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At least 1,639 records · Page 91
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Circular RNAs and systemic lupus erythematosus

Circular RNAs (circRNAs) are a large class of noncoding RNAs that form covalently closed RNA circles. The discovery of circRNAs discloses a new layer of gene regulation occurred post-transcriptionally. Identification of endogenous circRNAs benefits from the advance in high-throughput RNA sequencing and remains challenging. Many studies probing into the mechanisms of circRNAs formation occurred cotranscriptionally or posttranscriptionally emerge and conclude that canonical splicing mechanism, sequence properties, and certain regulatory factors are at play in the process. Although our knowledge on functions of circRNAs is rather limited, a few circRNAs are shown to sponge miRNA and regulate gene transcription. The clearest case is one circRNA CDR1as that serves as sponge of miR-7. Researches on circRNAs in human diseases such as cancers highlight the function and physical relevance of circRNAs. Given the implication of miRNAs in the initiation and progression of systemic lupus erythematosus (SLE) and the roles of circRNAs in sponging miRNA and gene regulation, it is appealing to speculate that circRNAs may associate with SLE and may be potential therapeutic targets for treatment of SLE. Future studies should attach more importance to the relationship between circRNAs and SLE. This review will concern identification, biogenesis, and function of circRNAs, introduce reports exploring the association of circRNAs with human diseases, and conjecture the potential roles of circRNAs in SLE. - Highlights: • Studies have discovered thousands of circRNAs and interpreted their biogenesis. • Cytoplasmic circRNAs sponge miRNA and nuclear circRNAs modulate gene transcription. • Aberrant expression of circRNAs has been observed in various cancers. • CircRNAs may partake in the pathogenesis of systemic lupus erythematosus.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The role and mechanism of KCa3.1 channels in human monocyte migration induced by palmitic acid

Highlights: • Palmitic acid upregulates K{sub Ca}3.1 channels in human monocytes. • TLR2/4, p38-MAPK and NF-κB pathway is involved in the effect of palmitic acid. • The migration of monocytes induced by palmitic acid is mediated by K{sub Ca}3.1 channels. • K{sub Ca}3.1 channels contribute to palmitic acid-induced MCP-1 production in monocytes. Monocyte migration into diseased tissues contributes to the pathogenesis of diseases. Intermediate-conductance Ca{sup 2+}-activated K{sup +} (K{sub Ca}3.1) channels play an important role in cell migration. However, the role of K{sub Ca}3.1 channels in mediating monocyte migration induced by palmitic acid (PA) is still unclear. Using cultured THP-1 cells and peripheral blood mononuclear cells from healthy subjects, we investigated the role and signaling mechanisms of K{sub Ca}3.1 channels in mediating the migration induced by PA. Using methods of Western blotting analysis, RNA interference, cell migration assay and ELISA, we found that PA-treated monocytes exhibited increment of the protein levels of K{sub Ca}3.1 channel and monocyte chemoattractant protein-1 (MCP-1), and the effects were reversed by co-incubation of PA with anti-TLR2/4 antibodies or by specific inhibitors of p38-MAPK, or NF-κB. In addition, PA increased monocyte migration, which was abolished by a specific K{sub Ca}3.1 channel blocker, TRAM-34, or K{sub Ca}3.1 small interfering RNA (siRNA). The expression and secretion of MCP-1 induced by PA was also similarly prevented by TRAM-34 and K{sub Ca}3.1 siRNA. These results demonstrate for the first time that PA upregulates K{sub Ca}3.1 channels through TLR2/4, p38-MAPK and NF-κB pathway to promote the expression of MCP-1, and then induce the trans-endothelial migration of monocytes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

miR-125a-5p regulation increases phosphorylation of FAK that contributes to imatinib resistance in gastrointestinal stromal tumors

