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1.  Genomic Analyses Reveal Broad Impact of miR-137 on Genes Associated with Malignant Transformation and Neuronal Differentiation in Glioblastoma Cells 
PLoS ONE  2014;9(1):e85591.
miR-137 plays critical roles in the nervous system and tumor development; an increase in its expression is required for neuronal differentiation while its reduction is implicated in gliomagenesis. To evaluate the potential of miR-137 in glioblastoma therapy, we conducted genome-wide target mapping in glioblastoma cells by measuring the level of association between PABP and mRNAs in cells transfected with miR-137 mimics vs. controls via RIPSeq. Impact on mRNA levels was also measured by RNASeq. By combining the results of both experimental approaches, 1468 genes were found to be negatively impacted by miR-137 – among them, 595 (40%) contain miR-137 predicted sites. The most relevant targets include oncogenic proteins and key players in neurogenesis like c-KIT, YBX1, AKT2, CDC42, CDK6 and TGFβ2. Interestingly, we observed that several identified miR-137 targets are also predicted to be regulated by miR-124, miR-128 and miR-7, which are equally implicated in neuronal differentiation and gliomagenesis. We suggest that the concomitant increase of these four miRNAs in neuronal stem cells or their repression in tumor cells could produce a robust regulatory effect with major consequences to neuronal differentiation and tumorigenesis.
doi:10.1371/journal.pone.0085591
PMCID: PMC3899048  PMID: 24465609
2.  Co-transcriptional production of RNA–DNA hybrids for simultaneous release of multiple split functionalities 
Nucleic Acids Research  2013;42(3):2085-2097.
Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA–DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA–RNA, RNA–DNA and DNA–DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates.
doi:10.1093/nar/gkt1001
PMCID: PMC3919563  PMID: 24194608
3.  Co-transcriptional Assembly of Chemically Modified RNA Nanoparticles Functionalized with siRNAs 
Nano letters  2012;12(10):5192-5195.
We report a generalized methodology for the one-pot production of chemically modified functional RNA nanoparticles during in vitro transcription with T7 RNA polymerase. The efficiency of incorporation of 2′-fluoro-dNTP in the transcripts by the wild type T7 RNA polymerase dramatically increases in the presence of manganese ions, resulting in a high-yield production of chemically modified RNA nanoparticles functionalized with siRNAs that are resistant to nucleases from human blood serum. Moreover, the unpurified transcription mixture can be used for functional ex vivo pilot experiments.
doi:10.1021/nl302302e
PMCID: PMC3498980  PMID: 23016824
RNA nanotechnology; self-assembly; siRNA; chemical modifications; transcription; T7 RNA polymerase
4.  Activation of different split functionalities upon re-association of RNA-DNA hybrids 
Nature nanotechnology  2013;8(4):296-304.
Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of the protein functions and improved detection sensitivity. Here we show a similar technique based on a pair of RNA-DNA hybrids that can be generally used for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept this work is mainly focused on activation of RNA interference; however the release of other functionalities (resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumors together with specific gene silencing. This split-functionality approach presents a new route in the development of “smart” nucleic acids based nanoparticles and switches for various biomedical applications.
doi:10.1038/nnano.2013.44
PMCID: PMC3618561  PMID: 23542902
5.  Coarse-graining RNA nanostructures for molecular dynamics simulations 
Physical biology  2010;7(3):036001.
A series of coarse-grained models have been developed for study of the molecular dynamics of RNA nanostructures. The models in the series have one to three beads per nucleotide and include different amounts of detailed structural information. Such a treatment allows us to reach, for systems of thousands of nucleotides, a time scale of microseconds (i.e. by three orders of magnitude longer than in full atomistic modeling) and thus to enable simulations of large RNA polymers in the context of bionanotechnology. We find that the three-beads-per-nucleotide models, described by a set of just a few universal parameters, are able to describe different RNA conformations and are comparable in structural precision to the models where detailed values of the backbone P-C4′ dihedrals taken from a reference structure are included. These findings are discussed in the context of RNA conformation classes.
doi:10.1088/1478-3975/7/3/036001
PMCID: PMC3767480  PMID: 20577037
6.  Computational strategies for the automated design of RNA nanoscale structures from building blocks using NanoTiler☆ 
One approach to designing RNA nanoscale structures is to use known RNA structural motifs such as junctions, kissing loops or bulges and to construct a molecular model by connecting these building blocks with helical struts. We previously developed an algorithm for detecting internal loops, junctions and kissing loops in RNA structures.
