Effects of codon usage on gene expression are promoter context dependent.

Codon usage bias is a universal feature of all genomes. Although codon usage has been shown to regulate mRNA and protein levels by influencing mRNA decay and transcription in eukaryotes, little or no genome-wide correlations between codon usage and mRNA levels are detected in mammalian cells, raising doubt on the significance of codon usage effect on gene expression. Here we show that gene-specific regulation reduces the genome-wide codon usage and mRNA correlations: Constitutively expressed genes exhibit much higher genome-wide correlations than differentially expressed genes from fungi to human cells. Using Drosophila S2 cells as a model system, we showed that the effect of codon usage on mRNA expression level is promoter-dependent. Regions downstream of the core promoters of differentially expressed genes can repress the codon usage effects on mRNA expression. An element in the Hsp70 promoter was identified to be necessary and sufficient for this inhibitory effect. The promoter-dependent codon usage effects on mRNA levels are regulated at the transcriptional level through modulation of histone modifications, nucleosome densities and premature termination. Together, our results demonstrate that promoters play a major role in determining whether codon usage influences gene expression and further establish the transcription-dependent codon usage effects on gene expression.

MicroRNAs mediate gene silencing via multiple different pathways in drosophila.

MicroRNAs (miRNAs) guide RNA-induced silencing complex (RISC) that contains an Argonaute family protein to complementary target messenger RNAs (mRNAs). Via RISC, miRNAs silence the expression of target mRNAs by shortening the poly(A) tail-which leads to mRNA decay-and by repressing translation. It has been suggested that GW182, an Argonaute-associating protein, plays the central role in such microRNA actions. Here we show that, although GW182 is obligatory for poly(A) shortening, translational repression by microRNAs occurs even in the absence of GW182. Yet, GW182 is also capable of inducing translational repression independently. Both of these translational repression mechanisms block formation of 48S and 80S ribosomal complexes. Thus microRNAs utilize at least three distinct silencing pathways: GW182-mediated deadenylation and GW182-dependent and -independent repression of early translation initiation. Differential contribution from these multiple pathways may explain previous, apparently contradictory observations of how microRNAs inhibit protein synthesis.

Rational design of novel N-alkyl-N capped biostable RNA nanostructures for efficient long-term inhibition of gene expression.

Computational techniques have been used to design a novel class of RNA architecture with expected improved resistance to nuclease degradation, while showing interference RNA activity. The in silico designed structure consists of a 24-29 bp duplex RNA region linked on both ends by N-alkyl-N dimeric nucleotides (BCn dimers; n = number of carbon atoms of the alkyl chain). A series of N-alkyl-N capped dumbbell-shaped structures were efficiently synthesized by double ligation of BCn-loop hairpins. The resulting BCn-loop dumbbells displayed experimentally higher biostability than their 3'-N-alkyl-N linear version, and were active against a range of mRNA targets. We studied first the effect of the alkyl chain and stem lengths on RNAi activity in a screen involving two series of dumbbell analogues targeting Renilla and Firefly luciferase genes. The best dumbbell design (containing BC6 loops and 29 bp) was successfully used to silence GRB7 expression in HER2+ breast cancer cells for longer periods of time than natural siRNAs and known biostable dumbbells. This BC6-loop dumbbell-shaped structure displayed greater anti-proliferative activity than natural siRNAs.

Cell-based high-throughput compound screening reveals functional interaction between oncofetal HMGA2 and topoisomerase I.

