Double-stranded Secondary Structures Research Articles

Abstract I005: Exploiting the immunoregulatory effect of double-stranded RNAs for cancer therapeutics

Abstract Introns are found in all eukaryotes and are one of the defining characteristics of eukaryotes. After transcription, pre-mRNAs undergo splicing, and introns are excised in the lariat structure. Since excised introns are degraded quickly after debranching, they have been considered as byproducts of gene expression, and their post-splicing role in the cells remains unclear. Here, we find that the inhibition of intron debranching process by depleting a debranching enzyme, DBR1, results in hyperactivation of protein kinase R (PKR) due to accumulation of intron lariat-derived transposable elements (TEs) that can adopt double-stranded secondary structure. Through in vitro PKR-binding assay, we show that PKR strongly binds to intron lariat RNAs harboring TEs, especially inverted Alu repeats. Interestingly, these TE-containing lariat RNAs are retained in the nucleus, but are released to the cytosol during mitosis, where they activate PKR, resulting in aberrant mitotic progression and apoptosis. We further exploit the lariat RNA-PKR interaction during mitosis and show that DBR1 depletion and anti-mitotic chemotherapy drugs can synergistically induce cancer cell death both in vitro and in vivo. Lastly, we perform docking simulation-based screening of DBR1-inhibitory small molecules. Collectively, our findings underscore the significance of intron turnover in avoiding innate immune activation and further suggest a potential strategy to enhance the efficacy of anti-mitotic drugs by targeting intron processing. Citation Format: Keonyong Lee, Jayoung Ku, Tria Asri Widowati, Namwook Kim, Soo Young Park, Han Suk Ryu,Yoosik Kim. Exploiting the immunoregulatory effect of double-stranded RNAs for cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I005.

Abstract LB-343: A Leu(CAG)-tRNA derived small RNA regulates ribosomal protein S28 after translation initiation in both human and mouse liver cancers

Abstract Transfer-RNA-derived small RNA (tsRNA) is implicated in regulating many cellular processes and is dysregulated in various diseases. We recently revealed that the 3′ end of the LeuCAG tRNA-derived small RNA (LeuCAG3′tsRNA) binds to two target sites in human RPS28 mRNA coding region and 3′ UTR, and unwinds their double-stranded secondary structure, which enhances RPS28 translation and subsequently regulates ribosome biogenesis. Inhibition of this tsRNA suppressed the growth of hepatocellular carcinoma in vivo, making it a bona-fide target for cancer therapeutics1. However, the functional conservation between species and the detailed mechanism by which the tsRNA regulates mRNA translation need to be determined in more detailed. Here, we showed that the target site in RPS28 coding region in human is highly conserved in many vertebrate species. While the 3′ UTR target site forms a double-stranded secondary structure with the translation initiation site (TIS) in human RPS28 mRNA, the functional 3′ UTR target site is not present in the species distantly related to human. We revealed that a conserved target site in the mouse Rps28 coding region was sufficient for tsRNA-regulated enhanced translation of the mRNA and cancer cell viability in mouse cell line. Moreover, we demonstrated that the tsRNA regulates mRNA translation after initiation in both human and mouse. Our results have implications for providing additional insights into the biological role of LeuCAG3'tsRNA in post-transcriptional gene regulation and cancer cell viability.1. Kim, HK, et al. (2017). A transfer-RNA-derived small RNA regulates ribosome biogenesis. Nature: 1-21doi:10.1038/nature25005. Citation Format: Hak Kyun Kim, Jianpeng Xu, Kirk Chu, Hyesuk Park, Hagoon jang, Pan Li, Paul Valdmanis, Qiangpeng Zhang, Mark Kay. A Leu(CAG)-tRNA derived small RNA regulates ribosomal protein S28 after translation initiation in both human and mouse liver cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-343.