Highlights: • Phosphorylation of FAK is regulated by PTPN18 and miR-125a-5p. • pFAK level is higher in imatinib-resistant than imatinib-sensitive GIST882 cells. • FAK inihibitor treatment in imatinib-resistant cells increases imatinib-induced apoptosis. • Blocking FAK phosphorylation rescues miR-125a-5p mediated imatinib resistance. The use of imatinib mesylate has greatly improved the clinical outcome for gastrointestinal stromal tumor (GIST) patients. However, imatinib resistance is still a major clinical challenge, and the molecular mechanisms are not fully understood. We have previously shown that miR-125a-5p and its mRNA target PTPN18 modulate imatinib response in GIST cells. Herein, we evaluated phosphorylated FAK (pFAK) as a candidate downstream target of PTPN18 and the possible association of this regulation with imatinib resistance in GIST. FAK and pFAK expressions were evaluated in GIST882 cells transfected with short hairpin RNA or short interfering RNA targeting PTPN18 or miR-125a-5p mimic, imatinib-resistant GIST882R subclones and clinical samples using Western blot analyses. FAK phosphorylation was blocked using the FAK inhibitor 14 (FAKi) and the effects on cell viability and apoptosis were evaluated using WST-1 assay and cleaved PARP expression. Clinical associations of FAK and pFAK expression with imatinib resistance, KIT mutation and patient outcome were assessed by Fisher's exact test or log-rank test. Over-expression of miR-125a-5p and silencing of PTPN18 increased pFAK, but not FAK, expression in GIST cells. Higher pFAK expression was observed in the GIST882R subclones with acquired imatinib resistance compared to their imatinib-sensitive parental cells. Treatment with FAKi in imatinib-resistant GIST882R cells reduced cell viability and increased apoptosis upon imatinib treatment. Additionally, FAKi could rescue the imatinib resistance effect mediated by miR-125a-5p over-expression. In clinical samples, high FAK and pFAK expressions were associated with KIT mutation status, and high FAK expression was also associated with metastasis in GIST. Higher pFAK was found in cases with shorter overall survival. Our findings highlight an important role for miR-125a-5p regulation and its downstream target pFAK for imatinib resistance in GIST. pFAK and FAK may have prognostic values in GIST.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

LncRNA embryonic stem cells expressed 1 (Lncenc1) is identified as a novel regulator in neuropathic pain by interacting with EZH2 and downregulating the expression of Bai1 in mouse microglia

LncRNA embryonic stem cells expressed 1 (Lncenc1), named after its high expression in naïve embryonic stem cells (nESCs), has been rarely studied in almost all pathological processes. Evidences suggest that Lncenc1 is likely to work in the form of RNA-protein complex. Here, we found that Lncenc1 in dorsal root ganglion (DRG) was significantly upregulated in response to mouse nerve injury caused by partial sciatic nerve ligation (pSNL). Overexpression of Lncenc1 mediated by adenoviral expression vector promoted the activation of microglia and the production of inflammatory cytokines including TNF-α, IL-1β and MCP-1. In contrast, knockdown of Lncenc1 suppressed activation of microglia and production of inflammatory cytokines. In the mechanism exploration, we found that Lncenc1 could bind with the RNA binding protein (RBP) enhancer of zeste homologue 2 (EZH2), an identified contributor in microglial activation and neuropathic pain. Lncenc1 interacted with EZH2 and downregulated the expression of brain-specific angiogenesis inhibitor 1 (BAI1). Either inhibition of EZH2 or overexpression of BAI1 could reverse the effects of Lncenc1 overexpression on microglial activation and neuroinflammation. Finally, the Lncenc1-siRNA was intrathecally injected into pSNL mice, and its effects on neuropathic pain were evaluated. Knockdown of Lncenc1 attenuated the development and maintenance of mechanical and thermal hyperalgesia of pSNL mice, accompanied by an increase in BAI1 expression and decrease in inflammatory cytokines. In conclusion, Lncenc1 contributes to neuropathic pain by interacting with EZH2 and downregulating the BAI1 gene in mouse microglia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

U2AF - Hypoxia-induced fas alternative splicing regulator

The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many disease states. Recent studies have revealed that tumorigenesis and hypoxia are involved in large-scale alterations in alternative pre-mRNA splicing. Fas pre-mRNA is alternatively spliced by excluding exon 6 to produce soluble Fas (sFas) protein that lacks a transmembrane domain and acts by inhibiting Fas mediated apoptosis. }In the present study we show that U2AF is involved in hypoxia dependent anti-apoptotic Fas mRNA isoform formation. Our performed studies show that U2AF-RNA interaction is reduced in hypoxic cells, leading to reduction of Fas and increased sFas mRNAs formation. Efficient U2AF-RNA interactions of both subunits are important for Fas exon 6 inclusion into forming mRNA in normoxic and hypoxic cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

LncRNA DLEU2 promotes cervical cancer cell proliferation by regulating cell cycle and NOTCH pathway

