Luciferase Coding Sequence Research Articles

NEAR INFRARED-RESPONSIVE HYDROGELS CONTAINING ADENOVIRAL VECTORS: APPLICATION IN BONE REGENERATION

We have developed plasmonic fibrin-based hydrogels that incorporate gold nanoparticles which transduce incident near-infrared (NIR) light into heat. Human adenovirus serotype type-5 vectors encoding a firefly luciferase (fLuc) coding sequence driven by a heat-inducible promoter were incorporated into the hydrogels. Transmission electronic microscopic analysis revealed that the adenoviral vectors were associated to the fibrin fibers. In vitro experiments in which human cells were cultured with plasmonic hydrogels showed that the adenoviral vectors can diffuse from the hydrogels, transduce the cells, and stimulate heat-induced transgene expression upon NIR irradiation. The hydrogels were implanted in 4.2 mm drill hole defects generated in the humerus of male rabbits. Three days after implantation, the defects were NIR-irradiated. Six h later, the animals were euthanized and samples from the bone defect zone were processed for immunohistochemical analyses using a specific fLuc antibody. The results showed strong expression of fLuc in tissues surrounding the implants of NIR-irradiated rabbits, while non- irradiated animals exhibited negligible expression. We next aimed to use the temperature increase to induce the production of transgenic bone morphogenetic protein 6 (BMP-6), using safe gene switches that can provide tighter control of in vivo transgene expression than heat-inducible promoters. These switches are only activated by heat in the presence of rapamycin and maintain a high level of targeted transgene expression for several days after heat activation. Adenoviral vectors encoding the safe switches that control the expression of BMP-6 were incorporated to the composites. The resulting NIR-responsive hydrogels were implanted in the bone defects generated in rabbits and used as a platform to transduce host cells, generate local hyperthermia and stimulate BMP-6 production.Acknowledgements: This research was supported by grants RTI2018-095159-B-I00 and PID2021-126325OB-I00 (MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”), by grant P2022/BMD- 7406 (Regional Government of Madrid). M.A.L-J. is the recipient of predoctoral fellowship PRE2019-090430 (MCIN/AEI/10.13039/501100011033).

Remote control of transgene expression using noninvasive near-infrared irradiation.

This study investigated whether noninvasive near-infrared (NIR) energy could be transduced into heat in deep-seated organs in which adenovirus type-5 vectors tend to accumulate, thereby activating heat shock protein (HSP) promoter-mediated transgene expression, without local administration of photothermal agents. NIR irradiation of the subdiaphragmatic and left dorsocranial part of the abdominal cavity of adult immunocompetent C3H/HeNRj mice with an 808-nm laser effectively increased the temperature of the irradiated regions of the liver and spleen, respectively, resulting in the accumulation of the heat-inducible HSP70 protein. Spatial control of transgene expression was achieved in the NIR-irradiated regions of the mice administered an adenoviral vector carrying a firefly luciferase (fLuc) coding sequence controlled by a human HSP70B promoter, as assessed by bioluminescence and immunohistochemistry analyses. Levels of reporter gene expression were modulated by controlling NIR power density. Spatial control of transgene expression through NIR-focused activation of the HSP70B promoter, as well as temporal regulation by administering rapamycin was achieved in the spleens of mice inoculated with an adenoviral vector encoding a rapamycin-dependent transactivator driven by the HSP70B promoter and an adenoviral vector carrying a fLuc coding sequence controlled by the rapamycin-activated transactivator. Mice that were administered rapamycin and exposed to NIR light expressed fLuc activity in the splenic region, whereas no activity was detected in mice that were only administered rapamycin or vehicle or only NIR-irradiated. Thus, in the absence of any exogenously supplied photothermal material, remote control of heat-induced transgene expression can be achieved in the liver and spleen by means of noninvasive NIR irradiation.