Here we present algorithms for automating or assisting many of the steps that are involved in creating RNA structures from building blocks: (1) assembling building blocks into nanostructures using either a combinatorial search or constraint satisfaction; (2) optimizing RNA 3D ring structures to improve ring closure; (3) sequence optimisation; (4) creating a unique non-degenerate RNA topology descriptor. This effectively creates a computational pipeline for generating molecular models of RNA nanostructures and more specifically RNA ring structures with optimized sequences from RNA building blocks. We show several examples of how the algorithms can be utilized to generate RNA tecto-shapes.
doi:10.1016/j.jmgm.2008.05.004
PMCID: PMC3744370  PMID: 18838281
RNA; Design; Nanotechnology; Building block; Topology; Ring closure
7.  RNA2D3D: A program for Generating, Viewing, and Comparing 3-Dimensional Models of RNA 
Using primary and secondary structure information of an RNA molecule, the program RNA2D3D automatically and rapidly produces a first-order approximation of a 3-dimensional conformation consistent with this information. Applicable to structures of arbitrary branching complexity and pseudoknot content, it features efficient interactive graphical editing for the removal of any overlaps introduced by the initial generating procedure and for making conformational changes favorable to targeted features and subsequent refinement. With emphasis on fast exploration of alternative 3D conformations, one may interactively add or delete base-pairs, adjacent stems can be coaxially stacked or unstacked, single strands can be shaped to accommodate special constraints, and arbitrary subsets can be defined and manipulated as rigid bodies. Compaction, whereby base stacking within stems is optimally extended into connecting single strands, is also available as a means of strategically making the structures more compact and revealing folding motifs. Subsequent refinement of the first-order approximation, of modifications, and for the imposing of tertiary constraints is assisted with standard energy refinement techniques. Previously determined coordinates for any part of the molecule are readily incorporated, and any part of the modeled structure can be output as a PDB or XYZ file. Illustrative applications in the areas of ribozymes, viral kissing loops, viral internal ribosome entry sites, and nanobiology are presented.
doi:10.1080/07391102.2008.10531240
PMCID: PMC3727907  PMID: 18399701
RNA secondary structure; RNA 3D structure; RNA modeling; molecular modeling; and nanobiology
8.  A Ribosome-Binding, 3′ Translational Enhancer Has a T-Shaped Structure and Engages in a Long-Distance RNA-RNA Interaction 
Journal of Virology  2012;86(18):9828-9842.
Many plant RNA viruses contain elements in their 3′ untranslated regions (3′ UTRs) that enhance translation. The PTE (Panicum mosaic virus-like translational enhancer) of Pea enation mosaic virus (PEMV) binds to eukaryotic initiation factor 4E (eIF4E), but how this affects translation from the 5′ end is unknown. We have discovered a three-way branched element just upstream of the PEMV PTE that engages in a long-distance kissing-loop interaction with a coding sequence hairpin that is critical for the translation of a reporter construct and the accumulation of the viral genome in vivo. Loss of the long-distance interaction was more detrimental than elimination of the adjacent PTE, indicating that the RNA-RNA interaction supports additional translation functions besides relocating the PTE to the 5′ end. The branched element is predicted by molecular modeling and molecular dynamics to form a T-shaped structure (TSS) similar to the ribosome-binding TSS of Turnip crinkle virus (TCV). The PEMV element binds to plant 80S ribosomes with a Kd (dissociation constant) of 0.52 μM and to 60S subunits with a Kd of 0.30 μM. Unlike the TCV TSS, the PEMV element also binds 40S subunits (Kd, 0.36 μM). Mutations in the element that suppressed translation reduced either ribosome binding or the RNA-RNA interaction, suggesting that ribosome binding is important for function. This novel, multifunctional element is designated a kl-TSS (kissing-loop T-shaped structure) to distinguish it from the TCV TSS. The kl-TSS has sequence and structural features conserved with the upper portion of most PTE-type elements, which, with the exception of the PEMV PTE, can engage in similar long-distance RNA-RNA interactions.
