RNA Hairpin Loops Research Articles

110 Refinement of force field torsion parameters for nucleic acids based on inclusion of conformation-dependent solvation effects

Accurate representation of nucleic acids in molecular dynamics simulations depends critically on the quality of the applied empirical force field. Among force field terms, the torsion parameters are known to strongly influence the conformational equilibria and molecular structures. Unfortunately, past several years witnessed severe problems in describing the torsion space in nucleic acids by current force fields and more problems continue emerging. In an attempt for improvement, we suggested a novel parameterization procedure that incorporates some previously neglected solvation-related effects, which proved to be essential for obtaining accurate torsion profiles. The suggested approach avoids double counting of solvation effects and provides parameters that may be used in combination with any of the widely used nonpolarizable discrete solvent models or with the continuum solvent models. Improvements are demonstrated for the latest AMBER force field for RNA simulations, ff10, which incorporates parameters for the glycosidic torsion (χOL3) developed by us using the above-described procedure (Banáš et al., 2010; Zgarbová et al., 2011). Resulting parameters are verified by extensive molecular dynamics simulations of canonical RNA duplexes and RNA hairpin loops. We show that our modification removes overstabilization of the high-anti region found in the ff99 force field and thus prevents formation of undesirable ‘ladder-like’ structural distortions in RNA simulations. In addition, we applied our parameterization approach to development of the glycosidic torsion in DNA (χOL4). This refinement focuses on adjusting description of the syn region and syn-anti balance of the χ potential. This modification exhibits a notable improvement of the description of the antiparallel G-DNA stem, which was not modeled correctly by the current ff99 force field (Krepl et al., 2012).

Cleavage Mediated by the Catalytic Domain of Bacterial RNase P RNA

Like other RNA molecules, RNase P RNA (RPR) is composed of domains, and these have different functions. Here, we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model RNA hairpin loop substrates. Compared to full-length Eco RPR, the rate constant, k(obs), of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13,000-fold depending on substrate. Specifically, the structural architecture of the -1/+73 played a significant role where a C(-1)/G(+73) pair had the most dramatic effect on k(obs). Substitution of A(248) (E. coli numbering), positioned near the cleavage site in the RNase P-substrate complex, with G in the CP RPR resulted in 30-fold improvement in rate. In contrast, strengthening the interaction between the RPR and the 3' end of the substrate only had a modest effect. Interestingly, although deleting the S-domain gave a reduction in the rate, it resulted in a less erroneous RPR with respect to cleavage site selection. These data support and extend our understanding of the coupling between the distal interaction between the S-domain and events at the active site. Our findings will also be discussed with respect to the structure of RPR derived from different organisms.

Effect of IGF-1 on expressions of VEGF under suppression of HIF-1α by shRNA in cultured human RPE cells

Background Proliferative vitreo-retinal disease (PVD)is one group of ocular complications marked by the enhanced proliferation of various cells included retinal pigment epithelial (RPE) cells.Insulin-like growth factor-1 (IGF-1)and vascular endothelial growth factor(VEGF) are implicated in the aberrant cell proliferation and pathological neovascularization that characterizes PVD,but the signaling mechanism is unclear now. Objective This study was to explore the effect of IGF-1 on VEGF in cultured human RPE cells under the small hairpin loop RNA (shRNA) keeping hypoxia-inducible factor-1 α ( HIF-1 α) silencing. Methods Human retinas were isolated from 4 healthy male donors,and the RPE cells were harvested and cultured.The ceils were identified using anti-human keratin antibody.The third to fifth generation of human RPE cells were used in the experiment.One target site of HIF-1α mRNA was chosen by certain design principle,and shRNA was designed and synthesized by the target site and transferred into the cells in vitro,and then the cells were cultivated with 50 μg/L IGF-1 for 24 hours.The mRNA and protein expressions of HIF-1α and VEGF were detected by RT-PCR and Western blot respectively. Results Cultured human RPE cells showed the flat irregularly multangular shape,and 97％ cells appeared the positive response for keratin.HIF-1α mRNA expression in human RPE cells was significantly lower in 50 μg/L IGF-1 group than the 0 pg/L IGF-1 group ( 1.49±0.18 vs 1.46±0.17 ) ( t =0.335,P =0.743 ),however,the expressing levels of HIF-1α protein( 1049.86±172.54 vs 0.00±0.00) and VEGF mRNA(0.95±0.15 vs 0.35±0.07) and VEGF protein (391.98±56.77 vs 214.36±37.15)were raised in the 50 μg/L IGF-I group compared with 0 μg/L IGF-1 group (t=16.098,9.935,6.928,P＜0.05).After the HIF-1α-specific shRNA was transferred into cultured RPE cells,the expressions of both HIF-1α mRNA and its protein significantly decreased in RPE cells under 50 μg/L IGF-1 concentration condition( F=68.679,89.904,P=0.000),moreover,the expression of VEGF mRNA and its protein were significantly lowed(F=21.770,6.205,P＜0.05). Conclusions IGF-1 promotes the accumulation of HIF-1α protein and induce the expression of VEGF in human RPE cells,which probably play a pivotal role in the development of PVD. Key words: Insulin-like growth factor-1; Hypoxia inducible factor-1α; Vascular endothelial growth factor; Retinal pigment epithelial cell; Proliferative vitreo-retinal disease