Modulating miRNA binding sites within circRNA for enhanced translation efficiency

Circular RNAs (circRNAs) are covalently closed circular RNAs, and some of them preserve translation potency. However, modulation of circRNA translation efficiency and its applications need to be explored. In this study, RNAs containing the translation initiation element CVB3 IRES and the coding sequence of luciferase protein were transcribed and circularized in vitro by T7 RNA polymerase and an optimized permutated intron‒exon (PIE) splicing strategy. The circularized RNAs were then transfected and translated into active luciferase in the cultured cells. Insertion of miRNA binding sites at the flanking region of the luciferase coding sequence significantly reduced the translation efficiency of the circRNAs. Mutations of the miRNA binding sites in the firefly luciferase coding sequence led to increased translation efficiency of synthetic circRNAs in cells. We also proved that mutations of the binding sites of specific miRNAs also enhanced the translation efficiency of synthetic circRNAs. Further in vivo experiments via bioluminescence imaging showed that synonymous mutation of the miRNA binding sites promoted synthetic circRNA translation in nude mice. This study demonstrates that the modulation of miRNA binding sites affects the translation efficiency of synthetic circRNAs in vitro and in vivo, which could be used as versatile tools for future applications in clinical imaging.

Inhibitor of Bruton's Tyrosine Kinase‐α (IBTKα) in the Unfolded Protein Response

Disruption of protein and membrane homeostasis in the endoplasmic reticulum (ER) occurs in response to diverse environmental and physiological stresses. The ensuing unfolded protein response (UPR) serves to alleviate stress damage by lowering the influx of nascent proteins into the stressed organelle concurrent with enhanced expression of stress adaptive proteins that serve to expand the ER and restore homeostasis. Phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2) is central to the translational and transcription regulation of the UPR. While eIF2α phosphorylation serves to dampen global translation during ER stress, selected mRNAs are preferentially translated. The selective translation is facilitated via specialized upstream open reading frames (uORFs) in the 5’ leaders of these mRNAs. Our laboratory previously showed that there is a significant increase in the expression of IBTKα mRNA and protein in response to ER stress. IBTKα is a large multi-domain protein that engages with CUL3 and is suggested to ubiquitylate proteins involved in the regulation of protein homeostasis. The 5’ leader of human IBTKα mRNA contains 4 uORFs, and uORF1 and uORF2 are phylogenetically conserved. To determine the translational regulation of IBTKα, we used a translational reporter expressing the 5’ leader of IBTKα mRNA fused to firefly luciferase coding sequence. Mutation of the uORFs start codon from ATG to AGG was made to individually disrupt their functions. Wild type and mutant versions of IBTKα vectors were transiently transfected in HEK293T cells and these cells were treated with a pharmacological inducer of ER stress, thapsigargin, or vehicle, and luciferase activity was determined as a measure of translation control. We determined that uORF2 serves as a major inhibitory uORF that can be selectively bypassed during ER stress to enhance translation of the IBTKα coding sequence. Mutational analyses of conserved RNA stem-loop structures downstream of uORF2 enhances IBTKα translation in a fashion suggested to be linked with reinitiation following the inhibitory uORF2. Our study provides an example of how the secondary RNA structure of specific mRNA can contribute to its regulation along with uORFs present in the 5’ leader of mRNA in the UPR. For IBTKα functional studies, we are utilizing ubiquitin enrichment analyses to determine downstream targets of the IBTKα-CUL3 complex in the UPR. Finally, we are generating a knockout of IBTKα in mice to further characterize the function of IBTKα in ER stress. Our studies will provide insights into the regulation and function of a new UPR member, IBTKα, in the UPR.

Effect of Prooxidative Natural Products: Comparison of the OSI1 (YKL071w) Promoter Luciferase Construct from Yeast with an Nrf2/Keap Reporter System

The oxidative stress response (OSR) in yeast is under the control of oxidation-sensitive cysteines in the Yap1p transcription factor, and fusion of the Yap1p-dependent OS-induced promoter of the YKL071w gene (OSI1) to a luciferase coding sequence makes a sensitive reporter for OS induced by electrophiles. In mammalian cells, the OSR induced by electrophiles is coordinated in a mechanistically similar way via oxidation-sensitive cysteines in the kelch-like ECH-associated protein 1 (Keap1)– nuclear factor erythroid 2-related factor 2 / antioxidant response element ( Nrf2/ARE) system. Many electrophilic oxidants have already been independently shown to trigger both the Yap1 and Keap1 systems. Here, we investigated the responses of Yap1 and Keap1 reporters to sulforaphane (SFN), allyl isothiocyanate (AITC), phenylethyl isothiocyanate (PEITC), previously known to stimulate Keap1–Nrf2/ARE but not known to activate Yap1, and as a positive control, allicin, previously reported to stimulate both Yap1 and Nrf2. We have compared the reciprocal responsiveness of the respective reporter systems and show that the yeast reporter system can have predictive value for electrophiles that stimulate the mammalian Keap1–Nrf2/ARE system.