doi:10.1128/JVI.00677-12
PMCID: PMC3446580  PMID: 22761367
9.  In Silico, In Vitro, and In Vivo Studies Indicate the Potential Use of Bolaamphiphiles for Therapeutic siRNAs Delivery 
Specific small interfering RNAs (siRNAs) designed to silence different oncogenic pathways can be used for cancer therapy. However, non-modified naked siRNAs have short half-lives in blood serum and encounter difficulties in crossing biological membranes due to their negative charge. These obstacles can be overcome by using siRNAs complexed with bolaamphiphiles, consisting of two positively charged head groups that flank an internal hydrophobic chain. Bolaamphiphiles have relatively low toxicities, long persistence in the blood stream, and most importantly, in aqueous conditions can form poly-cationic micelles thus, becoming amenable to association with siRNAs. Herein, two different bolaamphiphiles with acetylcholine head groups attached to an alkyl chain in two distinct configurations are compared for their abilities to complex with siRNAs and deliver them into cells inducing gene silencing. Our explicit solvent molecular dynamics (MD) simulations showed that bolaamphiphiles associate with siRNAs due to electrostatic, hydrogen bonding, and hydrophobic interactions. These in silico studies are supported by various in vitro and in cell culture experimental techniques as well as by some in vivo studies. Results demonstrate that depending on the application, the extent of siRNA chemical protection, delivery efficiency, and further intracellular release can be varied by simply changing the type of bolaamphiphile used.
doi:10.1038/mtna.2013.5
PMCID: PMC3615820  PMID: 23511334
bolaamphiphiles; cryo-EM; molecular dynamics simulations; FRET; poly-cationic micelles; RNA-based therapeutics; siRNA delivery; specific gene silencing
10.  Using cellzilla for plant growth simulations at the cellular level 
Cellzilla is a two-dimensional tissue simulation platform for plant modeling utilizing Cellerator arrows. Cellerator describes biochemical interactions with a simplified arrow-based notation; all interactions are input as reactions and are automatically translated to the appropriate differential equations using a computer algebra system. Cells are represented by a polygonal mesh of well-mixed compartments. Cell constituents can interact intercellularly via Cellerator reactions utilizing diffusion, transport, and action at a distance, as well as amongst themselves within a cell. The mesh data structure consists of vertices, edges (vertex pairs), and cells (and optional intercellular wall compartments) as ordered collections of edges. Simulations may be either static, in which cell constituents change with time but cell size and shape remain fixed; or dynamic, where cells can also grow. Growth is controlled by Hookean springs associated with each mesh edge and an outward pointing pressure force. Spring rest length grows at a rate proportional to the extension beyond equilibrium. Cell division occurs when a specified constituent (or cell mass) passes a (random, normally distributed) threshold. The orientation of new cell walls is determined either by Errera's rule, or by a potential model that weighs contributions due to equalizing daughter areas, minimizing wall length, alignment perpendicular to cell extension, and alignment perpendicular to actual growth direction.
doi:10.3389/fpls.2013.00408
PMCID: PMC3797531  PMID: 24137172
mathematical model; computational model; software; meristem; cellerator; cellzilla; wuschel; clavata
11.  Multi-Strand RNA Secondary Structure Prediction and Nanostructure Design including Pseudoknots 
ACS Nano  2011;5(12):9542-9551.
We are presenting NanoFolder, a method for the prediction of the base pairing of potentially pseudoknotted multi-strand RNA nanostructures. We show that the method outperforms several other structure prediction methods when applied to RNA complexes with non-nested base pairs. We extended this secondary structure prediction capability to allow RNA sequence design. Using native PAGE, we experimentally confirm that 4 in silico designed RNA strands corresponding to a triangular RNA structure form the expected stable complex.
doi:10.1021/nn202666w
PMCID: PMC3263976  PMID: 22067111
pseudoknot; RNA; secondary structure prediction; sequence design; tectoRNA
12.  Design and self-assembly of siRNA-functionalized RNA nanoparticles for use in automated nanomedicine 
Nature protocols  2011;6(12):2022-2034.