Highlights: • LncRNA DLEU2 is upregulated and related to tumor topography in cervical cancer. • LncRNA DLEU2 promotes cervical cancer cell proliferation. • LncRNA DLEU2 promotes the progression of the cell cycle. • LncRNA DLEU2 inhibits the activity of the Notch signaling pathway. Long noncoding RNAs (lncRNAs) are known to play a crucial role in the onset and progression of cervical cancer (CC). Here, the results of RNA microarray and RNA-sequencing dataset analysis showed that lncRNA DLEU2 was significantly upregulated in CC tissues. Clinicopathologic analysis indicated that lncRNA DLEU2 was closely related to tumor topography. Functional experiments and bioinformatics analysis revealed that lncRNA DLEU2 promoted CC cell proliferation and accelerated the cell cycle. Mechanistically, lncRNA DLEU2 promoted the progression of the cell cycle and inhibited the activity of the Notch signaling pathway by inhibiting p53 expression. Additionally, lncRNA DLEU2 probably interacted with ZFP36 Ring Finger Protein (ZFP36) to inhibit the expression of p53. In conclusion, this study revealed the function of lncRNA DLEU2 in CC tumorigenesis, suggesting new therapeutic targets in CC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

LncRNA LINC00680 promotes lung adenocarcinoma growth via binding to GATA6 and canceling GATA6-mediated suppression of SOX12 expression

Lung adenocarcinoma (LUAD) is a major subtype of non-small-cell lung cancers (NSCLC). LINC00680 has been characterized as a novel oncogenic lncRNA in LUAD, but its regulatory mechanisms remain largely unclear. This study aimed to explore the subcellular localization of LINC00680 in LUAD and its regulation on the transcriptional process. LUAD cell lines (A549, H1650, and H1299) were used for in vitro and in vivo studies. Results showed LINC00680 depletion resulted in G0/G1 phase arrest of LUAD cells and reduced CDK4 and cyclin D1 expression in H1650 and H1299 cells. LINC00680 overexpression promoted A549 cell proliferation and increased CDK4 and cyclin D1 expression. RNA-fluorescence in situ hybridization (FISH) assay showed that LINC00680 has both cytoplasmic and nuclear distribution in LUAD cells. RNA pulldown and western blotting assays confirmed a physical interaction between LINC00680 and GATA6. In LUAD cells, GATA6 overexpression only slightly suppressed SOX12 transcription. ChIP-qPCR and dual-luciferase assay showed that GATA6 only weakly bound to the SOX12 promoter and decreased its activity. However, when LINC00680 was depleted, these transcriptional suppressive effects were significantly enhanced. These findings suggested that LINC00680 forms a complex with GATA6 and weakens its transcriptional suppressive effect on SOX12 expression. In the nude mice model, LINC00680 overexpression partly abrogated the growth-suppressive effects of GATA6 on A549 derived tumors. In summary, this study revealed a novel LINC00680-GATA6-SOX12 axis in promoting LUAD cell cycle progression and proliferation. Future studies should be conducted for a better understanding of the complex networking of LINC00680 in LUAD.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Comprehensive analysis of differentially expressed mRNA and circRNA in Ankylosing spondylitis patients’ platelets

Highlights: • 4996 mRNAs and 2942 circRNAs were identified differentially expressed in platelets of AS patients. • Several platelet-derived immune mediators were upregulated to regulate the interaction between platelets and immune cells. • Two downregulated circRNA were identified, and the corresponding circRNA-miRNA-mRNA regulatory network was constructed. • mRNAs and circRNAs found herein could be used as diagnostic biomarkers or new therapeutic targets. Ankylosing spondylitis (AS) is a chronic inflammatory disease significantly decreasing the quality of life. Platelets play an important and active role in the development of AS. Accumulating evidence demonstrated platelets contain diverse RNA repository inherited from megakaryocytes or microvesicles. Platelet RNAs are dynamically affected by pathological conditions and could be used as diagnostic or prognostic biomarkers. However, the role of the platelet RNAs in AS is elusive. In this study, we compared mRNA and circRNA profiles in platelets between AS patients and healthy controls using RNA sequencing and bioinformatic analysis, and found 4996 mRNAs and 2942 circRNAs were differently expressed. The significantly over-expressed mRNAs in AS patients are involved in platelet activity, gap junction, focal adhesion, rap1 and toll and Imd signaling pathway. The previous identified platelet-derived immune mediators such as P2Y1, P2Y12, PF4, GPIbα, CD40L, ICAM2, CCL5 (RANTES), TGF-β (TGF-β1 and TGF-β2) and PDGF (PDGFB and PDGFA) are also included in these over expressed mRNAs, implying these factors may trigger inflammatory cascades and promote the development of AS. Additionally, we found two down-regulated circRNA (circPTPN22 and circFCHSD2) from the intersection analyses of platelets and spinal ligament tissues of AS patients. The circRNA-miRNA-mRNA regulatory network of these two circRNAs was constructed, and the target mRNAs were enriched in Th17 cell differentiation, inflammatory bowel disease, cell adhesion molecules, cytokine-cytokine receptor interaction, Jak-STAT and Wnt signaling pathway, all these pathways participate in the bone remodeling and pro/anti-inflammatory immune regulation in AS. Then, qRT-PCR was performed to validate the expression of selected key mRNAs and circRNAs and the results demonstrated that the expression levels of P2Y12, GPIbα, circPTPN22 and circFCHSD2 were consistent with the sequencing analysis. In addition, the high expression of five predicted miRNAs interacting with circPTPN22 and circFCHSD2 were also detected in AS by qRT-PCR. Taken together, our study presents a comprehensive overview of mRNAs and circRNAs in platelets in AS patients and offers new insight into the mechanisms of platelet involving in the pathogenesis of AS. The mRNAs and circRNAs identified in this study may serve as candidates for diagnosis and targeted treatment of AS.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