Establishment of 5'-3' interactions in mRNA independent of a continuous ribose-phosphate backbone.

Functions of eukaryotic mRNAs are characterized by intramolecular interactions between their ends. We have addressed the question whether 5′ and 3′ ends meet by diffusion-controlled encounter “through solution” or by a mechanism involving the RNA backbone. For this purpose, we used a translation system derived from Drosophila embryos that displays two types of 5′–3′ interactions: Cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug recognition elements (SREs) in the 3′ UTR. Chimeric RNAs were made consisting of one RNA molecule carrying a luciferase coding sequence and a second molecule containing SREs and a poly(A) tail; the two were connected via a protein linker. The poly(A) tail stimulated translation of such chimeras even when disruption of the RNA backbone was combined with an inversion of the 5′–3′ polarity between the open reading frame and poly(A) segment. Stimulation by the poly(A) tail also decreased with increasing RNA length. Both observations suggest that contacts between the poly(A) tail and the 5′ end are established through solution, independently of the RNA backbone. In the same chimeric constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the backbone is not involved in the repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting that the contact between the SREs in the 3′ UTR and the 5′ end of the RNA might be established in a manner that differs from the contact between the poly(A) tail and the cap.

The A allele of the UCP2 -866G/A polymorphism changes UCP2 promoter activity in HUVECs treated with high glucose.

The mitochondrial uncoupling protein 2 (UCP2) decreases reactive oxygen species (ROS) formation by mitochondria. Our group previously showed that the UCP2 -866A allele was associated with risk of diabetic retinopathy (DR), which is caused by hyperglycemia-induced oxidative stress. To date, it is still unclear if the -866A allele directly affects UCP2 expression in endothelial cells. Thus, we investigated the effect of the A allele on UCP2 promoter activity in HUVECs treated with high glucose (HG) or hydrogen peroxide (H2O2). To quantify UCP2 promoter activity, HUVECs were transfected with pGL3 plasmids containing the UCP2 promoter and the firefly luciferase coding sequence. Experimental groups were: (1) pGL3-866G-transfected cells and (2) pGL3-866A cells, both under normal (4mM) or HG (25mM) concentrations for 24h and 48h or incubated with H2O2 (0.1mM) for 1h. UCP2 promoter activity was monitored by Luminescent Dual-luciferase Assay. HG induced an upregulation of UCP2 promoter activity in PGL3-866G cells after 24h of treatment (P = 0.027), but not after 48h. Compared to pGL3-866G cells, pGL3-866A cells seems to have reduced UCP2 promoter activity following 24h and 48h of normal glucose treatment (P = 0.087 and P = 0.022). After HG treatment, pGL3-866A cells had more marked UCP2 downregulation (24h: - 3.2-folds, P < 0.001; and 48h: - 2.5-folds, P < 0.001 vs. G cells). Both pGL3-866G and pGL3-866A cells treated with H2O2 showed a ≅ 4-fold increase in UCP2 promoter activity (both P < 0.001). The -866A allele modifies UCP2 promoter activity in HUVECs under HG treatment but not in the H2O2 condition.

Novel Mechanism of Post-Transcriptional Regulation of PD-L1 Expression By 3'-UTR Binding Proteins