Individual genes can be targeted with siRNAs. The use of nucleic acid nanoparticles (NPs) is a convenient method for delivering combinations of specific siRNAs in an organized and programmable manner. We present three assembly protocols to produce two different types of RNA self-assembling functional NPs using processes that are fully automatable. These NPs are engineered based on two complementary nanoscaffold designs (nanoring and nanocube), which serve as carriers of multiple siRNAs. The NPs are functionalized by the extension of up to six scaffold strands with siRNA duplexes. The assembly protocols yield functionalized RNA NPs, and we show that they interact in vitro with human recombinant Dicer to produce siRNAs. Our design strategies allow for fast, economical and easily controlled production of endotoxin-free therapeutic RNA NPs that are suitable for preclinical development.
doi:10.1038/nprot.2011.418
PMCID: PMC3498981  PMID: 22134126
13.  Aging and Bone Health in Individuals with Developmental Disabilities 
Low bone mass density (BMD), a classical age-related health issue and a known health concern for fair skinned, thin, postmenopausal Caucasian women, is found to be common among individuals with developmental/intellectual disabilities (D/IDs). It is the consensus that BMD is decreased in both men and women with D/ID. Maintaining good bone health is important for this population as fractures could potentially go undetected in nonverbal individuals, leading to increased morbidity and a further loss of independence. This paper provides a comprehensive overview of bone health of adults with D/ID, their risk of fractures, and how this compares to the general aging population. We will specifically focus on the bone health of two common developmental disabilities, Down syndrome (DS) and cerebral palsy (CP), and will discuss BMD and fracture rates in these complex populations. Gaining a greater understanding of how bone health is affected in individuals with D/ID could lead to better customized treatments for these specific populations.
doi:10.1155/2012/469235
PMCID: PMC3408668  PMID: 22888344
14.  Use of RNA structure flexibility data in nanostructure modeling 
Methods (San Diego, Calif.)  2010;54(2):239-250.
In the emerging field of RNA-based nanotechnology there is a need for automation of the structure design process. Our goal is to develop computer methods for aiding in this process. Towards that end, we created the RNAJunction database, which is a repository of RNA junctions, i.e. internal, multi-branch and kissing loops with emanating stem stubs, extracted from the larger RNA structures stored in the PDB database. These junctions can be used as building blocks for nanostructures. Two programs developed in our laboratory, NanoTiler and RNA2D3D, can combine such building blocks with idealized fragments of A-form helices to produce desired 3D nanostructures. Initially, the building blocks are treated as rigid objects and the resulting geometry is tested against the design objectives. Experimental data, however, shows that RNA accommodates its shape to the constraints of larger structural contexts. Therefore we are adding analysis of the flexibility of our building blocks to the full design process. Here we present an example of RNA-based nanostructure design, putting emphasis on the need to characterize the structural flexibility of the building blocks to induce ring closure in the automated exploration. We focus on the use of kissing loops (KL) in nanostructure design, since they have been shown to play an important role in RNA self-assembly. By using an experimentally proven system, the RNA tectosquare, we show that considering the flexibility of the KLs as well as distortions of helical regions may be necessary to achieve a realistic design.
doi:10.1016/j.ymeth.2010.12.010
PMCID: PMC3107926  PMID: 21163354
RNA; Nanostructure; Design; Modeling; Flexibility; Molecular dynamics
15.  Self-assembling RNA nanorings based on RNAI/II inverse kissing complexes 
Nano letters  2011;11(2):878-887.
RNA is an attractive biopolymer for nanodesign of self-assembling particles for nanobiotechnology and synthetic biology. Here, we experimentally characterize by biochemical and biophysical methods the formation of thermostable and ribonuclease resistant RNA nanorings previously proposed by computational design. High yields of fully programmable nanorings were produced based on several RNAI/IIi kissing complex variants selected for their ability to promote polygon self-assembly. This self-assembly strategy relying on the particular geometry of bended kissing complexes has potential for developing siRNAs delivery agents.
