Sun Woo Hong

Phosphorylation of the RNA polymerase II C-terminal domain by TFIIH kinase is not essential for transcription of Saccharomyces cerevisiae genome

Ser-5 phosphorylation of the RNA polymerase II (Pol II) C-terminal domain by TFIIH kinase has been implicated in critical steps in mRNA synthesis, such as Pol II promoter escape and mRNA 5′-capping. However, the general requirement and precise role of TFIIH kinase in Pol II transcription still remain elusive. Here we use a chemical genetics approach to show that, for a majority of budding-yeast genes, specific inhibition of the yeast TFIIH kinase results in a dramatic reduction in both mRNA level and Ser-5 C-terminal domain phosphorylation. Surprisingly, inhibition of TFIIH kinase activity only partially affected both Pol II density and Ser-2 phosphorylation level. The discrepancy between mRNA level and Pol II density is attributed to the defective 5′-capping, which results in the destabilization of mRNAs. Therefore, contrary to the current belief, our study points strongly toward a minor role of TFIIH kinase in Pol II transcription, and a more significant role in mRNA capping in budding yeast.

Enhanced intracellular delivery and multi‐target gene silencing triggered by tripodal RNA structures

The development of gene interfering RNA (iRNA) molecules such as small interfering RNAs (siRNAs) and antagomirs provides promising therapeutic modalities for targeting specific mRNAs and microRNAs (miRNAs) involved in disease mechanisms. Therapeutic iRNA strategy against cancer or hypermutable viruses prefers targeting multiple genes simultaneously to achieve synergistic inhibition and to prevent resistance.

Long dsRNA-Mediated RNA Interference and Immunostimulation: A Targeted Delivery Approach Using Polyethyleneimine Based Nano-Carriers

RNA oligonucleotides capable of inducing controlled immunostimulation combined with specific oncogene silencing via an RNA interference (RNAi) mechanism provide synergistic inhibition of cancer cell growth. With this concept, we previously designed a potent immunostimulatory long double stranded RNA, referred to as liRNA, capable of executing RNAi mediated specific target gene silencing. In this study, we developed a highly effective liRNA based targeted delivery system to apply in the treatment of glioblastoma multiforme. A stable nanocomplex was fabricated by complexing multimerized liRNA structures with cross-linked branched poly(ethylene imine) (bPEI) via electrostatic interactions. We show clear evidence that the cross-linked bPEI was quite effective in enhancing the cellular uptake of liRNA on U87MG cells. Moreover, the liRNA-PEI nanocomplex provided strong RNAi mediated target gene silencing compared to that of the conventional siRNA-PEI complex. Further, the bPEI modification strategy with specific ligand attachment assisted the uptake of the liRNA-PEI complex on the mouse brain endothelial cell line (b.End3). Such delivery systems combining the beneficial elements of targeted delivery, controlled immunostimulation, and RNAi mediated target silencing have immense potential in anticancer therapy.

Efficient intracellular delivery and multiple-target gene silencing triggered by tripodal RNA based nanoparticles: a promising approach in liver-specific RNAi delivery.

RNA interference (RNAi) triggering oligonucleotides in unconventional structural format can offer advantages over conventional small interfering RNA (siRNA), enhanced cellular delivery and improved target gene silencing. With this concept, we present a well-defined tripodal-interfering RNA (tiRNA) structure that can induce simultaneous silencing of multiple target genes with improved potency. The tiRNA structure, formed by the complementary association of three single-stranded RNA units, was optimized for improved gene silencing efficacy. When combined with cationic polymers such as linear polyethyleneimine (PEI), tiRNA assembled to form a stable nano-structured complex through electrostatic interactions and induced stronger RNAi response over conventional siRNA-PEI complex. In combination with a liver-targeting delivery system, tripodal nucleic acid structure demonstrated enhanced fluorescent accumulation in mouse liver compared to standard duplex nucleic acid format. Tripodal RNA structure complexed with galactose-modified PEI could generate effective RNAi-mediated gene silencing effect on experimental mice models. Our studies demonstrate that optimized tiRNA structural format with appropriate polymeric carriers have immense potential to become an RNAi-based platform suitable for multi-target gene silencing.