Programmed cell death ligand 1 (PD-L1) plays a key role in tumor immune escape by negatively regulating cytotoxic T-cells (CTLs) via PD-1 receptors. Thus, those tumors with high PD-L1 expression are thought to be sensitive to PD-1/PD-L1 blockade, reactivating CTL reactions to tumors. However, the mechanism that regulates PD-L1 expression in tumor cells has not been fully elucidated, understanding of which would help to develop effective anti-tumor immunotherapy.Recently, we reported that disruption of 3'-UTR of PD-L1 led to a remarkably enhanced expression of PD-L1 in a wide variety of human cancers, particularly adult T-cell leukemia/lymphoma (ATL) and diffuse large B-cell lymphoma. In these cancers, stability of PD-L1 transcripts is negatively regulated via their 3'-UTR sequence, whose disruption thus, results in markedly elevated PD-L1 expression and immune evasion (Kataoka et al., Nature, 2016). It has been well established that non-coding regions (i.e., 5'- and 3'-untranslated regions (UTRs)) play important roles in the regulation of mRNA expression, which is accomplished by several transacting factors that bind to cis-regulatory elements within the UTR. Based on this knowledge, we investigated the mechanism that controls PD-L1 expression through 3'-UTR sequence, primarily focusing on those transacting RNA-binding proteins (RNA-BPs).To determine the relevant regions within the 3'-UTR which are capable of repressing PD-L1, several cell lines were transfected with luciferase reporter vectors, in which a luciferase coding sequence was concatenated to various deletion mutants of the PD-L1 3'-UTR (Panel A). We identified two critical sequences, segment 5 and 2 within the PD-L1 3'-UTR, whose existence significantly decreased luciferase expression (Panel B; 293T data, mean ± SD, * denotes t-test p < .05). Importantly, the deletion of these sequences showed a similar effect on gene expression in various cell lines derived from different tissues. To confirm this finding, we used a CRISPR-mediated tiled 3'-UTR editing in situ technique. We designed all possible single-guide (sg) RNAs in the PD-L1 3'-UTR. The library was virally infected into cells and those with high expression of PD-L1 were concentrated by FACS, and the enrichment of each sgRNA was evaluated by high-throughput sequencer. The enriched sgRNAs localized to positions compatible with the luciferase assay, confirming that the two regions are actually responsible for the regulation of PD-L1 expression.Next, we searched RNA-BPs that bind to these regions in mass spectrometry, utilizing flag-peptide-tagged RNA pull-down method (Panel C). In brief, we synthesized PD-L1 3'-UTR RNA segments along with β-actin mRNA as control in vitro, and conjugated a flag-peptide to their 3'-ends. Using these RNA-baits, an immunoprecipitation experiment was performed in 293T cells and the co-immunoprecipitated proteins were analyzed by mass spectrometry. The data obtained from different segments were compared to each other. In addition to those proteins binding to multiple regions within the PD-L1 3'-UTR, we found proteins that specifically interacted with the repressive segments (segment 5 and 2) identified through luciferase assays.Finally, to confirm that the proteins detected by mass spectrometry actually suppress PD-L1 expression, we performed knock-down experiments using siRNA designed for the RNA-BPs that are presumed to interact with the segment 5 and 2. Three siRNA constructs per gene were transfected to 293T cells and their effect on PD-L1 expression was evaluated by RQ-PCR. Panel D shows relative PD-L1 expressions for each siRNA targets with median line, which are grouped according to relevant PD-L1 3'-UTR segments. The negative regulatory effect of these RNA-PBs on PD-L1 expression was largely confirmed. PD-L1 protein level was also increased, when these genes were knocked out by CRISPR/Cas9 system. Expression of these genes in ATL and other lymphomas was also evaluated.In summary, we identified critical sequences within the PD-L1 3'-UTR and RNA-BPs that bind to these sequences and negatively regulate PD-L1 expression. Our findings should not only provide novel insight into the molecular mechanisms by which PD-L1 expression in tumor cell is regulated but also help to identify potential targets for immune therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The coding sequence of firefly luciferase reporter gene affects specific hyperexpression in Arabidopsis thaliana cpl1 mutant

ABSTRACTForward genetic screening of mutants using firefly luciferase (LUC) reporter gene became a standard practice in plant research. Such screenings frequently identified alleles of CPL1 (Carboxyl-terminal Phosphatase-Like 1) regardless of promoters or pathways studied. Expression of the corresponding endogenous genes often shows the minimal difference between wild type and cpl1. Here we show that the LUC coding sequence is responsible for the high expression in cpl1, using a classical RD29a-LUC. Deletion of the LUC 3′-UTR did not change hyperactivation of LUC in cpl1. However, a codon-modified LUC (LUC2) produced similar expression levels both in wild type and in cpl1. These results indicate that the coding region of LUC is responsible for the cpl1-specific LUC overexpression uncoupled with the expression of the endogenous counterpart.

Visualization and Quantification of Browning Using a Ucp1-2A-Luciferase Knock-in Mouse Model.