doi:10.1021/nl104271s
PMCID: PMC3036768  PMID: 21229999
RNA architectonics; bionanotechnology; silencing RNA; supramolecular assembly; RNA interaction; RNA motif
16.  Understanding the effects of carbocyclic sugars constrained to north and south conformations on RNA nanodesign 
Relatively new types of the modified nucleotides, namely carbocyclic sugars that are constrained to north or south (C2′ or C3′ exo) conformations, can be used for RNA nanoparticle design to control their structures and stability by rigidifying nucleotides and altering the helical properties of RNA duplexes. Two RNA structures, an RNA dodecamer and an HIV kissing loop complex where several nucleotides were replaced with north or south constrained sugars, were studied by molecular dynamics (MD) simulations. The substituted south constrained nucleotides in the dodecamer widened the major groove and narrowed and deepened the minor groove thus inducing local conformational changes that resemble a B-form DNA helix. In the HIV kissing loop complex, north and south constrained nucleotides were substituted into flanking bases and stems. The modified HIV kissing loop complex showed a lower RMSD value than the normal kissing loop complex. The overall twist angle was also changed and its standard deviation was reduced. In addition, the modified RNA dodecamer and HIV kissing loop complex were characterized by principal component analysis (PCA) and steered molecular dynamics (SMD). PCA results showed that the constrained sugars stabilized the overall motions. The results of the SMD simulations indicated that as the backbone δ angles were increased by elongation, more force was applied to the modified RNA due to the constrained sugar analogues.
doi:10.1016/j.jmgm.2010.11.007
PMCID: PMC3040123  PMID: 21159533
RNA; North constrained sugar; South constrained sugar; Molecular dynamics simulations; RNA structure deformations; RNA nanodesign
17.  In vitro Assembly of Cubic RNA-Based Scaffolds Designed in silico 
Nature nanotechnology  2010;5(9):676-682.
The organization of biological materials into versatile three-dimensional assemblies could be used to build multifunctional therapeutic scaffolds for use in nanomedicine. Here we report a strategy to design three-dimensional nanoscale scaffolds that can be self-assembled from RNA with precise control over their shape, size and composition. These cubic nanoscaffolds are only ~13 nm in diameter and are composed of short oligonucleotides making them amenable to chemical synthesis, point modifications and further functionalization. Nanocube assembly is verified by gel assays, dynamic light scattering and cryogenic electron microscopy. Formation of functional RNA nanocubes is also demonstrated by incorporation of a light-up fluorescent RNA aptamer that is optimally active only upon full RNA assembly. Moreover, we show the RNA nano-scaffolds can self-assemble in isothermal conditions (37°C) during in vitro transcription, which opens a route towards the construction of sensors, programmable packaging and cargo delivery systems for biomedical applications.
doi:10.1038/nnano.2010.160
PMCID: PMC2934861  PMID: 20802494
18.  A Method for Helical RNA Global Structure Determination in Solution Using Small Angle X-ray Scattering and NMR Measurements 
Journal of molecular biology  2009;393(3):717-734.
We report a “top-down” method that uses mainly duplexes' global orientations and overall molecular dimension and shape restraints, which were extracted from experimental NMR and small angle X-ray scattering (SAXS) data respectively, to determine global architectures of RNA molecules consisting of mostly A-form like duplexes. The method is implemented in the G2G (from Global measurement to Global Structure) toolkit of programs. We demonstrate the efficiency and accuracy of the method by determining the global structure of a 71-nucleotide RNA using experimental data. The backbone root-mean-square-deviation (RMSD) of the ensemble of the calculated global structures relative to the X-ray crystal structure using the experimental data is 3.0 ± 0.3 Å, and the RMSD is only 2.5 ± 0.2 Å for the three duplexes that were orientation-restrained during the calculation. The global structure simplifies interpretation of multi-dimensional nuclear Overhauser spectra for high resolution structure determination. The potential general application of the method for RNA structure determination is discussed.