HMGA2 is an important chromatin factor that interacts with DNA via three AT-hook domains, thereby regulating chromatin architecture and transcription during embryonic and fetal development. The protein is absent from differentiated somatic cells, but aberrantly re-expressed in most aggressive human neoplasias where it is causally linked to cell transformation and metastasis. DNA-binding also enables HMGA2 to protect cancer cells from DNA-damaging agents. HMGA2 therefore is considered to be a prime drug target for many aggressive malignancies. Here, we have developed a broadly applicable cell-based reporter system which can identify HMGA2 antagonists targeting functionally important protein domains, as validated with the known AT-hook competitor netropsin. In addition, high-throughput screening can uncover functional links between HMGA2 and cellular factors important for cell transformation. This is demonstrated with the discovery that HMGA2 potentiates the clinically important topoisomerase I inhibitor irinotecan/SN-38 in trapping the enzyme in covalent DNA-complexes, thereby attenuating transcription.

False responses of Renilla luciferase reporter control to nuclear receptor TR4.

Renilla luciferase reporter is a widely used internal control in dual luciferase reporter assay system, where its transcription is driven by a constitutively active promoter. However, the authenticity of the Renilla luciferase response in some experimental settings has recently been questioned. Testicular receptor 4 (TR4, also known as NR2C2) belongs to the subfamily 2 of nuclear receptors. TR4 binds to a direct repeat regulatory element in the promoter of a variety of target genes and plays a key role in tumorigenesis, lipoprotein regulation, and central nervous system development. In our experimental system using murine pituitary corticotroph tumor AtT20 cells to investigate TR4 actions on POMC transcription, we found that overexpression of TR4 resulted in reduced Renilla luciferase expression whereas knockdown TR4 increased Renilla luciferase expression. The TR4 inhibitory effect was mediated by the TR4 DNA-binding domain and behaved similarly to the GR and its agonist, Dexamethasone. We further demonstrated that the chimeric intron, commonly present in various Renilla plasmid backbones such as pRL-Null, pRL-SV40, and pRL-TK, was responsible for TR4's inhibitory effect. The results suggest that an intron-free Renilla luciferase reporter may provide a satisfactory internal control for TR4 at certain dose range. Our findings advocate caution on the use of Renilla luciferase as an internal control in TR4-directed studies to avoid misleading data interpretation.

An alternative approach in regulation of expression of a transgene by endogenous miR-145 in carcinoma and normal breast cell lines.

MicroRNAs are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidences indicate that miRNAs have different expression profiles and play crucial roles in numerous cellular processes. Delivery and expression of transgenes for cancer therapy must be specific for tumors to avoid killing of healthy tissues. Many investigators have shown that transgene expression can be suppressed in normal cells using vectors that are responsive to microRNA regulation. To overcome this problem, miR-145 that exhibits downregulation in many types of cancer cells was chosen for posttranscriptional regulatory systems mediated by microRNAs. In this study, a psiCHECK-145T vector carrying four tandem copies of target sequences of miR-145 into 3'-UTR of the Renilla luciferase gene was constructed. Renilla luciferase activity from the psiCHECK-145T vector was 57% lower in MCF10A cells with high miR-145 expression as compared to a control condition. Additionally, overexpression of miR-145 in MCF-7 cells with low expression level of miR-145 showed more than 76% reduction in the Renilla luciferase activity from the psiCHECK-145T vector. Inclusion of miR-145 target sequences into the 3'-UTR of the Renilla luciferase gene is a feasible strategy for restricting transgene expression in a breast cancer cell line while sparing a breast normal cell line.

The dynamics of MAPK inactivation at fertilization in mouse eggs.

Egg activation at fertilization in mammals is initiated by prolonged Ca(2+) oscillations that trigger the completion of meiosis and formation of pronuclei. A fall in mitogen-activated protein kinase (MAPK) activity is essential for pronuclear formation, but the precise timing and mechanism of decline are unknown. Here, we have measured the dynamics of MAPK pathway inactivation during fertilization of mouse eggs using novel chemiluminescent MAPK activity reporters. This reveals that the MAPK activity decrease begins during the Ca(2+) oscillations, but MAPK does not completely inactivate until after pronuclear formation. The MAPKs present in eggs are Mos, MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) and MAPK3 and MAPK1 (ERK1 and ERK2, respectively). Notably, the MAPK activity decline at fertilization is not explained by upstream destruction of Mos, because a decrease in the signal from a Mos-luciferase reporter is not associated with egg activation. Furthermore, Mos overexpression does not affect the timing of MAPK inactivation or pronuclear formation. However, the late decrease in MAPK could be rapidly reversed by the protein phosphatase inhibitor, okadaic acid. These data suggest that the completion of meiosis in mouse zygotes is driven by an increased phosphatase activity and not by a decline in Mos levels or MEK activity.