A Multiscale Approach to RNA 3D Structure Prediction

One of the key issues in the theoretical prediction of RNA stability and structure is how to predict the loop free energy. Experimental results have shown strong sequence-dependence of the loop free energy. However, most currently available models only account for the loop length-dependence of the loop free energy. We recently developed a three-bead coarse-grained model to generate the three-dimensional conformations for RNA hairpin loops. Based on the pseudo-torsion angles for the coarse-grained structures, we can extract a set of pseudo-torsion angle-based statistical potential parameters from the known structures. The statistical potential parameters enable folding predictions of the low-energy coarse-grained 3D structures from the sequence. Further molecular dynamics computations for an ensemble of decoy conformations about the predicted coarse-grained structures lead to the final all-atom structures. A notable advantage of the approach is the use of the statistical potential for evaluating the loop free energies and guiding the search for the low-energy coarse-grained structures from the sequence.

Short Communication: A Single Step Assay for Rapid Evaluation of Inhibitors Targeting HIV Type 1 Tat-Mediated Long Terminal Repeat Transactivation

Human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter-mediated gene expression is regulated by the viral Tat protein that relieves a block to viral transcription elongation after binding with a viral hairpin loop RNA structure called the trans-activation-responsive region (TAR). Tat protein significantly up-regulates viral genome transcription and hence it has long been considered a potential target for antiretrovirals. Here we report the construction of a plasmid containing an HIV-1 LTR-driven reporter cassette with a colinear tat gene under control of a viral promoter and thus conditionally configured for constitutive expression of reporter genes. Inhibition of luciferase reporter expression in a cell line harboring the plasmid in the presence of tat-targeted shRNA confirmed the specificity of the assay and a dose-dependent reporter activity inhibition by the fluoroquinoline derivative K-37, a class of small RNA binding molecule that inhibits Tat and other RNA-dependent transactivations, further validated the method. Subsequently we also made a lentiviral vector (LV) containing the same transcription units and derived a stable cell line using the said LV and similar dose-dependent inhibition was documented using K-37. This quick and sensitive reporter-based method is the simplest screening assay for putative inhibitors of HIV-1 Tat-induced LTR-driven gene expression requiring test material addition as the only manipulation.

Estimating RNA Loop Entropies Using a New Nucleobase Model and Sequential Monte Carlo Method

We report a new scheme that is designed to accurately and efficiently compute the entropy of RNA loops. The scheme is based on a new RNA nucleobase discrete state (RNAnbds) model and a Sequential Monte Carlo (SMC) method. The novelty of the RNAnbds model is that it directly represents the conformation of the RNA nucleobases, instead of the RNA backbones. To test the performance of this new scheme, we calculate the entropies for RNA hairpin loops and compare the results with the exact computational values obtained by an enumeration strategy and with the experimental data. It is found that the SMC method gives almost indistinguishable results from enumerations for short loops. For long hairpin loops, it also provides a good estimation that agrees with experiments.