doi:10.1016/j.jmb.2009.08.001
PMCID: PMC2760655  PMID: 19666030
RNA structure; RDC; SAXS; NMR; duplex orientation; RDC waves
19.  Sequence signatures and mRNA concentration can explain two-thirds of protein abundance variation in a human cell line 
We provide a large-scale dataset on absolute protein and matching mRNA concentrations from the human medulloblastoma cell line Daoy. The correlation between mRNA and protein concentrations is significant and positive (Rs=0.46, R2=0.29, P-value<2e16), although non-linear.Out of ∼200 tested sequence features, sequence length, frequency and properties of amino acids, as well as translation initiation-related features are the strongest individual correlates of protein abundance when accounting for variation in mRNA concentration.When integrating mRNA expression data and all sequence features into a non-parametric regression model (Multivariate Adaptive Regression Splines), we were able to explain up to 67% of the variation in protein concentrations. Half of the contributions were attributed to mRNA concentrations, the other half to sequence features relating to regulation of translation and protein degradation. The sequence features are primarily linked to the coding and 3′ untranslated region. To our knowledge, this is the most comprehensive predictive model of human protein concentrations achieved so far.
mRNA decay, translation regulation and protein degradation are essential parts of eukaryotic gene expression regulation (Hieronymus and Silver, 2004; Mata et al, 2005), which enable the dynamics of cellular systems and their responses to external and internal stimuli without having to rely exclusively on transcription regulation. The importance of these processes is emphasized by the generally low correlation between mRNA and protein concentrations. For many prokaryotic and eukaryotic organisms, <50% of variation in protein abundance variation is explained by variation in mRNA concentrations (de Sousa Abreu et al, 2009).
Given the plethora of regulatory mechanisms involved, most studies have focused so far on individual regulators and specific targets. Particularly in human, we currently lack system-wide, quantitative analyses that evaluate the relative contribution of regulatory elements encoded in the mRNA and protein sequence. Existing studies have been carried out only in bacteria and yeast (Nie et al, 2006; Brockmann et al, 2007; Tuller et al, 2007; Wu et al, 2008). Here, we present the first comprehensive analysis on the impact of translation and protein degradation on protein abundance variation in a human cell line. For this purpose, we experimentally measured absolute protein and mRNA concentrations in the Daoy medulloblastoma cell line, using shotgun proteomics and microarrays, respectively (Figure 1). These data comprise one of the largest such sets available today for human. We focused on sequence features that likely impact protein translation and protein degradation, including length, nucleotide composition, structure of the untranslated regions (UTRs), coding sequence, composition of the translation initiation site, presence of upstream open reading frames putative target sites of miRNAs, codon usage, amino-acid composition and protein degradation signals.
Three types of tests have been conducted: (a) we examined partial Spearman's rank correlation of numerical features (e.g. length) with protein concentration, accounting for variation in mRNA concentrations; (b) for numerical and categorical features (e.g. function), we compared two extreme populations with Welch's t-test and (c) using a Multivariate Adaptive Regression Splines model, we analyzed the combined contributions of mRNA expression and sequence features to protein abundance variation (Figure 1). To account for the non-linearity of many relationships, we use non-parametric approaches throughout the analysis.
We observed a significant positive correlation between mRNA and protein concentrations, larger than many previous measurements (de Sousa Abreu et al, 2009). We also show that the contribution of translation and protein degradation is at least as important as the contribution of mRNA transcription and stability to the abundance variation of the final protein products. Although variation in mRNA expression explains ∼25–30% of the variation in protein abundance, another 30–40% can be accounted for by characteristics of the sequences, which we identified in a comparative assessment of global correlates. Among these characteristics, sequence length, amino-acid frequencies and also nucleotide frequencies in the coding region are of strong influence (Figure 3A). Characteristics of the 3′UTR and of the 5′UTR, that is length, nucleotide composition and secondary structures, describe another part of the variation, leaving 33% expression variation unexplained. The unexplained fraction may be accounted for by mechanisms not considered in this analysis (e.g. regulation by RNA-binding proteins or gene-specific structural motifs), as well as expression and measurement noise.
Our combined model including mRNA concentration and sequence features can explain 67% of the variation of protein abundance in this system—and thus has the highest predictive power for human protein abundance achieved so far (Figure 3B).