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

Doxycycline inhibits pre-rRNA processing and mature rRNA formation in E. coli

In bacteria, RNase III cleaves the initial long primary ribosomal RNA transcripts/precursors (pre-rRNAs), thereby releasing the pre-16S and pre-23S rRNAs for maturation. This cleavage is specified by the double-stranded secondary structures flanking the mature rRNAs, and not necessarily by the nucleotide sequences. Inhibition of this cleavage would lead to a build-up of pre-rRNA molecules. Doxycycline has earlier been shown to bind synthetic double-stranded RNAs and inhibit their cleavage by RNase III. Since bacterial rRNA processing is primarily dependent on RNase III cleavage (which is inhibited by doxycycline), doxycycline could therefore inhibit the normal processing of bacterial rRNA. In this study, the effect of doxycycline on bacterial rRNA processing was investigated by analyzing the amounts of various rRNAs in growing Escherichia coli cells treated with doxycycline. The results showed a doxycycline dose-dependent decrease in mature 16S and 23S rRNAs, concurrent with an accumulation of the initial rRNA transcripts and long precursors. Morphologically, treated cells were elongated at low drug concentrations, while nucleoid degeneration indicative of cell death occurred at higher drug concentrations. These observations suggest that doxycycline inhibits the cleavage and processing of bacterial rRNA transcripts/precursors, leading to impaired formation of mature rRNAs, and the consequent inhibition of protein synthesis for which the tetracycline group of antibiotics are renowned. Since rRNA structure and processing pathway is conserved among bacterial species, this mechanism may account for the broad spectrum of antibiotic activity and selective microbial protein synthesis inhibition of doxycycline and the tetracyclines.

Widespread cis-regulation of RNA editing in a large mammal.

Post-transcriptional RNA editing may regulate transcript expression and diversity in cells, with potential impacts on various aspects of physiology and environmental adaptation. A small number of recent genome-wide studies in Drosophila, mouse, and human have shown that RNA editing can be genetically modulated, highlighting loci that quantitatively impact editing of transcripts. The potential gene expression and physiological consequences of these RNA-editing quantitative trait loci (edQTL), however, are almost entirely unknown. Here, we present analyses of RNA editing in a large domestic mammal (Bos taurus), where we use whole-genome and high-depth RNA sequencing to discover, characterize, and conduct genetic mapping studies of novel transcript edits. Using a discovery population of nine deeply sequenced cows, we identify 2413 edit sites in the mammary transcriptome, the majority of which are adenosine to inosine edits (98.6%). Most sites are predicted to reside in double-stranded secondary structures (85.1%), and quantification of the rates of editing in an additional 355 cows reveals editing is negatively correlated with gene expression in the majority of cases. Genetic analyses of RNA editing and gene expression highlight 152 cis-regulated edQTL, of which 15 appear to cosegregate with expression QTL effects. Trait association analyses in a separate population of 9989 lactating cows also shows 12 of the cis-edQTL coincide with at least one cosegregating lactation QTL. Together, these results enhance our understanding of RNA-editing dynamics in mammals, and suggest mechanistic links by which loci may impact phenotype through RNA editing mediated processes.

U1-RNP and TLR receptors in the pathogenesis of mixed connective tissue diseasePart I. The U1-RNP complex and its biological significance in the pathogenesis of mixed connective tissue disease.

Mixed connective tissue disease (MCTD) is a rare autoimmune syndrome, signified by complex interactions between disease-related phenomena, including inflammation, proliferative vascular arteriopathy, thrombotic events and humoral autoimmune processes. It is still controversial whether MCTD is a distinct clinical entity among systemic connective tissue diseases, although several authors consider that it is distinct and underline characteristic, distinct clinical, serological and immunogenetic features. The putative target of autoimmunity in MCTD is U1-RNP, which is a complex of U1-RNA and small nuclear RNP. Both the U1-RNA component and the specific proteins, particularly U1-70K, engage immune cells and their receptors in a complex network of interactions that ultimately lead to autoimmunity, inflammation, and tissue injury. U1-RNA is capable of inducing manifestations consistent with TLR activation. Stimulation of innate immunity by native RNA molecules with a double-stranded secondary structure may help explain the high prevalence of autoimmunity to RNA binding proteins.

Intrinsic Nucleic Acid-Binding Activity of Chp1 Chromodomain Is Required for Heterochromatic Gene Silencing

Centromeric heterochromatin assembly in fission yeast requires the RNAi pathway. Chp1, a chromodomain (CD) protein, forms the Ago1-containing RNA-induced transcriptional silencing (RITS) complex and recruits siRNA-bound RITS to methylated histone H3 lysine 9 (H3K9me) via its CD. Here, we show that the CD of Chp1 (Chp1-CD) possesses unique nucleic acid-binding activities that are essential for heterochromatic gene silencing. Detailed electrophoretic-mobility shift analyses demonstrated that Chp1 binds to RNA via the CD in addition to its central RNA-recognition motif. Interestingly, robust RNA- and DNA-binding activity of Chp1-CD was strongly enhanced when it was bound to H3K9me, which was revealed to involve a positively charged domain within the Chp1-CD by structural analyses. These results demonstrate a role for the CD that provides a link between RNA, DNA, and methylated histone tails to ensure heterochromatic gene silencing.