In vitro translation in a hybrid cell free lysate with exogenous cellular ribosomes.

Cell free protein synthesis systems (CFPS) have been widely used to express proteins and to explore the pathways of gene expression. In the present manuscript, we describe the design of a novel adaptable hybrid in vitro translation system which is assembled with ribosomes isolated from many different origins. We first show that this hybrid system exhibits all important features such as efficiency, sensitivity, reproducibility and the ability to translate specialized mRNAs in less than 1 h. In addition, the unique design of this cell free assay makes it highly adaptable to utilize ribosomes isolated from many different organs, tissues or cell types.

miR-Synth: a computational resource for the design of multi-site multi-target synthetic miRNAs.

RNAi is a powerful tool for the regulation of gene expression. It is widely and successfully employed in functional studies and is now emerging as a promising therapeutic approach. Several RNAi-based clinical trials suggest encouraging results in the treatment of a variety of diseases, including cancer. Here we present miR-Synth, a computational resource for the design of synthetic microRNAs able to target multiple genes in multiple sites. The proposed strategy constitutes a valid alternative to the use of siRNA, allowing the employment of a fewer number of molecules for the inhibition of multiple targets. This may represent a great advantage in designing therapies for diseases caused by crucial cellular pathways altered by multiple dysregulated genes. The system has been successfully validated on two of the most prominent genes associated to lung cancer, c-MET and Epidermal Growth Factor Receptor (EGFR). (See http://microrna.osumc.edu/mir-synth).

A novel function for the survival motoneuron protein as a translational regulator.

SMN1, the causative gene for spinal muscular atrophy (SMA), plays a housekeeping role in the biogenesis of small nuclear RNA ribonucleoproteins. SMN is also present in granular foci along axonal projections of motoneurons, which are the predominant cell type affected in the pathology. These so-called RNA granules mediate the transport of specific mRNAs along neurites and regulate mRNA localization, stability, as well as local translation. Recent work has provided evidence suggesting that SMN may participate in the assembly of RNA granules, but beyond that, the precise nature of its role within these structures remains unclear. Here, we demonstrate that SMN associates with polyribosomes and can repress translation in an in vitro translation system. We further identify the arginine methyltransferase CARM1 as an mRNA that is regulated at the translational level by SMN and find that CARM1 is abnormally up-regulated in spinal cord tissue from SMA mice and in severe type I SMA patient cells. We have previously characterized a novel regulatory pathway in motoneurons involving the SMN-interacting RNA-binding protein HuD and CARM1. Thus, our results suggest the existence of a potential negative feedback loop in this pathway. Importantly, an SMA-causing mutation in the Tudor domain of SMN completely abolished translational repression, a strong indication for the functional significance of this novel SMN activity in the pathology.

Caveolin-1 mediates gene transfer and cytotoxicity of polyethyleneimine in mammalian cell lines.