Transcription, mRNA decay, translation and protein degradation are essential processes during eukaryotic gene expression, but their relative global contributions to steady-state protein concentrations in multi-cellular eukaryotes are largely unknown. Using measurements of absolute protein and mRNA abundances in cellular lysate from the human Daoy medulloblastoma cell line, we quantitatively evaluate the impact of mRNA concentration and sequence features implicated in translation and protein degradation on protein expression. Sequence features related to translation and protein degradation have an impact similar to that of mRNA abundance, and their combined contribution explains two-thirds of protein abundance variation. mRNA sequence lengths, amino-acid properties, upstream open reading frames and secondary structures in the 5′ untranslated region (UTR) were the strongest individual correlates of protein concentrations. In a combined model, characteristics of the coding region and the 3′UTR explained a larger proportion of protein abundance variation than characteristics of the 5′UTR. The absolute protein and mRNA concentration measurements for >1000 human genes described here represent one of the largest datasets currently available, and reveal both general trends and specific examples of post-transcriptional regulation.
doi:10.1038/msb.2010.59
PMCID: PMC2947365  PMID: 20739923
gene expression regulation; protein degradation; protein stability; translation
20.  CyloFold: secondary structure prediction including pseudoknots 
Nucleic Acids Research  2010;38(Web Server issue):W368-W372.
Computational RNA secondary structure prediction approaches differ by the way RNA pseudoknot interactions are handled. For reasons of computational efficiency, most approaches only allow a limited class of pseudoknot interactions or are not considering them at all. Here we present a computational method for RNA secondary structure prediction that is not restricted in terms of pseudoknot complexity. The approach is based on simulating a folding process in a coarse-grained manner by choosing helices based on established energy rules. The steric feasibility of the chosen set of helices is checked during the folding process using a highly coarse-grained 3D model of the RNA structures. Using two data sets of 26 and 241 RNA sequences we find that this approach is competitive compared to the existing RNA secondary structure prediction programs pknotsRG, HotKnots and UnaFold. The key advantages of the new method are that there is no algorithmic restriction in terms of pseudoknot complexity and a test is made for steric feasibility. Availability: The program is available as web server at the site: http://cylofold.abcc.ncifcrf.gov.
doi:10.1093/nar/gkq432
PMCID: PMC2896150  PMID: 20501603
21.  Molecular dynamics study of the RNA ring nanostructure: a phenomenon of self-stabilization 
Physical biology  2009;6(4):46003.
We study mechanical and thermodynamic properties of RNA nanostructures focusing on a hexagonal nanoring discussed in Yingling and Shapiro (2007 Nano Lett. 7 2328). We are concerned with the following main issues: (i) the stability of the nanoring versus temperature; (ii) the effect of the environment (solvent, counterions) on its stability; (iii) conformations and dynamics under external force. The process of evaporation of the ions from the ring upon temperature drop has been found, demonstrating a surprising feature—the uptake of ions by the nanoring increases with the temperature. The connection of this behavior to the dielectric constant of water, hydration and structural changes in the nanoring is discussed. Several properties of the nanoring, such as elastic and transport coefficients, have been determined. A measure of the tensile elasticity of the ring against its uniform 2D in-plane compression has been given, as Keff ≤ 0.01 GPa, which is a much lower value compared to typical values found for soft matter other than RNA.
doi:10.1088/1478-3975/6/4/046003
PMCID: PMC2790865  PMID: 19741282
22.  An Intergenic Non-Coding rRNA Correlated with Expression of the rRNA and Frequency of an rRNA Single Nucleotide Polymorphism in Lung Cancer Cells 
PLoS ONE  2009;4(10):e7505.
Background
Ribosomal RNA (rRNA) is a central regulator of cell growth and may control cancer development. A cis noncoding rRNA (nc-rRNA) upstream from the 45S rRNA transcription start site has recently been implicated in control of rRNA transcription in mouse fibroblasts. We investigated whether a similar nc-rRNA might be expressed in human cancer epithelial cells, and related to any genomic characteristics.
Methodology/Principal Findings
Using quantitative rRNA measurement, we demonstrated that a nc-rRNA is transcribed in human lung epithelial and lung cancer cells, starting from approximately −1000 nucleotides upstream of the rRNA transcription start site (+1) and extending at least to +203. This nc-rRNA was significantly more abundant in the majority of lung cancer cell lines, relative to a nontransformed lung epithelial cell line. Its abundance correlated negatively with total 45S rRNA in 12 of 13 cell lines (P = 0.014). During sequence analysis from −388 to +306, we observed diverse, frequent intercopy single nucleotide polymorphisms (SNPs) in rRNA, with a frequency greater than predicted by chance at 12 sites. A SNP at +139 (U/C) in the 5′ leader sequence varied among the cell lines and correlated negatively with level of the nc-rRNA (P = 0.014). Modelling of the secondary structure of the rRNA 5′-leader sequence indicated a small increase in structural stability due to the +139 U/C SNP and a minor shift in local configuration occurrences.