Early In Vitro Transcription Termination in Human H5 Influenza Viral RNA Synthesis

Rapid diagnostic identification of the human H5 influenza virus is a strategic cornerstone for outbreak prevention. We recently reported a method for direct detection of viral RNA from a highly pathogenic human H5 influenza strain (A/Hanoi/30408/2005(H5N1)), which necessarily was transcribed in vitro from non-viral sources. This article provides an in-depth analysis of the reaction conditions for in vitro transcription (IVT) of full-length influenza H5 RNA, which is needed for diagnostic RNA production, for the T7 and SP6 phage promoter systems. Gel analysis of RNA transcribed from plasmids containing the H5 sequence between a 5' SP6 promoter and 3' restriction site (BsmBI) showed that three sequence-verified bands at 1,776, 784, and 591 bases were consistently produced, whereas only one 1,776-base band was expected. These fragments were not observed in H1 or H3 influenza RNA transcribed under similar conditions. A reverse complement of the sequence produced only a single band at 1,776 bases, which suggested either self-cleavage or early termination. Aliquots of the IVT reaction were quenched with EDTA to track the generation of the bands over time, which maintained a constant concentration ratio. The H5 sequence was cloned with T7 and SP6 RNA polymerase promoters to allow transcription in either direction with either polymerase. The T7 transcription product from purified, restricted plasmids in the vRNA direction only produced the 1,776-base full-length sequence and the 784-base fragment, instead of the three bands generated by the SP6 system, suggesting an early termination mechanism. Additionally, the T7 system produced a higher fraction of full-length vRNA transcripts than the SP6 system did under similar reaction conditions. By sequencing we identified a type II RNA hairpin loop terminator, which forms in a transcription direction-dependent fashion. Variation of the magnesium concentration produced the greatest impact on termination profiles, where some reaction mixtures were unable to produce full-length transcripts. Optimized conditions are presented for the T7 and SP6 phage polymerase systems to minimize these early termination events during in vitro transcription of H5 influenza vRNA.

Performance of Molecular Mechanics Force Fields for RNA Simulations: Stability of UUCG and GNRA Hairpins.

The RNA hairpin loops represent important RNA topologies with indispensable biological functions in RNA folding and tertiary interactions. 5'-UNCG-3' and 5'-GNRA-3' RNA tetraloops are the most important classes of RNA hairpin loops. Both tetraloops are highly structured with characteristic signature three-dimensional features and are recurrently seen in functional RNAs and ribonucleoprotein particles. Explicit solvent molecular dynamics (MD) simulation is a computational technique which can efficiently complement the experimental data and provide unique structural dynamics information on the atomic scale. Nevertheless, the outcome of simulations is often compromised by imperfections in the parametrization of simplified pairwise additive empirical potentials referred to also as force fields. We have pointed out in several recent studies that a force field description of single-stranded hairpin segments of nucleic acids may be particularly challenging for the force fields. In this paper, we report a critical assessment of a broad set of MD simulations of UUCG, GAGA, and GAAA tetraloops using various force fields. First, we utilized the three widely used variants of Cornell et al. (AMBER) force fields known as ff94, ff99, and ff99bsc0. Some simulations were also carried out with CHARMM27. The simulations reveal several problems which show that these force fields are not able to retain all characteristic structural features (structural signature) of the studied tetraloops. Then we tested four recent reparameterizations of glycosidic torsion of the Cornell et al. force field (two of them being currently parametrized in our laboratories). We show that at least some of the new versions show an improved description of the tetraloops, mainly in the syn glycosidic torsion region of the UNCG tetraloop. The best performance is achieved in combination with the bsc0 parametrization of the α/γ angles. Another critically important region to properly describe RNA molecules is the anti/high-anti region of the glycosidic torsion, where there are significant differences among the tested force fields. The tetraloop simulations are complemented by simulations of short A-RNA stems, which are especially sensitive to an appropriate description of the anti/high-anti region. While excessive accessibility of the high-anti region converts the A-RNA into a senseless "ladder-like" geometry, excessive penalization of the high-anti region shifts the simulated structures away from typical A-RNA geometry to structures with a visibly underestimated inclination of base pairs with respect to the helical axis.