Polyethyleneimine (PEI) is a cost-effective and non-viral vector for gene transfer, but the factors determining gene transfer efficiency and cytotoxicity of PEI in different mammalian cell lines remain largely unknown. In the present study, three different cell lines were chosen for investigation. Using pEGFP DNA and PEI, 21.5, 29.2, and 92.1 % of GFP-positive cells were obtained in BMSC, Hela, and 293T, respectively. In luciferase reporter assay, similar results were obtained (for luciferase activity, BMSC < Hela < 293T cells). By MTT test and cell apoptotic marker analysis, we demonstrated that high gene transfer efficiency is accompanied with high cytotoxicity of PEI. Moreover, we found that high expression level of caveolin-1 was accompanied with high gene transfer efficiency and cytotoxicity of PEI in 293T cells. More convincingly, caveolin-1 silencing in 293T could reduce both gene transfer efficiency and cytotoxicity of PEI. In contrast, caveolin-1 overexpression in BMSCs increases both gene transfer efficiency and cytotoxicity of PEI. Taken together, our study suggests that caveolin-1 may at least in part determine gene transfer efficiency and cytotoxicity of PEI in mammalian cell lines, providing caveolin-1 as a potential target for improving gene transfer efficiency when applying positively charged polyplexes to cell transfection.

CREB regulates the expression of neuronal glucose transporter 3: a possible mechanism related to impaired brain glucose uptake in Alzheimer's disease.

Impaired brain glucose uptake and metabolism precede the appearance of clinical symptoms in Alzheimer disease (AD). Neuronal glucose transporter 3 (GLUT3) is decreased in AD brain and correlates with tau pathology. However, what leads to the decreased GLUT3 is yet unknown. In this study, we found that the promoter of human GLUT3 contains three potential cAMP response element (CRE)-like elements, CRE1, CRE2 and CRE3. Overexpression of CRE-binding protein (CREB) or activation of cAMP-dependent protein kinase significantly increased GLUT3 expression. CREB bound to the CREs and promoted luciferase expression driven by human GLUT3-promoter. Among the CREs, CRE2 and CRE3 were required for the promotion of GLUT3 expression. Full-length CREB was decreased and truncation of CREB was increased in AD brain. This truncation was correlated with calpain I activation in human brain. Further study demonstrated that calpain I proteolysed CREB at Gln28-Ala29 and generated a 41-kDa truncated CREB, which had less activity to promote GLUT3 expression. Importantly, human brain GLUT3 was correlated with full-length CREB positively and with activation of calpain I negatively. These findings suggest that overactivation of calpain I caused by calcium overload proteolyses CREB, resulting in a reduction of GLUT3 expression and consequently impairing glucose uptake and metabolism in AD brain.

Area-specific cell stimulation via surface-mediated gene transfer using apatite-based composite layers.

Surface-mediated gene transfer systems using biocompatible calcium phosphate (CaP)-based composite layers have attracted attention as a tool for controlling cell behaviors. In the present study we aimed to demonstrate the potential of CaP-based composite layers to mediate area-specific dual gene transfer and to stimulate cells on an area-by-area basis in the same well. For this purpose we prepared two pairs of DNA-fibronectin-apatite composite (DF-Ap) layers using a pair of reporter genes and pair of differentiation factor genes. The results of the area-specific dual gene transfer successfully demonstrated that the cells cultured on a pair of DF-Ap layers that were adjacently placed in the same well showed specific gene expression patterns depending on the gene that was immobilized in the underlying layer. Moreover, preliminary real-time PCR results indicated that multipotential C3H10T1/2 cells may have a potential to change into different types of cells depending on the differentiation factor gene that was immobilized in the underlying layer, even in the same well. Because DF-Ap layers have a potential to mediate area-specific cell stimulation on their surfaces, they could be useful in tissue engineering applications.

Transcription factor Foxo3a prevents apoptosis by regulating calcium through the apoptosis repressor with caspase recruitment domain.

