From structure prediction to genomic screens for novel non-coding RNAs

Non-coding RNAs (ncRNAs) are receiving more and more attention not only as an abundant class of genes, but also as regulatory structural elements (some located in mRNAs). A key feature of RNA function is its structure. Computational methods were developed early for folding and prediction of RNA structure with the aim of assisting in functional analysis. With the discovery of more and more ncRNAs, it has become clear that a large fraction of these are highly structured. Interestingly, a large part of the structure is comprised of regular Watson-Crick and GU wobble base pairs. This and the increased amount of available genomes have made it possible to employ structure-based methods for genomic screens. The field has moved from folding prediction of single sequences to computational screens for ncRNAs in genomic sequence using the RNA structure as the main characteristic feature. Whereas early methods focused on energy-directed folding of single sequences, comparative analysis based on structure preserving changes of base pairs has been efficient in improving accuracy, and today this constitutes a key component in genomic screens. Here, we cover the basic principles of RNA folding and touch upon some of the concepts in current methods that have been applied in genomic screens for de novo RNA structures in searches for novel ncRNA genes and regulatory RNA structure on mRNAs. We discuss the strengths and weaknesses of the different strategies and how they can complement each other.     

Proteomic analysis of apoptosis related proteins regulated by proto‐oncogene protein DEK

A nuclear phosphoprotein, DEK, is implicated in certain human diseases, such as leukemia and antoimmune disorders, and a major component of metazoan chromatin. Basically as a modulator of chromatin structure, it can involve in various DNA and RNA‐dependent processes and function as either an activator or repressor. Despite of numerous efforts to suggest the biological role of DEK, direct target proteins of DEK in different physiological status remains elusive. To investigate if DEK protein triggers the changes in certain protein networks, DEK was knocked down at both types of cell clones using siRNA expression. Here we provide a catalogue of proteome profiles in total cell lysates derived from normal HeLa and DEK knock‐down HeLa cells and a good in vitro model system for dissecting the protein networks due to this proto‐oncogenic DEK protein. In this biological context, we compared total proteome changes by the combined methods of two‐dimensional gel electrophoresis, quantitative image analysis and MALDI‐TOF MS analysis. There were a large number of targets for DEK, which were differentially expressed in DEK knock‐down cells and consisted of 58 proteins (41 up‐regulated and 17 down‐regulated) differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. In the identified 58 spots, 16% of proteins are known to be associated with apoptosis. Among others, we identified apoptosis related proteins such as Annexins, Enolase1, Lamin A, and Glutathione‐S‐transferase omega 1. These results are consistent with recent studies indicating the crucial role of DEK in apoptosis pathway. We further demonstrated by ChIP analysis that knock‐down of DEK caused hyperacetylation of histones around Prx VI promoter which is upregulated in our profile. Using immunoblotting analysis, we have demonstrated the modulation of other caspase‐dependent apoptosis related proteins by DEK knock‐down and further implicate its role in apoptosis pathway. J. Cell. Biochem. 106: 1048–1059, 2009. © 2009 Wiley‐Liss, Inc.     

Conserved RNA secondary structures in Picornaviridae genomes

The family Picornaviridae contains important pathogens including, for example, hepatitis A virus and foot-and-mouth disease virus. The genome of these viruses is a single messenger-active (+)-RNA of 7200–8500 nt. Besides coding for the viral proteins, it also contains functionally important RNA secondary structures, among them an internal ribosomal entry site (IRES) region towards the 5′-end. This contribution provides a comprehensive computational survey of the complete genomic RNAs and a detailed comparative analysis of the conserved structural elements in seven of the currently nine genera in the family Picornaviridae. Compared with previous studies we find: (i) that only smaller sections of the IRES region than previously reported are conserved at single base-pair resolution and (ii) that there is a number of significant structural elements in the coding region. Furthermore, we identify potential cis-acting replication elements in four genera where this feature has not been reported so far.     

Conserved RNA secondary structures in viral genomes: a survey

SUMMARY: The genomes of RNA viruses often carry conserved RNA structures that perform vital functions during the life cycle of the virus. Such structures can be detected using a combination of structure prediction and co-variation analysis. Here we present results from pilot studies on a variety of viral families performed during bioinformatics computer lab courses in past years.     

Predicting RNA 3D structure using a coarse-grain helix-centered model

A 3D model of RNA structure can provide information about its function and regulation that is not possible with just the sequence or secondary structure. Current models suffer from low accuracy and long running times and either neglect or presume knowledge of the long-range interactions which stabilize the tertiary structure. Our coarse-grained, helix-based, tertiary structure model operates with only a few degrees of freedom compared with all-atom models while preserving the ability to sample tertiary structures given a secondary structure. It strikes a balance between the precision of an all-atom tertiary structure model and the simplicity and effectiveness of a secondary structure representation. It provides a simplified tool for exploring global arrangements of helices and loops within RNA structures. We provide an example of a novel energy function relying only on the positions of stems and loops. We show that coupling our model to this energy function produces predictions as good as or better than the current state of the art tools. We propose that given the wide range of conformational space that needs to be explored, a coarse-grain approach can explore more conformations in less iterations than an all-atom model coupled to a fine-grain energy function. Finally, we emphasize the overarching theme of providing an ensemble of predicted structures, something which our tool excels at, rather than providing a handful of the lowest energy structures.     

Point mutations as an optimal search process in biological evolution

Point mutations are pictured as jumps in a phase space representing the sequences of amino acids or nucleotides as discrete points. It is shown that this space can be given a natural metric by quantifying common physical and chemical properties of amino acid constituents in terms of a natural measure. Evolution through point mutations is simulated by the search for points in the phase space representing amino acid sequences of high survival fitness. Due to the local compactness of the distribution of these functionally allowed points in phase space any successful search procedure has characteristics qualitatively different from those in the case of a random distribution. This is demonstrated by model calculations. A specified distribution of allowed points is generated with subsequent evaluation of the success of the retrieval process as a function of the jump probabilities between lattice sites. The results of such simulations are compared with data obtained from the analysis of the DNA or mRNA sequences coding related proteins. By counting silent and expressed nucleotide replacement frequencies one can draw conclusions as to the efficacy of the natural evolutionary search processes in the phase space of amino acid sequences. There are cases, where the highest possible information gain of one bit per accepted point mutation is achieved. In general the information gain is found to be somewhat sub-maximal due to functional requirements.     

Characterization of singlet oxygen-accumulating mutants isolated in a screen for altered oxidative stress response in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Background  

When photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast. In Chlamydomonas reinhardtii singlet oxygen was shown to act as a specific signal inducing the expression of the nuclear glutathione peroxidase gene GPXH/GPX5 during high light stress, but little is known about the cellular mechanisms involved in this response. To investigate components affecting singlet oxygen signaling in C. reinhardtii, a mutant screen was performed.     

Hybridization thermodynamics of NimbleGen Microarrays

Background  

While microarrays are the predominant method for gene expression profiling, probe signal variation is still an area of active research. Probe signal is sequence dependent and affected by probe-target binding strength and the competing formation of probe-probe dimers and secondary structures in probes and targets.