Stability of mRNA/DNA and DNA/DNA Duplexes Affects mRNA Transcription

Nucleic acids, due to their structural and chemical properties, can form double-stranded secondary structures that assist the transfer of genetic information and can modulate gene expression. However, the nucleotide sequence alone is insufficient in explaining phenomena like intron-exon recognition during RNA processing. This raises the question whether nucleic acids are endowed with other attributes that can contribute to their biological functions. In this work, we present a calculation of thermodynamic stability of DNA/DNA and mRNA/DNA duplexes across the genomes of four species in the genus Saccharomyces by nearest-neighbor method. The results show that coding regions are more thermodynamically stable than introns, 3′-untranslated regions and intergenic sequences. Furthermore, open reading frames have more stable sense mRNA/DNA duplexes than the potential antisense duplexes, a property that can aid gene discovery. The lower stability of the DNA/DNA and mRNA/DNA duplexes of 3′-untranslated regions and the higher stability of genes correlates with increased mRNA level. These results suggest that the thermodynamic stability of DNA/DNA and mRNA/DNA duplexes affects mRNA transcription.

Double‐stranded secondary structures on mRNA induce type I interferon (IFN α/β) production and maturation of mRNA‐transfected monocyte‐derived dendritic cells

The development of dendritic cell (DC)-based vaccines using antigen-encoding mRNA requires identification of the critical parameters for efficient ex vivo loading of DCs. Exogenously delivered mRNA can induce DC activation, but the molecular mechanisms involved are unknown. The aim of the present study was to identify the means by which mRNA-dependent activation of DCs occurs. In vitro transcribed mRNA molecules were delivered into porcine monocyte-derived DCs (MoDCs) using different non-viral gene transfer procedures. Using the green fluorescent protein (GFP) as reporter gene, as well as rhodamine-labeled RNA, intracellular delivery and transfection efficiency were assessed by confocal microscopy and flow cytometry. DC activation was monitored in terms of MHC class II and CD80/86 upregulation, as well as the production of type I interferon (IFN-alpha/beta). mRNA-lipofected MoDCs produced type I IFN and upregulated MHC class II and CD80/86. Computational analysis of the mRNA molecules predicted highly ordered secondary structures forming double-stranded RNA (dsRNA). This dsRNA was also detectable by immunofluorescence in mRNA-lipofected cells, using antibody specific for dsRNA. Digestion of the mRNA prior to lipofection with a double-strand-specific RNase, but not a single-strand-specific RNase, abrogated DC activation. Impairment of protein kinase R (PKR) with 2-aminopurine also interfered with the activation. Double-stranded secondary structures on mRNA delivered by lipofection can activate MoDCs. This could have important implications for mRNA-based immunomodulation of DCs, DC-based immunotherapy, and formulation of RNA-based vaccines. In addition, this report describes the first in vitro steps towards development of a novel large animal model system to evaluate DC-based vaccines against infectious diseases.

U1 RNA induces innate immunity signaling

The U1-70-kd RNP is a prominent target of autoimmunity in connective tissue diseases. In this study, we explored whether its endogenous ligand, U1 RNA, mediates a proimmune signal and may be immunogenic. We assayed the proliferation of control and MyD88-knockout splenocytes in response to in vitro-synthesized U1 RNA, and measured interleukin-6 (IL-6) and IL-8 secretion induced by U1 RNA in a human cell line competent for signaling through Toll-like receptor 3 (TLR-3) and TLR-5. Treatment with U1 RNA or with poly(I-C), a known agonist of TLR-3, induced approximately twice as much control splenocyte proliferation as did treatment with RNase-digested U1 RNA. Proliferation in response to either poly(I-C) or U1 RNA by MyD88-knockout splenocytes was similarly attenuated. Similar to poly(I-C), U1 RNA induced significant secretion of both IL-6 and IL-8 from a TLR-3-expressing human cell line; in contrast, the TLR-5 agonist flagellin induced predominantly IL-8 secretion. Pretreatment of U1 RNA with RNase abolished IL-6 and IL-8 secretion. U1 RNA is capable of inducing manifestations consistent with TLR-3 activation. The ability of U1 RNA (which has a substantial double-stranded secondary structure) to activate TLR-3 may contribute to the immunogenicity of the U1-70-kd autoantigen. Stimulation of innate immunity by native RNA molecules with a double-stranded secondary structure may help explain the high prevalence of autoimmunity to RNA binding proteins.

Sequence and secondary structure requirements in a highly conserved element for foot-and-mouth disease virus internal ribosome entry site activity and eIF4G binding.

