Estimating the Fraction of Non-Coding RNAs in Mammalian Transcriptomes

Recent studies of mammalian transcriptomes have identified numerous RNA transcripts that do not code for proteins; their identity, however, is largely unknown. Here we explore an approach based on sequence randomness patterns to discern different RNA classes. The relative z-score we use helps identify the known ncRNA class from the genome, intergene and intron classes. This leads us to a fractional ncRNA measure of putative ncRNA datasets which we model as a mixture of genuine ncRNAs and other transcripts derived from genomic, intergenic and intronic sequences. We use this model to analyze six representative datasets identified by the FANTOM3 project and two computational approaches based on comparative analysis (RNAz and EvoFold). Our analysis suggests fewer ncRNAs than estimated by DNA sequencing and comparative analysis, but the verity of our approach and its prediction requires more extensive experimental RNA data

Low P66shc with High SerpinB3 Levels Favors Necroptosis and Better Survival in Hepatocellular Carcinoma.

Cell proliferation and escape from apoptosis are important pathological features of hepatocellular carcinoma (HCC), one of the tumors with the highest mortality rate worldwide. The aim of the study was to evaluate the expression of the pro-apoptotic p66shc and the anti-apoptotic SerpinB3 in HCCs in relation to clinical outcome, cell fate and tumor growth. p66shc and SerpinB3 were evaluated in 67 HCC specimens and the results were correlated with overall survival. Proliferation and cell death markers were analyzed in hepatoma cells overexpressing SerpinB3, under different stress conditions. p66shc-/- mice and xenograft models were also used to assess the effects of p66shc and SerpinB3 on tumor growth. In patients with HCC, the best survival was observed in the subgroup with p66shc levels below median values and SerpinB3 levels above median values. Mice p66shc-/- showed high levels of SerpinB3, while in HepG2 cells overexpressing SerpinB3, p66shc expression was trivial. HepG2 overexpressing SerpinB3 cells were more prone to die after oxidizing treatments, such as diamide or high concentration H2O2. These cells injected in nude mice developed tumors five times smaller than those from control HepG2 cells. Tumors originating from HepG2 overexpressing SerpinB3 cells showed decreased activated Caspase-8, with concomitant increase of RIP3K and decreased levels of cleaved RIP3K, typical features of necroptosis. In conclusion, in patients affected by HCC, the pattern characterized by p66shc downregulation and elevated SerpinB3 levels was associated with markedly better survival. This pattern favored necroptosis in experimental high-stress conditions

Dynamic Energy Landscapes of Riboswitches Help Interpret Conformational Rearrangements and Function

Riboswitches are RNAs that modulate gene expression by ligand-induced conformational changes. However, the way in which sequence dictates alternative folding pathways of gene regulation remains unclear. In this study, we compute energy landscapes, which describe the accessible secondary structures for a range of sequence lengths, to analyze the transcriptional process as a given sequence elongates to full length. In line with experimental evidence, we find that most riboswitch landscapes can be characterized by three broad classes as a function of sequence length in terms of the distribution and barrier type of the conformational clusters: low-barrier landscape with an ensemble of different conformations in equilibrium before encountering a substrate; barrier-free landscape in which a direct, dominant “downhill” pathway to the minimum free energy structure is apparent; and a barrier-dominated landscape with two isolated conformational states, each associated with a different biological function. Sharing concepts with the “new view” of protein folding energy landscapes, we term the three sequence ranges above as the sensing, downhill folding, and functional windows, respectively. We find that these energy landscape patterns are conserved in various riboswitch classes, though the order of the windows may vary. In fact, the order of the three windows suggests either kinetic or thermodynamic control of ligand binding. These findings help understand riboswitch structure/function relationships and open new avenues to riboswitch design

Proposed folding pathways for the GEMM (a) and <i>moaA</i> (b) riboswitches.

<p>See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002368#pcbi-1002368-g002" target="_blank">figure 2</a> caption for description of figure elements. For full description of energy landscape characteristics see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002368#pcbi.1002368.s005" target="_blank">Figure S5</a>.</p

Proposed folding pathway for the TPP riboswitches <i>tenA</i> (a) and <i>thiM</i> (b).

<p>Structures formed in the sensing windows are represented in red boxes; downhill folding window structures are found in blue boxes; and functional window structures are represented inside the green boxes. Double-head arrows represent structures that can interchange. Broken-line structural elements in downhill folding window (blue box) represent structural elements that would be coerced to form in the presence of ligand. Colored circles adjacent to structures are marked by their points on the respective energy landscape to the right. Yellow arrows represent the series of structures accessed in the presence of ligand. For all sequence lengths inside of a window, the energy landscape repeatedly displays similar patterns (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002368#s4" target="_blank">Materials and Methods</a>). The specific sequence length corresponding to the window shown is given following the length range. For full description of energy landscape characteristics see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002368#pcbi.1002368.s005" target="_blank">Figure S5</a>.</p

Proposed folding pathway for the S-adenosylmethionine (SAM) <i>metI</i> riboswitch.

<p>See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002368#pcbi-1002368-g002" target="_blank">figure 2</a> caption for description of figure elements. For full description of energy landscape characteristics see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002368#pcbi.1002368.s005" target="_blank">Figure S5</a>.</p

Proposed folding pathway for the <i>pbuE</i> (a) and <i>xpt</i> (b) purine riboswitches.

<p>See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002368#pcbi-1002368-g002" target="_blank">figure 2</a> caption for description of figure elements. For full description of energy landscape characteristics see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002368#pcbi.1002368.s005" target="_blank">Figure S5</a>.</p

Riboswitch classes in present study.

<p>Thermodynamic and kinetic control of ligand binding is defined in the Introduction. Briefly, if the time required for equilibrium between ligand and aptamer is equal to or less than the amount of time it takes for the RNA to be fully transcribed, the switch will approximate thermodynamic (equilibrium) control. However, if the time required for RNA-ligand equilibrium is long, the switch is under kinetic control.</p><p>*K_D values listed are for the aptamer sequence at 298K, but ranges depend on experimental conditions.</p>‡<p>Investigators proposed that these riboswitches could function under thermodynamic control if there were transcriptional pause sites of a couple seconds or changes from standard temperature.</p>§<p>These riboswitches are known to require pseudoknot interactions for activity.</p