Both mammals and adult humans possess classic brown adipocytes and beige adipocytes, and the amount and activity of these adipocytes are considered key factors in combating obesity and its associated metabolic diseases. Uncoupling protein 1 (Ucp1) is the functional marker of both brown and beige adipocytes. To facilitate a reliable, easy, and sensitive measurement of Ucp1 expression both in vivo and in vitro, we generated a Ucp1-2A-luciferase knock-in mouse by deleting the stop codon for the mouse Ucp1 gene and replacing it with a 2A peptide. This peptide was followed by the luciferase coding sequence to recapitulate the expression of the Ucp1 gene at the transcriptional and translational levels. With this mouse, we discovered a cold-sensitive brown/beige adipose depot underneath the skin of the ears, which we named uBAT. Because of the sensitivity and high dynamic range of luciferase activity, the Ucp1-2A-luciferase mouse is useful for both in vitro quantitative determination and in vivo visualization of nonshivering thermogenesis. With the use of this model, we identified and characterized axitinib, an oral small-molecule tyrosine kinase inhibitor, as an effective browning agent.

IL-1-induced post-transcriptional mechanisms target overlapping translational silencing and destabilizing elements in IκBζ mRNA.

During further molecular analysis concerning the function of the translational silencing element (TSE) identified in our publication, we noticed that a plasmid different from the one intended had been used erroneously in one part of the study. The mistake led to the use of a plasmid that contained the TSE sequence in antisense instead of sense orientation, downstream of the luciferase coding sequence in the bicistronic vector employed for experiments in Fig. 8A. Therefore, the conclusions drawn from this experiment that the TSE does not silence cap-independent translation and may function in a spatially restricted fashion (see under “Abstract,” line 10; see under “Results,” p. 29173, right column) have no basis. The discussion of this point (see under “Discussion,” p. 29176, left column) is obsolete. We are very sorry for this error and apologize for any problems it may have caused. Other conclusions and results of the publication are not affected by it. Concerning the question addressed by the experiment in Fig. 8, our most recent experiments demonstrate that the TSE does indeed silence even IRES-dependent translation.

Abstract 4479: Unveiling the relationship between the SWI/SNF chromatin remodeling complex and noncoding RNAs

Abstract Chromatin remodeling complexes are crucial for the viability of the cells due to their role in regulating interactions between DNA and histones and, therefore, modifying the accessibility of the genetic information to the transcriptional machinery. This relevance can also been seen in the SWI/SNF complex that has been associated with cancer in the last deep-sequencing efforts on tumoral genomes. BRG1 is the helicase/ATPase catalytic subunit of the SWI/SNF complex and it is frequently lost in NSCLC cell lines with a high mutation rate. In primary tumors, the loss of expression of BRG1 is also frequent, however it cannot be explained by mutations or by promoter hypermethylation. In this study we have focused on the regulation of the expression of BRG1 by microRNAs. These are small RNA molecules that do not code for any protein but they have key roles in gene expression regulation by binding to the 3’UTR of the transcripts. In addition, their levels have been found altered in several diseases including cancer. Our hypothesis is that the deregulation observed in BRG1 levels in primary tumors can be associated with changes in the microRNAs that control its expression. We have studied the 3’UTR of BRG1 by performing a Rapid Amplification of 3’ cDNA Ends (3’ RACE) and we have cloned the resulting 3’UTRs after a luciferase coding sequence of a plasmid in other to use the luminescence signal as a method to analyze the binding of the microRNAs tested to the 3’UTR of BRG1. Previous bioinformatic analyses were performed to choose the best microRNAs that could potentially regulate BRG1. Our preliminary results indicate that some microRNAs are able to bind to the 3’UTRs and successfully repress the expression of catalytic subunit of SWI/SNF. Importantly, one of these microRNAs is well known for developing oncogenic activities in lung cancer and it has been related with poor prognosis. In conclusion, the activity of the SWI/SNF complex can be regulated by microRNAs and this regulation may be relevant during lung carcinogenesis. From the other perspective, SWI/SNF complex could also impact in lung carcinogenesis regulating microRNAs expression, an aspect that we are currently evaluating. Citation Format: Paola Peinado Fernández, Isabel Fernández Coira, Eva E. Rufino Palomares, Octavio A. Romero, Chanatip Metheetrairut, Laura Boyero Corral, Julián Carretero, Esther Farez Vidal, Marta Cuadros Celorrio, Fernando Reyes Zurita, Victoria Sánchez Martín, Carlos Baliñas Gavira, Jose A. Lupiáñez Cara, Montse Sánchez Céspedes, Frank Slack, Pedro P. Medina. Unveiling the relationship between the SWI/SNF chromatin remodeling complex and noncoding RNAs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4479.