Abstract 2326: Loss of prostate-specific antigen slows growth and alters the extracellular proteome of LNCaP human prostate cancer cells

Abstract Prostate-specific antigen (PSA) is the most extensively used biomarker in cancer. There continues to be intense debate regarding the use of PSA in screening for prostate cancer (PCa) and how it may be used for optimal benefit. The role PSA plays in reproductive biology and PCa has been an area of ongoing interest. As a serine protease with novel substrate preferences, PSA continues to be secreted at high levels (mg/mL) even by the majority of advanced prostatic carcinomas. This protease has been shown to affect the growth or viability of a variety of cell types, including epithelial cells, osteoclasts, T-cells, and human PCa cell lines. In this study, we stably reduced endogenous PSA gene expression using lentiviral short hairpin loop RNA (shRNA) technology in the LNCaP human PCa cell line. We then investigated any resulting changes to LNCaP growth rates in vitro and ex vivo. Acting on the hypothesis that any PSA-induced changes to LNCaP behavior are the result of the enzyme's extracellular activity, we used a high-throughput shot-gun mass spectrometry-based (MS) approach to analyze the in vitro extracellular LNCaP proteome in control and PSA-targeted shRNA populations. Having demonstrated that our shRNA strategy targeted to PSA caused as much as a 90% reduction in PSA protein production compared to control cells, we next showed that in vitro doubling times are slowed from 2 days to 11 days. Subcutaneous tumors displayed an almost 50% reduction in take rate, with the final weights of those remaining only reaching 10% of controls after 8 weeks of growth. Serum-free media conditioned for 24 hours by control or PSA-targeted LNCaP populations were then subjected to global semi-quantitative proteomics analysis using the high resolution LTQ-Orbitrap mass spectrometer (Thermo Electron, San Jose, CA). In one 200 minute LTQ-Orbitrap analysis, in conjunction with the Mascot Search algorithm (Matrix Science, Boston, MA), we were able to identify 1784 proteins in the LNCaP conditioned media. Of these, 686 were specific to the control populations, 527 were specific to the reduced PSA populations, and 571 were common to both. These results demonstrate the utility of high-throughput proteomic strategies as an unbiased approach for the identification of candidate mechanisms behind novel observations in cancer biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2326.

Free Energy Profile of RNA Hairpins: A Molecular Dynamics Simulation Study

RNA hairpin loops are one of the most abundant secondary structure elements and participate in RNA folding and protein-RNA recognition. To characterize the free energy surface of RNA hairpin folding at an atomic level, we calculated the potential of mean force (PMF) as a function of the end-to-end distance, by using umbrella sampling simulations in explicit solvent. Two RNA hairpins containing tetraloop cUUCGg and cUUUUg are studied with AMBER ff99 and CHARMM27 force fields. Experimentally, the UUCG hairpin is known to be significantly more stable than UUUU. In this study, the calculations using AMBER force field give a qualitatively correct description for the folding of two RNA hairpins, as the calculated PMF confirms the global stability of the folded structures and the resulting relative folding free energy is in quantitative agreement with the experimental result. The hairpin stabilities are also correctly differentiated by the more rapid molecular mechanics-Poisson Boltzmann-surface area approach, but the relative free energy estimated from this method is overestimated. The free energy profile shows that the native state basin and the unfolded state plateau are separated by a wide shoulder region, which samples a variety of native-like structures with frayed terminal basepair. The calculated PMF lacks major barriers that are expected near the transition regions, and this is attributed to the limitation of the 1-D reaction coordinate. The PMF results are compared with other studies of small RNA hairpins using kinetics method and coarse grained models. The two RNA hairpins described by CHARMM27 are significantly more deformable than those represented by AMBER. Compared with the AMBER results, the CHARMM27 calculated ΔG fold for the UUUU tetraloop is in better agreement with the experimental results. However, the CHARMM27 calculation does not confirm the global stability of the experimental UUCG structure; instead, the extended conformations are predicted to be thermodynamically stable in solution. This finding is further supported by separate unrestrained CHARMM27 simulations, in which the UUCG hairpin unfolds spontaneously within 10 ns. The instability of the UUCG hairpin originates from the loop region, and propagates to the stem. The results of this study provide a molecular picture of RNA hairpin unfolding and reveal problems in the force field descriptions for the conformational energy of certain RNA hairpin.