New CRISPR–Cas systems from uncultivated microbes

We present that CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Lastly, interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Architecture of the U6 snRNP reveals specific recognition of 3'-end processed U6 snRNA

The spliceosome removes introns from precursor messenger RNA (pre-mRNA) to produce mature mRNA. Prior to catalysis, spliceosomes are assembled de novo onto pre-mRNA substrates. During this assembly process, U6 small nuclear RNA (snRNA) undergoes extensive structural remodeling. The early stages of this remodeling process are chaperoned by U6 snRNP proteins Prp24 and the Lsm2–8 heteroheptameric ring. We now report a structure of the U6 snRNP from Saccharomyces cerevisiae. The structure reveals protein–protein contacts that position Lsm2–8 in close proximity to the chaperone “active site” of Prp24. The structure also shows how the Lsm2–8 ring specifically recognizes U6 snRNA that has been post-transcriptionally modified at its 3' end, thereby elucidating the mechanism by which U6 snRNPs selectively recruit 3' end-processed U6 snRNA into spliceosomes. Additionally, the structure reveals unanticipated homology between the C-terminal regions of Lsm8 and the cytoplasmic Lsm1 protein involved in mRNA decay.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Direct observation of tRNA-chaperoned folding of a dynamic mRNA ensemble

T-box riboswitches are multi-domain noncoding RNAs that surveil individual amino acid availabilities in most Gram-positive bacteria. T-boxes directly bind specific tRNAs, query their aminoacylation status to detect starvation, and feedback control the transcription or translation of downstream amino-acid metabolic genes. Most T-boxes rapidly recruit their cognate tRNA ligands through an intricate three-way stem I-stem II-tRNA interaction, whose establishment is not understood. Using single-molecule FRET, SAXS, and time-resolved fluorescence, we find that the free T-box RNA assumes a broad distribution of open, semi-open, and closed conformations that only slowly interconvert. tRNA directly binds all three conformers with distinct kinetics, triggers nearly instantaneous collapses of the open conformations, and returns the T-box RNA to their pre-binding conformations upon dissociation. This scissors-like dynamic behavior is enabled by a hinge-like pseudoknot domain which poises the T-box for rapid tRNA-induced domain closure. This study reveals tRNA-chaperoned folding of flexible, multi-domain mRNAs through a Venus flytrap-like mechanism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Investigating the roles of MicroRNAs in biotic stress response induced by Rhizoctonia solani in rice

Sheath blight disease, caused by Rhizoctonia solani 1802/KB was screened on two rice varieties, Oryza sativaindica cultivar MR219 and Oryza sativa indica cultivar UKMRC9. The disease symptom was severe in MR219 compared to UKMRC9. Total RNA from R. solani 1802/KB, infected rice leaves of MR219 and infected rice leaves of UKMRC9 were extracted using TRIzol reagent, purified and sent for small RNA sequencing. Three miRNA libraries were generated and analyzed. The libraries generated 65 805, 78 512 and 81 325 known miRNAs respectively. The structure of miRNA of these samples was predicted. The up-regulated and down-regulated of miRNAs target gene prediction and its target functions were discovered and were mainly related to the growth and development of metabolism, protein transport, transcriptional regulation, stress response, and hormone signaling and electron transfer. Sheath blight-induced differential expression of known miRNAs tends to targetMYB transcription factor, F-box proteins, NBS-LRR, leucine-rich repeat receptor protein kinases and zinc finger proteins. Detecting new miRNAs and measuring the expression profiles of known miRNAs is an important tasks required for a better understanding of various biological conditions. Therefore, further analysis using Gene Ontology Slim will be conducted to deduce some biological information from the datasets obtained.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Single-molecule-sensitive fluorescence resonance energy transfer in freely-diffusing attoliter droplets