Genetic Screening for Arabidopsis Mutants Defective in STA1 Regulation under Thermal Stress Implicates the Existence of Regulators of Its Specific Expression, and the Genetic Interactions in the Stress Signaling Pathways.

To cope with environmental stresses, plants have developed various stress tolerance mechanisms that involve the induction of many stress responsive genes through stress-specific and common signaling pathways. Stress-specific/common transcription factors, rather than general basal factors, were considered important in this stress tolerance. The Arabidopsis STABILIZED1 (STA1) gene encodes a putative pre-mRNA splicing factor that is similar to the human U5 snRNP-associated 102-kDa protein and the yeast pre-mRNA splicing factors, PRP1p and Prp6p. As pre-mRNA splicing is a necessary process for proper gene expression in eukaryotes, STA1 is expected to be constantly functional in all conditions. Interestingly, STA1 expression is induced by temperature stresses, and STA1 recessive mutation (sta1-1) resulted in temperature stress-specific hypersensitivity. This suggests STA1’s stress specific function in addition to its presumed “housekeeping” role. In order to establish the genetic system to understand the regulation of STA1 expression in temperature stresses, we generated a bioluminescent Arabidopsis plant harboring the STA1 promoter fused to the firefly luciferase coding sequence (STA1p-LUC). Through genetic analysis, the bioluminescent Arabidopsis homozygous for one-copy STA1p-LUC was isolated and characterized. In this STA1p-LUC line, the expression patterns of STA1p-LUC were similar to those of the endogenous STA1 gene under cold and heat stresses. The STA1p-LUC line was then chemically mutagenized and screened to isolate the genetic loci of STA1 regulators under cold or heat stresses. Mutants with altered STA1p-LUC luminescence were identified and further confirmed through luminescence imaging in the next generation and analysis of endogenous STA1 expression. The categorization of STA1p-LUC deregulated mutants implicated the existence of cold or heat stress-specific as well as common genetic regulators for STA1 expression. Interestingly, some mutants showed opposite-directional deregulation of STA1 expression depending on the type of thermal stress, suggesting that the loci may represent important switch factors which determine the direction of signaling pathways for STA1 expression in response to temperature.

Abstract 429: MicroRNA Control of the Pacemaker Channel and Training-induced Bradycardia

Background: Athletes are prone to bradyarrythmias and have a higher-than-normal incidence of pacemaker implantation but the underlying mechanisms are poorly understood. In previous work we demonstrated a training-induced electrical remodelling of the sinoatrial node (SAN), including a down regulation of the key pacemaker channel HCN4 that carries the pacemaker current I f . Here we investigated post-transcriptional regulation of HCN4 by microRNAs (miRNAs) in the trained SN. Methods and Results: Mice subjected to 60-min swimming twice daily for 28 days (TM) were compared to parallel sedentary mice (SM).TM were bradycardic (cycle length in vivo : SM, 81±1.3 ms; TM, 102±3 ms; n=6, p &lt;0.05). The cycle length of the isolated, denervated SAN (intrinsic heart rate) was also prolonged by exercise training (SM, 110±3 ms; TM, 150±5 ms; n=7, p &lt;0.05). Deep sequencing for miRNAs on SAN punch biopsies revealed 10 differentially expressed miRNAs in TM vs SM (FDR adjusted P &lt;0.05); 8 miRNAs were upregulated, 2 were downregulated. These findings were further confirmed by real-time PCR. We used the computation prediction tools Targetscan and Ingenuity Pathway Analysis to identify miR-423-5p and miR-486-3p as putative miRNAs participating in the post-transcriptional repression of HCN4 in the trained SAN. Both miRNAs had conserved seed sequences in the HCN4 3’UTR. To validate whether these miRNAs directly recognise the 3’UTR of HCN4, they were co-transfected with a construct containing HCN4 3’UTR fused downstream to a luciferase coding sequence in H9c2 cells. Transient transfection of precursor miR-423-5p and miR-486-3p decreased HCN4 3’UTR luciferase reporter activity in H9C2 cells by 47% and 22% respectively, compared with unaltered luciferase activity on co-transfection with control miR or miR-27a. Conclusions: This is the first report of miRNA dysregulation in the trained SAN. We describe a novel molecular mechanism whereby an altered miRNA profile in the trained SAN promotes HCN4 downregulation and altered sinus automaticity. Data suggest miR-423-5p as a potential therapeutic target for bradyarrythmias in the athlete.