First-Principles Prediction of the Sequence-Dependent Stability of RNA Hairpin Loop

We develop a statistical mechanical model to predict the sequence-dependent folding stability from the sequence for simple RNA hairpin loops. We use a recently developed 3-vector virtual bond-based RNA folding model which enables rigorous computation of RNA chain entropy. Enumeration of all the possible arrangements of base pairs and the corresponding conformational entropies, through exhaustive self-avoiding random walks of the virtual bonds in a diamond lattice, gives the partition function and free energy for a given RNA sequence. The new model developed here has two unique advantages. First, the model is based on the complete conformational ensemble, including loop conformations that contain all the possible intra-loop base pairs and/or terminal mismatches. Therefore, intra-loop base pairs and mismatches are the results predicted from the model instead of known input information for the model. Second, the model gives accurate estimation for the dramatic entropic changes caused by the formation of the intra-loop base pairs and mismatches. Our first principles calculations for the chain entropy for each given set of base pairs, in combination with the empirical energy function provided from Turner rules, provide ab initio predictions for the sequence-dependent loop stability from RNA sequence. Tests against experimental data indicate that the theory can give improved predictions for the sequence-dependent RNA hairpin loop stability than the nearest-neighbor model.

Binding of the Bacteriophage P22 N-Peptide to the boxB RNA Motif Studied by Molecular Dynamics Simulations

Protein-RNA interactions are important for many cellular processes. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics simulations were used to investigate the dynamics of the P22 N-peptide-boxB complex and to elucidate the energetic contributions to binding. In addition, the free-energy changes of RNA and peptide conformational adaptation to the bound forms, as well as the role of strongly bound water molecules at the peptide-RNA interface, were studied. The influence of peptide amino acid substitutions and the salt dependence of interaction were investigated and showed good agreement with available experimental results. Several tightly bound water molecules were found at the RNA-binding interface in both the presence and absence of N-peptide. Explicit consideration of the waters resulted in shifts of calculated contributions during the energetic analysis, but overall similar binding energy contributions were found. Of interest, it was found that the electrostatic field of the RNA has a favorable influence on the coil-to-α-helix transition of the N-peptide already outside of the peptide-binding site. This result may have important implications for understanding peptide-RNA complex formation, which often involves coupled folding and association processes. It indicates that electrostatic interactions near RNA molecules can lead to a shift in the equilibrium toward the bound form of an interacting partner before it enters the binding pocket.

Two-dimensional combinatorial screening and the RNA Privileged Space Predictor program efficiently identify aminoglycoside–RNA hairpin loop interactions

Herein, we report the identification of RNA hairpin loops that bind derivatives of kanamycin A, tobramycin, neamine, and neomycin B via two-dimensional combinatorial screening, a method that screens chemical and RNA spaces simultaneously. An arrayed aminoglycoside library was probed for binding to a 6-nucleotide RNA hairpin loop library (4096 members). Members of the loop library that bound each aminoglycoside were excised from the array, amplified and sequenced. Sequences were analyzed with our newly developed RNA Privileged Space Predictor (RNA-PSP) program, which analyzes selected sequences to identify statistically significant trends. RNA-PSP identified the following unique trends: 5′UNNNC3′ loops for the kanamycin A derivative (where N is any nucleotide); 5′UNNC3′ loops for the tobramycin derivative; 5′UNC3′ loops for the neamine derivative; and 5′UNNG3′ loops for the neomycin B derivative. The affinities and selectivities of a subset of the ligand–hairpin loop interactions were determined. The selected interactions have Kd values ranging from 10 nM to 605 nM. Selectivities ranged from 0.4 to >200-fold. Interestingly, the results from RNA-PSP are able to qualitatively predict specificity based on overlap between the RNA sequences selected for the ligands. These studies expand the information available on small molecule–RNA motif interactions, which could be useful to design ligands targeting RNA.

Contribution of the Closing Base Pair to Exceptional Stability in RNA Tetraloops: Roles for Molecular Mimicry and Electrostatic Factors

Hairpins are common RNA secondary structures that play multiple roles in nature. Tetraloops are the most frequent RNA hairpin loops and are often phylogenetically conserved. For both the UNCG and GNRA families, CG closing base pairs (cbps) confer exceptional thermodynamic stability but the molecular basis for this has remained unclear. We propose that, despite having very different overall folds, these two tetraloop families achieve stability by presenting the same functionalities to the major groove edge of the CG cbp. Thermodynamic contributions of this molecular mimicry were investigated using substitutions at the nucleobase and functional group levels. By either interrupting or deleting loop-cbp electrostatic interactions, which were identified by solving the nonlinear Poisson-Boltzmann (NLPB) equation, stability changed in a manner consistent with molecular mimicry. We also observed a linear relationship between DeltaG(o)(37) and log[Na(+)] for both families, and loops with a CG cbp had a decreased dependence of stability on salt. NLPB calculations revealed that, for both UUCG and GAAA tetraloops, the GC cbp form has a higher surface charge density, although it arises from changes in loop compaction for UUCG and changes in loop configuration for GAAA. Higher surface charge density leads to stronger interactions of GC cbp loops with solvent and salt, which explains the correlation between experimental and calculated trends of free energy with salt. Molecular mimicry as evidenced in these two stable but otherwise unrelated tetraloops may underlie common functional roles in other RNA and DNA motifs.

