miR-125 is induced independently of ecdysone in Drosophila, raising the possibility of a temporal signal that is distinct from the well-characterized ecdysone-EcR pathway.

Animals↗
PubMed2004

Functional silencing of hepatic microsomal glucose-6-phosphatase gene expression in vivo by adenovirus-mediated delivery of short hairpin RNA.

An expression cassette containing mouse U6 polymerase III promoter directing expression of short hairpin RNA (shRNA) targeting murine microsomal glucose-6-phosphatase (G6P) transcript was generated. This construct was packaged into an adenoviral (AdV) backbone and viral stocks generated. Mice injected intravenously with AdV-G6PshRNA exhibited a significant reduction in postprandial glucose levels and had significantly elevated steady-state hepatic glycogen stores. Target gene silencing was confirmed by measurements demonstrating a significant reduction in both hepatic G6P transcript level and phosphohydrolase activity. These findings provide evidence that AdV delivery of expressed shRNA can be a productive tool to explore gene function in vivo.

Adenoviridae↗
PubMed2004

Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference.

The severe acute respiratory syndrome (SARS) has been one of the most epidemic diseases threatening human health all over the world. Based on clinical studies, SARS-CoV (the SARS-associated coronavirus), a novel coronavirus, is reported as the pathogen responsible for the disease. To date, no effective and specific therapeutic method can be used to treat patients suffering from SARS-CoV infection. RNA interference (RNAi) is a process by which the introduced small interfering RNA (siRNA) could cause the degradation of mRNA with identical sequence specificity. The RNAi methodology has been used as a tool to silence genes in cultured cells and in animals. Recently, this technique was employed in anti-virus infections in human immunodeficiency virus and hepatitis C/B virus. In this study, RNAi technology has been applied to explore the possibility for prevention of SARS-CoV infection. We constructed specific siRNAs targeting the S gene in SARS-CoV. We demonstrated that the siRNAs could effectively and specifically inhibit gene expression of Spike protein in SARS-CoV-infected cells. Our study provided evidence that RNAi could be a tool for inhibition of SARS-CoV.

Animals↗
PubMed2004

RNA interfering approach for clarifying the PPARgamma pathway using lentiviral vector expressing short hairpin RNA.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in adipocyte differentiation and insulin sensitivity. Although PPARgamma also appears to regulate diverse cellular processes in other cell types such as lymphocytes, the detailed mechanisms remain unclear. In this study, we established a lentivirus-mediated short hairpin RNA expression system and identified a potent short hairpin RNA which suppresses PPARgamma expression, resulting in marked inhibition of preadipocyte-to-adipocyte differentiation in 3T3-L1 cells. Our PPARgamma-knockdown method will serve to clarify the PPARgamma pathway in various cell types in vivo and in vitro, and will facilitate the development of therapeutic applications for a variety of diseases.

3T3-L1 Cells↗
PubMed2003

Regulation by RNA.

In recent years, noncoding RNAs (ncRNAs) have been shown to constitute key elements implicated in a number of regulatory mechanisms in the cell. They are present in bacteria and eukaryotes. The ncRNAs are involved in regulation of expression at both transcriptional and posttranscriptional levels, by mediating chromatin modifications, modulating transcription factor activity, and influencing mRNA stability, processing, and translation. Noncoding RNAs play a key role in genetic imprinting, dosage compensation of X-chromosome-linked genes, and many processes of differentiation and development.

Animals↗
PubMed2005

Mechanistic insights aid computational short interfering RNA design.

RNA interference is widely recognized for its utility as a functional genomics tool. In the absence of reliable target site selection tools, however, the impact of RNA interference (RNAi) may be diminished. The primary determinants of silencing are influenced by highly coordinated RNA-protein interactions that occur throughout the RNAi process, including short interfering RNA (siRNA) binding and unwinding followed by target recognition, cleavage, and subsequent product release. Recently developed strategies for identification of functional siRNAs reveal that thermodynamic and siRNA sequence-specific properties are crucial to predict functional duplexes (Khvorova et al., 2003; Reynolds et al., 2004; Schwarz et al., 2003). Additional assessments of siRNA specificity reveal that more sophisticated sequence comparison tools are also required to minimize potential off-target effects (Jackson et al., 2003; Semizarov et al., 2003). This chapter reviews the biological basis for current computational design tools and how best to utilize and assess their predictive capabilities for selecting functional and specific siRNAs.

Algorithms↗