ZC3H12D attenuated inflammation responses by reducing mRNA stability of proinflammatory genes.

Infection in airspaces and lung parenchyma may cause acute lung injury and multiple organ dysfunction syndrome due to acute inflammatory response, leading to organ failure and high mortality. ZC3H12D has been shown to modulate Toll-like receptor signaling. This study aimed to investigate the change of ZC3H12D during acute lung injury and its role in inflammation processes. Mice were challenged with lipopolysaccharides (LPS) intratracheally. The expression levels of Zc3h12d, NF-κB, and cytokines were analyzed by quantitative real-time PCR (qPCR), ELISA, and Western blot. The mRNA stability was assessed by qPCR after cells were treated with actinomycin D for specified times. The 3' untranslated region (3'-UTR) of c-fos was cloned immediately downstream of the luciferase coding sequence driven by CMV promoter and luciferase activity was measured with a Luciferase Assay kit. Upon LPS treatment, ZC3H12D levels were reduced in mouse immune cells, whereas levels of NF-κB, IL-6, and TNF-α were significantly increased. Knockdown Zc3h12d in THP1 cells resulted in the upregulation of NF-κB while overexpression of Zc3h12d inhibited NF-κB expression. Ectopic Zc3h12d significantly reduced the mRNA stability of c-fos, NF-κB, TNF-α, IL-1β, and IL-6. Attachment of the c-fos 3'-UTR made luciferase expression levels sensitive to levels of ZC3H12D. The data indicated that ZC3H12D could suppress both the initial inflammation storm and chronic inflammation by targeting the mRNA of cytokines as well as NF-κB and c-fos.

9B.02

Objective: We previously showed that a blunted expression of the Twik-related Acid-Sensitive K+ channel 2 (TASK-2) is a common feature of Aldosterone Producing Adenoma (APA). Thus, we aimed at investigating the presence of mutations in the promoter region of the TASK-2 gene (KCNK5) in APA. Design and method: We sequenced the entire TASK-2 promoter region of 88 APA and 98 primary hypertensive patients (controls). We next tested the in vitro effects of the most frequently detected mutation by fusing the mutated and wild type TASK-2 promoter region to the luciferase coding sequence and transfecting the reporter vectors in H295R cells. Results: We detected only one mutation (C999T) in one of 98 primary hypertensive patients. Seven novel mutations were found in 16% of APA: the C999T variant was found in 6% of APA, the G595A in 3.5% of APA, the G36A in 2% of APAs, while the Gins466, G263C, C1247T and G1140T mutations in 1% of APA. None was germline. By site-direct mutagenesis we demonstrated that the C999T mutation significantly decreased the TASK-2 transcription activity and luciferase signal of the reporter vector in transfected H295R cells (fold change of normalized luciferase signal: 0.55 ± 0.22, p Conclusions: Thus, we demonstrated that 16% of APA have seven recurrent mutations in the promoter region of TASK-2 gene. One of these TASK-2 genetic variants blunts the transcription of the TASK-2 in human adrenal cells, thus suggesting a possible molecular mechanism contributing to the autonomous aldosterone secretion typical of APA.