Apoptosis can occur in the myocardium under a variety of pathological conditions, including myocardial infarction and heart failure. The forkhead family of transcription factor Foxo3a plays a pivotal role in apoptosis; however, its role in regulating cardiac apoptosis remains to be fully elucidated. We showed that enforced expression of Foxo3a inhibits cardiomyocyte apoptosis, whereas knockdown of endogenous Foxo3a sensitizes cardiomyocytes to undergo apoptosis. The apoptosis repressor with caspase recruitment domain (ARC) is a potent anti-apoptotic protein. Here, we demonstrate that it attenuates the release of calcium from the sarcoplasmic reticulum and inhibits calcium elevations in the cytoplasm and mitochondria provoked by oxidative stress in cardiomyocytes. Furthermore, Foxo3a is shown to maintain cytoplasmic and mitochondrial calcium homeostasis through ARC. We observed that Foxo3a knock-out mice exhibited enlarged myocardial infarction sizes upon ischemia/reperfusion, and ARC transgenic mice demonstrated reduced myocardial infarction and balanced calcium levels in mitochondria and sarcoplasmic reticulum. Moreover, we showed that Foxo3a activates ARC expression by directly binding to its promoter. This study reveals that Foxo3a maintains calcium homeostasis and inhibits cardiac apoptosis through trans-activation of the ARC promoter. These findings provided novel evidence that Foxo3a and ARC constitute an anti-apoptotic pathway that regulates calcium homeostasis in the heart.

Expression of PRPF31 and TFPT: regulation in health and retinal disease.

PRPF31, a gene located at chromosome 19q13.4, encodes the ubiquitous splicing factor PRPF31. The gene lies in a head-to-head arrangement with TFPT, a poorly characterized gene with a role in cellular apoptosis. Mutations in PRPF31 have been implicated in autosomal dominant retinitis pigmentosa (adRP), a frequent and important cause of blindness worldwide. Disease associated with PRPF31 mutations is unusual, in that there is often non-penetrance of the disease phenotype in affected families, caused by differential expression of PRPF31. This study aimed to characterize the basic promoter elements of PRPF31 and TFPT. Luciferase reporter constructs were made, using genomic DNA from an asymptomatic individual with a heterozygous deletion of the entire putative promoter region. Fragments were tested by the dual-luciferase reporter assay in HeLa and RPE-1 cell lines. A comparison was made between the promoter regions of symptomatic and asymptomatic mutation-carrying individuals. A patient (CAN493) with adRP was identified, harbouring a regulatory region mutation; both alleles were assayed by the dual-luciferase reporter assay. Luciferase assays led to the identification of core promoters for both PRPF31 and TFPT; despite their shared gene architecture, the two genes appear to be controlled by slightly different regulatory regions. One functional polymorphism was identified in the PRPF31 promoter that increased transcriptional activation. The change was not, however, consistent with the observed symptomatic-asymptomatic phenotypes in a family affected by PRPF31-adRP. Analysis of the mutant promoter fragment from CAN493 showed a >50% reduction in promoter activity, suggesting a disease mechanism of functional haploinsufficiency-the first report of this disease mechanism in adRP.

TRBP alters human precursor microRNA processing in vitro.

MicroRNAs play central roles in controlling gene expression in human cells. Sequencing data show that many miRNAs are produced at different levels and as multiple isoforms that can vary in length at their 5' or 3' ends, but the biogenesis and functional significance of these RNAs are largely unknown. We show here that the human trans-activation response (TAR) RNA binding protein (TRBP), a known molecular partner of the miRNA processing enzyme Dicer, changes the rates of pre-miRNA cleavage in an RNA-structure-specific manner. Furthermore, TRBP can trigger the generation of iso-miRNAs (isomiRs) that are longer than the canonical sequence by one nucleotide. We show that this change in miRNA processing site can alter guide strand selection, resulting in preferential silencing of a different mRNA target. These results implicate TRBP as a key regulator of miRNA processing and targeting in humans.

A guide to design and optimization of reporter assays for 3' untranslated region mediated regulation of mammalian messenger RNAs.