HIF-1α-dependent gene expression program during the nucleic acid-triggered antiviral innate immune responses

Recent studies suggest a novel role of HIF-1α under non-hypoxic conditions, including antibacterial and antiviral innate immune responses. However, the identity of the pathogen-associated molecular pattern which triggers HIF-1α activation during the antiviral response remains to be identified. Here, we demonstrate that cellular administration of double-stranded nucleic acids, the molecular mimics of viral genomes, results in the induction of HIF-1α protein level as well as the increase in HIF-1α target gene expression. Whole-genome DNA microarray analysis revealed that double-stranded nucleic acid treatment triggers induction of a number of hypoxia-inducible genes, and induction of these genes are compromised upon siRNA-mediated HIF-1α knock-down. Interestingly, HIF-1α knock-down also resulted in down-regulation of a number of genes involved in antiviral innate immune responses. Our study demonstrates that HIF-1α activation upon nucleic acid-triggered antiviral innate immune responses plays an important role in regulation of genes involved in not only hypoxic response, but also immune response.

bZIPDB : A database of regulatory information for human bZIP transcription factors

BackgroundBasic region-leucine zipper (bZIP) proteins are a class of transcription factors (TFs) that play diverse roles in eukaryotes. Malfunctions in these proteins lead to cancer and various other diseases. For detailed characterization of these TFs, further public resources are required.DescriptionWe constructed a database, designated bZIPDB, containing information on 49 human bZIP TFs, by means of automated literature collection and manual curation. bZIPDB aims to provide public data required for deciphering the gene regulatory network of the human bZIP family, e.g., evaluation or reference information for the identification of regulatory modules. The resources provided by bZIPDB include (1) protein interaction data including direct binding, phosphorylation and functional associations between bZIP TFs and other cellular proteins, along with other types of interactions, (2) bZIP TF-target gene relationships, (3) the cellular network of bZIP TFs in particular cell lines, and (4) gene information and ontology. In the current version of the database, 721 protein interactions and 560 TF-target gene relationships are recorded. bZIPDB is annually updated for the newly discovered information.ConclusionbZIPDB is a repository of detailed regulatory information for human bZIP TFs that is collected and processed from the literature, designed to facilitate analysis of this protein family. bZIPDB is available for public use at http://biosoft.kaist.ac.kr/bzipdb.

Target Gene Abundance Contributes to the Efficiency of siRNA-Mediated Gene Silencing

The gene-silencing activity of a small interfering RNA (siRNA) is determined by various factors. Considering that RNA interference (RNAi) is an unparalleled technology in both basic research and therapeutic applications, thorough understanding of the factors determining RNAi activity is critical. This report presents observations that siRNAs targeting KRT7 show cell-line-dependent activity, which correlates with the expression level of KRT7 mRNA. By modulating the target mRNA level, it was confirmed that highly expressed genes are more susceptible to siRNA-mediated gene silencing. Finally, several genes that show different expression levels in a cell-line dependent manner were tested, which verified the expression-level-dependent siRNA activities. These results strongly suggest that the abundance of target mRNA is a critical factor that determines the efficiency of the siRNA-mediated gene silencing in a given cellular context. This report should provide practical guidelines for designing RNAi experiments and for selecting targetable genes in RNAi therapeutics studies.

Selection and optimization of asymmetric siRNA targeting the human c-MET gene

The silencing of specific oncogenes via RNA interference (RNAi) holds great promise for the future of cancer therapy. RNAi is commonly carried out using small interfering RNA (siRNA) composed of a 19 bp duplex region with a 2-nucleotide overhang at each 3′ end. This classical siRNA structure, however, can trigger non-specific effects, which has hampered the development of specific and safe RNAi therapeutics. Previously, we developed a novel siRNA structure, called asymmetric shorter-duplex siRNA (asiRNA), which did not cause the non-specific effects triggered by conventional siRNA, such as off-target gene silencing mediated by the sense strand. In this study, we first screened potent asiRNA molecules targeting the human c-MET gene, a promising anticancer target. Next, the activity of a selected asiRNA was further optimized by introducing a locked nucleic acid (LNA) to maximize the gene silencing potency. The optimized asiRNA targeted to c-MET may have potential as a specific and safe anticancer RNAi therapeutic.

RNA interference-mediated simultaneous silencing of four genes using cross-shaped RNA

The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.

Gene expression profiling of Bangpungtongseong-san (Bofutsushosan) and Bangkihwangki-tang (Boiogito) administered individuals

Bangpungtongseong-san (Bofutsushosan, BTS) and Bangkihwangki-tang (Boiogito, BOT) are widely prescribed Korean, Japanese and Chinese traditional herbal medicines used to clinically prevent or improve obesity. In this study, we investigated genomewide transcriptional response to administration of either BTS or BOT to the Korean obese adults. From the blood samples, peripheral blood mononuclear cells (PBMCs) were isolated and RNA samples extracted from PBMCs were processed to conduct DNA microarray. Analysis of DNA microarray data revealed that administration of the both BTS and BOT evokes gene expression profile changes of PBMCs. In addition, comparative analysis between the drugs and placebo identified sets of specific genes regulated by BTS or BOT. Interestingly, analysis of gene list using functional annotation tool revealed that PMBCs of BOT-prescribed subjects have increased expression level of genes which are localized to mitochondria. The fact that mitochondrion is the main organelle of regulating cellular energy metabolism suggests that induced expressions of mitochondrial genes by BOT administration might be responsible for the anti-obesity effect of the medicine. We expect that further study based on our finding will provide molecular mechanisms of BTS and BOT effects on obesity.