Comprehensive luciferase-based reporter gene assay reveals previously masked up-regulatory effects of miRNAs.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the majority of the transcriptome at a post-transcriptional level. Because of this critical role, it is important to ensure that the assays used to determine their functionality are robust and reproducible. Typically, the reporter gene assay in cell-based systems has been the first-line method to study miRNA functionality. In order to overcome some of the potential errors in interpretation that can be associated with this assay, we have developed a detailed protocol for the luciferase reporter gene assay that has been modified for miRNAs. We demonstrate that normalization against the effect of the miRNA and cellular factors on the luciferase coding sequence is essential to obtain the specific impact of the miRNA on the 3'UTR (untranslated region) target. Our findings suggest that there is a real possibility that the roles for miRNA in transcriptome regulation may be misreported due to inaccurate normalization of experimental data and also that up-regulatory effects of miRNAs are not uncommon in cells. We propose to establish this comprehensive method as standard for miRNA luciferase reporter assays to avoid errors and misinterpretations in the functionality of miRNAs.

Modulating and measuring Wingless signalling

The main Wnt ligand of Drosophila activates a conserved canonical signalling pathway to regulate a plethora of cellular activities during development, regeneration and nervous system function. Here I first describe experimental means of measuring and modulating Wingless signalling in Drosophila cell culture. Various reporters have been devised by placing TCF-binding sites or DNA fragments from known target genes upstream of luciferase-coding sequences. Signalling can be activated in cells by addition of Wingless conditioned medium, treatment with a chemical inhibitor of Shaggy/GSK3 or transfection with a plasmid encoding activated Armadillo (Drosophila β-catenin). Measuring Wingless signalling in intact tissue is somewhat more challenging than in cell culture. Synthetic transgenic reporters have been devised but further improvements are needed to achieve sensitive responsiveness to Wingless at all times and places. As an alternative, gene traps in frizzled3 and notum/wingful, two context-independent endogenous targets, can be used as reporters. It is hoped that further modification of these loci could lead to more versatile and sensitive means of detecting signalling. Many genetic tools are available to trigger ectopic signalling or prevent endogenous signalling. These mostly rely on RNAi-producing transgenes or the generation of mutant patches by mitotic recombination. New developments in genome engineering are opening further means of manipulating the components of Wingless signalling with exquisite temporal and spatial precision.

Mapping of internal ribosome entry sites (IRES).

The dicistronic luciferase reporter gene system is the most common method to isolate and characterize internal ribosome entry sites (IRES). It is based on the expression of a dicistronic RNA comprising two independent reporter genes in 3' and 5'cistrons, and the putative IRES inserted into intercistronic region. The most convenient aspect of using Renilla and firefly luciferase genes is that both gene products can be detected in a single assay using Dual-Luciferase(®) Reporter Assay System from Promega. The Renilla luciferase coding sequence is often inserted into the 5'cistron and serves as internal control. It is translated cap dependently, as it is close to the cap structure at the 5' end. The 3'cistron located far downstream to the cap structure can only be translated by a cap-independent mechanism when the intercistronic sequence is capable of ribosome binding and re-initiation of translation. Expression level of the 3'cistron is usually normalized to the expression of 5'cistron to estimate the relative IRES activity of intercistronic sequences.

Role of microRNA-136-3p on the Expression of Luteinizing Hormone-Human Chorionic Gonadotropin Receptor mRNA in Rat Ovaries1

MicroRNAs (miRNAs) are small noncoding RNAs that interact with mRNAs and trigger either translation repression or RNA cleavage of target genes. In this study, we investigated whether miRNA was involved in down-regulation of the luteinizing hormone receptor (LHR) in rat ovaries. An miRNA microarray was used to analyze the overall miRNA expression profile of rat ovaries in association with the down-regulation of LHR mRNA. We found that 23 miRNAs were highly expressed during this period. Combining these results with data from a bioinformatics database, clustering analysis led us to focus on miR-136-3p for further analysis. In both in vivo and in vitro studies, miR-136-3p expression levels were increased at 6 h after human chorionic gonadotropin (hCG) administration, concurrent with down-regulation of LHR mRNA. Moreover, transfection of cultured granulosa cells with miR-136-3p resulted in a significant decrease in LHR mRNA levels in comparison with those of cells transfected with negative control. In contrast, transfection with a miR-136-3p inhibitor increased LHR mRNA levels. Finally, cotransfection of granulosa cells with a miR-136-3p inhibitor and a reporter vector containing the 3'-untranslated region (UTR) of LHR mRNA and Renilla luciferase coding sequence revealed that miR-136-3p bound directly to the 3'-UTR of LHR mRNA. These data demonstrated that miR-136-3p participated in the down-regulation of LHR mRNA by binding directly to LHR mRNA.