Post-transcriptional regulatory mechanisms are pervasive in the control of gene expression. Regulatory sequences within transcripts can control RNA processing, localization, translation efficiency, and stability of the RNA. Regulation is mediated by a diverse set of RNA binding regulators, including proteins and RNAs, which interact with specific mRNA sequences that are often found in untranslated regions. The potential for vast post-transcriptional control exists: mammalian mRNAs contain extensive untranslated regions and their genomes encode many hundreds of RNA binding proteins and non-coding RNAs. Facile quantitative methods are necessary to study the activities and mechanisms of regulatory sequences and the RNA binding factors that recognize them. Here we discuss the design and implementation of luciferase-based reporter assays to measure the effect of regulatory RNA sequences on protein and RNA expression. Protocols are described for transfection of the reporter into cells, measurement of protein expression levels with luciferase activity assays, RNA purification, and measurement of mRNA levels by reverse-transcription and quantitative polymerase chain reaction. For each assay, troubleshooting of common problems and critical controls are discussed. We present our optimized techniques and data from studies that measure specific and direct repression (i.e. negative regulation) of mRNAs by members of the PUF family of RNA binding proteins in cultured human cells.

Nanoparticle-DNA-polymer composites for hepatocellular carcinoma cell labeling, sensing, and magnetic resonance imaging.

This paper describes comparative studies and protocols in (1) self-assembling of ultrasmall superparamagnetic iron oxide nanoparticle (NP), circular plasmid DNA, and branched polyethylenimine (PEI) composites; (2) magnetofection; (3) gene delivery, (4) magnetic resonance imaging (MRI), and (5) cytotoxicity of the composites toward hepatocellular carcinoma HepG2 cells.

Dissecting non-coding RNA mechanisms in cellulo by Single-molecule High-Resolution Localization and Counting.

Non-coding RNAs (ncRNAs) recently were discovered to outnumber their protein-coding counterparts, yet their diverse functions are still poorly understood. Here we report on a method for the intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) of microRNAs (miRNAs), a conserved, ubiquitous class of regulatory ncRNAs that controls the expression of over 60% of all mammalian protein coding genes post-transcriptionally, by a mechanism shrouded by seemingly contradictory observations. We present protocols to execute single particle tracking (SPT) and single-molecule counting of functional microinjected, fluorophore-labeled miRNAs and thereby extract diffusion coefficients and molecular stoichiometries of micro-ribonucleoprotein (miRNP) complexes from living and fixed cells, respectively. This probing of miRNAs at the single molecule level sheds new light on the intracellular assembly/disassembly of miRNPs, thus beginning to unravel the dynamic nature of this important gene regulatory pathway and facilitating the development of a parsimonious model for their obscured mechanism of action.

Differential effects of diverse p53 isoforms on TAp73 transcriptional activity and apoptosis.

The p53 activities are due, at least in part, to its ability to form oligomers that bind to specific DNA sequences and activate transcription. Since some mutant p53 proteins and ΔNp73 isoforms form heterocomplexes with TAp73, we asked whether p53 isoforms can do the same and potentially act as dominant-negative inhibitors of TAp73. Moreover, it has already been found that some isoforms form complex with wtp53 and some of them inhibit p53 tumor-suppressor functions. Therefore, we studied the complex formation and co-immunoprecipitation assays show that all six p53 isoforms examined can form complexes with TAp73ß, whereas only Δ133p53α/ß/γ isoforms form complex with TAp73α. All p53 isoforms counteract TAp73ß transactivation function but with different efficiency and in a promoter-dependent manner. Furthermore, apoptotic activity of TAp73ß was augmented by coexpression of p53ß, whereas Δ133p53α and ß inhibit its apoptotic activity most efficiently. We have determined the half-life of different p53 isoforms: p53γ isoform has the shortest half-life, whereas Δ133p53γ has the longest half-life. Inhibitory interactions of two proteins in complex often lead to their stabilization. However, only three isoforms (Δ133p53α, Δ133p53ß and Δ40p53α) stabilize TAp73ß. We are convinced that defining the interactions between p53/p73 would give a new insight into how the p53 isoforms modulate the p73 functions in tumorigenesis.