Two-Dimensional Combinatorial Screening Identifies Specific 6′-Acylated Kanamycin A− and 6′-Acylated Neamine−RNA Hairpin Interactions

Herein, we report the RNA hairpin loops from a six-nucleotide hairpin library that bind 6'-acylated kanamycin A (1) and 6'-acylated neamine (2) identified by two-dimensional combinatorial screening (2DCS). Hairpins selected to bind 1 have K(d)'s ranging from 235 to 1035 nM, with an average K(d) of 618 nM. For 2, the selected hairpins bind with K(d)'s ranging from 135 to 2300 nM, with an average K(d) of 1010 nM. The selected RNA hairpin-ligand interactions are also specific for the ligand that they were selected to bind compared with the other arrayed ligand. For example, the mixture of hairpins selected for 1 on average bind 33-fold more tightly to 1 than to 2, while the mixtures of hairpins selected for 2 on average bind 11-fold more tightly to 2 than to 1. Secondary structure prediction of the selected sequences was completed to determine the motifs that each ligand binds, and the hairpin loop preferences for 1 and 2 were computed. For 1, the preferred hairpin loops contain an adenine separated by at least two nucleotides from a cytosine, for example, ANNCNN (two-tailed p-value = 0.0010) and ANNNCN (two-tailed p-value <0.0001). For 2, the preferred hairpin loops contain both 5'GC and 5'CG steps (two-tailed p-value <0.0001). These results expand the information available on the RNA hairpin loops that bind small molecules and could prove useful for targeting RNA.

Small hairpin loop RNA targeting HIF-1α down-regulates VEGF and up-regulates PEDF in human retinal pigment epithelial cells under hypoxic condition

The aim of this study was to explore the effect of small hairpin loop RNA (shRNA) silencing hypoxia-induced factor 1alpha (HIF-1alpha) gene on the expression of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in human retinal pigment epithelium (RPE) cells under hypoxic condition. Two target sites of HIF-1alpha mRNA were chosen and two kinds of shRNA were designed and synthesized against the target sites. Then the two kinds of shRNA were transfected into human RPE cells in vitro, respectively. These cells were cultured under hypoxic condition that was simulated by using 150 mumol/L CoCl(2). The mRNA expressions of HIF-1alpha, VEGF and PEDF were tested by semi-quantitative reverse transcription PCR (RT-PCR). The protein levels of HIF-1alpha, VEGF and PEDF were analyzed by Western blotting. After the two kinds of HIF-1alpha-specific shRNA were transfected into RPE cells respectively, the expression of HIF-1alpha mRNA and the levels of HIF-1alpha protein were decreased significantly in RPE cells under hypoxic condition. The expression of VEGF mRNA and the levels of protein significantly were also decreased. However, the levels of PEDF protein was significantly increased, but the expression of PEDF mRNA showed no significant changes. In conclusion, HIF-1alpha-specific shRNA can effectively silence the HIF-1alpha gene, and consequently down-regulate VEGF and up-regulate PEDF expression against hypoxia. These results reveal that HIF-1 is associated with posttranslational mechanism for down-regulating PEDF under hypoxia and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios. These results also suggest that HIF-1 is one of the key cytokines to retinal neovascularization.

ISFOLD: Structure Prediction of Base Pairs in Non-Helical RNA Motifs from Isostericity Signatures in Their Sequence Alignments

The existence and identity of non-Watson-Crick base pairs (bps) within RNA bulges, internal loops, and hairpin loops cannot reliably be predicted by existing algorithms. We have developed the Isfold (Isosteric Folding) program as a tool to examine patterns of nucleotide substitutions from sequence alignments or mutation experiments and identify plausible bp interactions. We infer these interactions based on the observation that each non-Watson-Crick bp has a signature pattern of isosteric substitutions where mutations can be made that preserve the 3D structure. Isfold produces a dynamic representation of predicted bps within defined motifs in order of their probabilities. The software was developed under Windows XP, and is capable of running on PC and MAC with Matlab 7.1 (SP3) or higher. A PC standalone version that does not require Matlab also is available. This software and a user manual are freely available at www.ucsf.edu/frankel/isfold.