Foot-and-mouth disease virus (FMDV) and other picornaviruses initiate translation of their positive-strand RNA genomes at the highly structured internal ribosome entry site (IRES), which mediates ribosome recruitment to an internal site of the virus RNA. This process is facilitated by eukaryotic translation initiation factors (eIFs), such as eIF4G and eIF4B. In the eIF4G-binding site, a characteristic, discontinuous sequence element is highly conserved within the cardio- and aphthovirus subgroup (including FMDV) of the picornaviruses. This conserved element was mutated in order to investigate its primary sequence and secondary structure requirements for IRES function. Both binding of eIF4G to the IRES and IRES-directed translation are seriously impaired by mutations in two unpaired dinucleotide stretches that are exposed from the double-stranded (ds)RNA. In the base-paired regions of the conserved element, maintenance of the double-stranded secondary structure is essential, whilst in some cases, the primary sequence within the dsRNA regions is also important for IRES function. Extra eIF4F added to the translation reaction does not restore full IRES activity or eIF4G binding, indicating that disturbances in the structure of this conserved element cannot be overcome by increased initiation factor concentrations.

Characterization of the Solution Complex between the Interferon-induced, Double-stranded RNA-activated Protein Kinase and HIV-I Trans-activating Region RNA

The antiviral activity of the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (PKR) is mediated through dsRNA binding leading to PKR autophosphorylation and subsequent inhibition of protein synthesis. Previous biochemical studies have suggested that autophosphorylation of PKR occurs via a protein-protein interaction and that PKR can form dimers in vitro. Using four independent biophysical and biochemical methods, we have characterized the solution complex formed between PKR and trans-activating region (TAR) RNA, a 57-nucleotide RNA species with double-stranded secondary structure derived from the human immunodeficiency virus type I genome. Chemical cross-linking and gel filtration analyses of PKR.TAR RNA complexes reveals that TAR RNA addition increases PKR dimerization and results in the formation of a solution complex with a molecular weight of approximately 150,000. Addition of TAR RNA to PKR results in a quenching of tryptophan fluorescence, indicative of a conformational shift. Through small angle neutron scattering analysis, we show that PKR exists in solution predominantly as a dimer, and has an elongated solution structure. Addition of TAR RNA to PKR causes a significant conformational shift in the protein at a 2:1 stoichiometric ratio of protein to RNA. Taken together, these data indicate that the PKR activation complex consists of a protein dimer bound cooperatively to one dsRNA molecule.

Alternative polyadenylation of the gene transcripts encoding a rat DNA polymerase β

Rat cells produce two different transcripts of DNA polymerase β (β-Pol). The low-molecular-weight transcript (1.4 kb) was already sequenced. We report here the cloning and sequencing of the full-length cDNA, corresponding to the high-molecular-weight (HMW) transcript (4.0 kb) of β-Pol. Sequence data strongly suggest that both transcripts are produced from a single gene by alternative polyadenylation. The HMW transcript contains the entire 1.4 kb transcript sequence and the additional 2.2 kb on the 3' end. The 3' UTR of the HMW transcript contains some regulatory sequences which are not present in the 1.4-kb transcript. The A + U-rich fragment and (GU) 21 sequence are believed to influence the stability of the mRNA. The functional significance of the A-rich region locally destabilizing double-stranded secondary structure remains unknown.

Folding of the MS2 Coat Protein in Escherichia coli is Modulated by Translational Pauses Resulting from mRNA Secondary Structure and Codon Usage: A Hypothesis

Possible translational pauses within the coat protein of the RNA bacteriophage MS2 were located on the basis of a distribution plot of rare codons and RNA secondary structure. It appeared that the position of certain codon pauses corresponds with the size of some nascent polypeptide intermediates, which have been isolated from MS2-infected cells. Other accumulated polypeptide intermediates seemed to be related to RNA regions, where double-stranded secondary structures occur, which probably impede the movement of ribosomes during chain elongation. We assume that a discontinuous translation rate is designed to allow optimal folding of this (and other) polypeptide(s).

The physical properties of the deoxyribonucleic acid from N 4 coliphage

1. 1. Purified preparations of N4 bacteriophage were treated with cold phenol and the DNA was subjected to a series of physical and chemical investigations. 2. 2. The extracted DNA was shown to be homogeneous by ultracentrifugation and chromatography on magnesium pyrophosphate gel. 3. 3. The size of N4 DNA, as determined from sedimentation, viscometry and light-scattering studies, was found to be in the range of 40·10 6 daltons. Since the molecular weight of N4 bacteriophage is 83·10 6, there is probably only one DNA molecule per virus particle. 4. 4. Thermal denaturation studies provided evidence for a double-stranded secondary structure of N4 DNA. The transition temperature was 87.2° corresponding to a mean guanine plus cytosine content of 44 moles %. 5. 5. The buoyant density of native N4 DNA in CsCl density gradients was found to be 1.707 g/ml and an increase of 0.015 g/ml was observed for thermally denatured samples banded at the isopycnic point.