Fluorescence resonance energy transfer (FRET) from individual, dye-labeled RNA molecules confined in freely-diffusing attoliter-volume aqueous droplets is carefully compared to FRET from unconfined RNA in solution. The use of freely-diffusing droplets is a remarkably simple and high-throughput technique that facilitates a substantial increase in signal-to-noise for single-molecular-pair FRET measurements. We show that there can be dramatic differences between FRET in solution and in droplets, which we attribute primarily to an altered pH in the confining environment. We also demonstrate that a sufficient concentration of a non-ionic surfactant mitigates this effect and restores FRET to its neutral-pH solution value. At low surfactant levels, even accounting for pH, we observe differences between the distribution of FRET values in solution and in droplets which remain unexplained. Our results will facilitate the use of nanoemulsion droplets as attoliter volume reactors for use in biophysical and biochemical assays, and also in applications such as protein crystallization or nanoparticle synthesis, where careful attention to the pH of the confined phase is required.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2015

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Intracellular effects of atmospheric-pressure plasmas on melanoma cancer cells

Gas discharge plasmas formed at atmospheric pressure and near room temperature have recently been shown as a promising tool for cancer treatment. The mechanism of the plasma action is attributed to generation of reactive oxygen and nitrogen species, electric fields, charges, and photons. The relative importance of different modes of action of atmospheric-pressure plasmas depends on the process parameters and specific treatment objects. Hence, an in-depth understanding of biological mechanisms that underpin plasma-induced death in cancer cells is required to optimise plasma processing conditions. Here, the intracellular factors involved in the observed anti-cancer activity in melanoma Mel007 cells are studied, focusing on the effect of the plasma treatment dose on the expression of tumour suppressor protein TP73. Over-expression of TP73 causes cell growth arrest and/or apoptosis, and hence can potentially be targeted to enhance killing efficacy and selectivity of the plasma treatment. It is shown that the plasma treatment induces dose-dependent up-regulation of TP73 gene expression, resulting in significantly elevated levels of TP73 RNA and protein in plasma-treated melanoma cells. Silencing of TP73 expression by means of RNA interference inhibited the anticancer effects of the plasma, similar to the effect of caspase inhibitor z-VAD or ROS scavenger N-acetyl cysteine. These results confirm the role of TP73 protein in dose-dependent regulation of anticancer activity of atmospheric-pressure plasmas.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗
DOE OSTI2015

Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. Here, the tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structure of pre-miR-31 reveals an active role in Dicer–TRBP complex processing

As an essential posttranscriptional regulator of gene expression, microRNA (miRNA) levels must be strictly maintained. The biogenesis of many miRNAs is mediated by trans-acting protein partners through a variety of mechanisms, including remodeling of the RNA structure. miR-31 functions as an oncogene in numerous cancers, and interestingly, its biogenesis is not known to be regulated by protein-binding partners. Therefore, the intrinsic structural properties of the precursor element of miR-31 (pre-miR-31) can provide a mechanism by which its biogenesis is regulated. We determined the solution structure of pre-miR-31 to investigate the role of distinct structural elements in regulating processing by the Dicer–TRBP complex. We found that the presence or absence of mismatches within the helical stem does not strongly influence Dicer–TRBP processing of the pre-miRNAs. However, both the apical loop size and structure at the Dicing site are key elements for discrimination by the Dicer–TRBP complex. Interestingly, our NMR-derived structure reveals the presence of a triplet of base pairs that link the Dicer cleavage site and the apical loop. Mutational analysis in this region suggests that the stability of the junction region strongly influences processing by the Dicer–TRBP complex. Our results enrich our understanding of the active role that RNA structure plays in regulating miRNA biogenesis, which has direct implications for the control of gene expression.

59 BASIC BIOLOGICAL SCIENCES↗