Conclusions/Significance
The results demonstrate occurrence of a sense nc-rRNA in human lung epithelial and cancer cells, and imply a role in regulation of the rRNA gene, which may be affected by a +139 SNP in the 5′ leader sequence of the primary rRNA transcript.
doi:10.1371/journal.pone.0007505
PMCID: PMC2759515  PMID: 19838300
23.  Predicting Consensus Structures for RNA Alignments Via Pseudo-Energy Minimization 
Thermodynamic processes with free energy parameters are often used in algorithms that solve the free energy minimization problem to predict secondary structures of single RNA sequences. While results from these algorithms are promising, an observation is that single sequence-based methods have moderate accuracy and more information is needed to improve on RNA secondary structure prediction, such as covariance scores obtained from multiple sequence alignments. We present in this paper a new approach to predicting the consensus secondary structure of a set of aligned RNA sequences via pseudo-energy minimization. Our tool, called RSpredict, takes into account sequence covariation and employs effective heuristics for accuracy improvement. RSpredict accepts, as input data, a multiple sequence alignment in FASTA or ClustalW format and outputs the consensus secondary structure of the input sequences in both the Vienna style Dot Bracket format and the Connectivity Table format. Our method was compared with some widely used tools including KNetFold, Pfold and RNAalifold. A comprehensive test on different datasets including Rfam sequence alignments and a multiple sequence alignment obtained from our study on the Drosophila X chromosome reveals that RSpredict is competitive with the existing tools on the tested datasets. RSpredict is freely available online as a web server and also as a jar file for download at http://datalab.njit.edu/biology/RSpredict.
PMCID: PMC2808183  PMID: 20140072
RNA secondary structure prediction; Drosophila secondary structure; Rfam sequence alignments; normalized energy
24.  Correction: Role of 3′UTRs in the Translation of mRNAs Regulated by Oncogenic eIF4E—A Computational Inference 
PLoS ONE  2009;4(4):10.1371/annotation/6a44204e-5d20-45bf-bbce-358dc9e2de27.
doi:10.1371/annotation/6a44204e-5d20-45bf-bbce-358dc9e2de27
PMCID: PMC2674774
25.  Role of 3′UTRs in the Translation of mRNAs Regulated by Oncogenic eIF4E—A Computational Inference 
PLoS ONE  2009;4(3):e4868.
Eukaryotic cap-dependent mRNA translation is mediated by the initiation factor eIF4E, which binds mRNAs and stimulates efficient translation initiation. eIF4E is often overexpressed in human cancers. To elucidate the molecular signature of eIF4E target mRNAs, we analyzed sequence and structural properties of two independently derived polyribosome recruited mRNA datasets. These datasets originate from studies of mRNAs that are actively being translated in response to cells over-expressing eIF4E or cells with an activated oncogenic AKT: eIF4E signaling pathway, respectively. Comparison of eIF4E target mRNAs to mRNAs insensitive to eIF4E-regulation has revealed surprising features in mRNA secondary structure, length and microRNA-binding properties. Fold-changes (the relative change in recruitment of an mRNA to actively translating polyribosomal complexes in response to eIF4E overexpression or AKT upregulation) are positively correlated with mRNA G+C content and negatively correlated with total and 3′UTR length of the mRNAs. A machine learning approach for predicting the fold change was created. Interesting tendencies of secondary structure stability are found near the start codon and at the beginning of the 3′UTR region. Highly upregulated mRNAs show negative selection (site avoidance) for binding sites of several microRNAs. These results are consistent with the emerging model of regulation of mRNA translation through a dynamic balance between translation initiation at the 5′UTR and microRNA binding at the 3′UTR.
doi:10.1371/journal.pone.0004868
PMCID: PMC2654073  PMID: 19290046

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