Down-regulation of vascular endothelial growth factor in human retinal pigment epithelial cells by small hairpin loop RNA targeting hypoxia inducible factor-1 alpha under hypoxia condition

To explore the effect of hypoxia inducible factor-1 alpha (HIF-1 alpha) gene on the expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (hRPE) cells under hypoxia conditions by using small hairpin loop RNA (shRNA) to silence HIF-1 alpha. CoCl(2) (150 micromol/L) was used to simulate the hypoxia environment for hRPE cells. After choosing a target site of HIF-1 alpha mRNA, shRNA was designed and synthesized by this target site. hRPE cells were transfected by this shRNA in vitro. Then, these cells were cultured under hypoxia conditions (150 micromol/L CoCl(2)). The mRNA expression of HIF-1 alpha and VEGF was measured by semi-quantitative reverse transcription PCR (RT-PCR). The protein level of HIF-1 alpha and VEGF was studied by western blot analysis. After hRPE cells were transfected by HIF-1 alpha-specific shRNA, RT-PCR showed that the expression of HIF-1 alpha mRNA was inhibited by 77.1%, and western blot analysis showed that the level of HIF-1 alpha protein was significantly decreased in hRPE cells under hypoxia conditions. Moreover, the expression of VEGF mRNA was inhibited by 27.8% and the level of VEGF protein was also significantly decreased in transfected hRPE cells under hypoxia conditions. Under hypoxia conditions, HIF-1 alpha-specific shRNA effectively keeps HIF-1 alpha gene silenced, and consequently down-regulates VEGF expression against hypoxia. These results suggest that HIF-1 alpha is one of the most important cytokines for retinal neovascularization.

Heat Shock Factor 2 (HSF2) Contributes to Inducible Expression of hsp Genes through Interplay with HSF1

The heat shock response is a defense reaction activated by proteotoxic damage induced by physiological or environmental stress. Cells respond to the proteotoxic damage by elevated expression of heat shock proteins (Hsps) that function as molecular chaperones and maintain the vital homeostasis of protein folds. Heat shock factors (HSFs) are the main transcriptional regulators of the stress-induced expression of hsp genes. Mammalian HSF1 was originally identified as the transcriptional regulator of the heat shock response, whereas HSF2 has not been implicated a role in the stress response. Previously, we and others have demonstrated that HSF1 and HSF2 interact through their trimerization domains, but the functional consequence of this interaction remained unclear. We have now demonstrated on chromatin that both HSF1 and HSF2 were able to bind the hsp70 promoter not only in response to heat shock but also during hemin-induced differentiation of K562 erythroleukemia cells. In both cases an intact HSF1 was required in order to reach maximal levels of promoter occupancy, suggesting that HSF1 influences the DNA binding activity of HSF2. The functional consequence of the HSF1-HSF2 interplay was demonstrated by real-time reverse transcription-PCR analyses, which showed that HSF2 was able to modulate the HSF1-mediated expression of major hsp genes. Our results reveal, contrary to the predominant model, that HSF2 indeed participates in the transcriptional regulation of the heat shock response.

Eukaryotic RNase P RNA mediates cleavage in the absence of protein

The universally conserved ribonucleoprotein RNase P is involved in the processing of tRNA precursor transcripts. RNase P consists of one RNA and, depending on its origin, a variable number of protein subunits. Catalytic activity of the RNA moiety so far has been demonstrated only for bacterial and some archaeal RNase P RNAs but not for their eukaryotic counterparts. Here, we show that RNase P RNAs from humans and the lower eukaryote Giardia lamblia mediate cleavage of four tRNA precursors and a model RNA hairpin loop substrate in the absence of protein. Compared with bacterial RNase P RNA, the rate of cleavage (k(obs)) was five to six orders of magnitude lower, whereas the affinity for the substrate (appK(d)) was reduced approximately 20- to 50-fold. We conclude that the RNA-based catalytic activity of RNase P has been preserved during evolution. This finding opens previously undescribed ways to study the role of the different proteins subunits of eukaryotic RNase P.