Identification of sequence features that predict competition potency of siRNAs

Small interfering RNAs (siRNAs) specifically knock-down target mRNAs via RNA interference (RNAi) mechanism. During this process, introduction of excess amount of exogenous siRNAs could lead to the saturation of cellular RNAi machinery. One consequence of RNAi machinery saturation is the competition between two simultaneously introduced siRNAs, during which one siRNA loses gene silencing activity. Although competition phenomena have been well characterized, the molecular and sequence features of siRNAs that specify the competition potency remain poorly understood. Here, for the first time, we performed a large-scale siRNA competition potency analysis by measuring the competition potency of 56 different siRNAs and ranking them based on their competition potency. We have also established an algorithm to predict the competition potency of siRNAs based upon the conserved sequence features of strong and weak competitor siRNAs. The present study supports our hypothesis that the competition potency of siRNAs is specified by the 5′-half antisense sequence and provides a useful guideline to design siRNAs with minimal RNAi machinery saturation.     

Absolute quantitation of specific mRNAs in cell and tissue samples by comparative PCR.

A comparative PCR assay, for the absolute quantitation of specific mRNAs in cell and tissue samples, has been designed to overcome problems with previous techniques. cDNAs made from the RNAs are co-amplified with "competitor" plasmid templates under conditions in which reagents are not limiting at the equivalence point, thereby preventing competition between target and competitor templates and distinguishing the assay from competitive PCR assays. The cDNAs are serially diluted, and competitor templates concentrations are kept constant, rather than vice versa, as occurs in competitive PCR assays. Products from target and competitor templates are resolved by electrophoresis and measured by phosphorescent or fluorescent imagery. Both products are measured to minimize errors in the competitor:target ratio. A synthetic external standard RNA is included in the tissue lysis solution and co-purified with endogenous mRNAs, thereby being subjected to identical losses of yield during subsequent procedures. The determination of the number of copies of external standard cDNA allows inefficiencies of RNA extraction and cDNA synthesis to be taken into account. Standard concentrations of plasmids containing the endogenous target sequences are also measured, so that corrections can be made for discrepancies due to unequal amplification of target and competitor sequences. These corrections, together with the use of an external standard and the PCR conditions chosen, allow for the accurate, specific and sensitive determination of the absolute number of mRNA copies in a sample.     

Competition and coexistence in regional habitats

Habitat heterogeneity plays a key role in the dynamics and structures of communities. In this article, a two-species metapopulation model that includes local competitive dynamics is analyzed to study the population dynamics of two competing species in spatially structured habitats. When local stochastic extinction can be ignored, there are, as in Lotka-Volterra equations, four outcomes of interspecific competition in this model. The outcomes of competition depend on the competitive intensity between the competing pairs. An inferior competitor and a superior competitor, or two strongly competing species, can never stably coexist, whereas two weak competitors (even if they are very similar species) may coexist over the long term in such environments. Local stochastic extinction may greatly affect the outcomes of interspecific competition. Two competing species can or cannot stably coexist depending not only on the competitive intensity between the competing pairs but also on their precompetitive distributions. Two weak competitors that have similar precompetitive distributions can always regionally coexist. Two strongly competing species that competitively exclude each other in more stable habitats may be able to stably coexist in highly heterogenous environments if they have similar precompetitive distributions. There is also a chance for an inferior competitor to coexist regionally or even to exclude a superior competitor when the superior competitor has a narrow precompetitive distribution and the inferior competitor has a wide precompetitive distribution.     

Competition and species coexistence in a metapopulation model: Can fast asymmetric migration reverse the outcome of competition in a homogeneous environment?

We investigate whether asymmetric fast migration can modify the predictions of classical competition theory and, in particular revert species dominance. We consider a model of two species competing for an implicit resource on a habitat divided into two patches. Both patches are connected through constant migration rates and in each patch local dynamics are driven by a Lotka-Volterra competition system.Local competition is asymmetric with the same superior competitor in both patches. Migration is asymmetric, species dependent and fast in comparison to local competitive interactions. The species and patches are taken to be otherwise similar: in both patches we assume the same carrying capacities for both species, and the same growth rates and pair-wise competition coefficients for each species.We show that global dynamics can be described by a classical Lotka-Volterra competition model. We found that by modifying the ratio of intraspecific migration rates for both species all possible combinations of global species relative dominance can be achieved. We find specific conditions for which the local superior competitor is globally excluded. This is to our knowledge the first study showing that fast asymmetric migration can lead to inferior competitor dominance in a homogeneous environment. We conclude that disparity of temporal scales between migration and local dynamics may have important consequences for the maintenance of biodiversity in spatially structured populations.     

The effects of competition on fitness depend on the sex of both competitors

In intraspecific competition, the sex of competing individuals is likely to be important in determining the outcome of competitive interactions and the way exposure to conspecifics during development influences adult fitness traits. Previous studies have explored differences between males and females in their response to intraspecific competition. However, few have tested how the sex of the competitors, or any interactions between focal and competitor sex, influences the nature and intensity of competition. We set up larval seed beetles Callosobruchus maculatus to develop either alone or in the presence of a male or female competitor and measured a suite of traits: development time, emergence weight; male ejaculate mass, copulation duration, and lifespan; and female lifetime fecundity, offspring egg–adult survival, and lifespan. We found effects of competition and competitor sex on the development time and emergence weight of both males and females, and also of an interaction between focal and competitor sex: Females emerged lighter when competing with another female, while males did not. There was little effect of larval competition on male and female adult fitness traits, with the exception of the effect of a female competitor on a focal female's offspring survival rate. Our results highlight the importance of directly measuring the effects of competition on fitness traits, rather than distant proxies for fitness, and suggest that competition with the sex with the greater resource requirements (here females) might play a role in driving trait evolution. We also found that male–male competition during development resulted in shorter copulation times than male–female competition, a result that remained when controlling for the weight of competitors. Although it is difficult to definitively tease apart the effects of social environment and access to resources, this result suggests that something about the sex of competitors other than their size is driving this pattern.     

Competing endogenous RNA database

A given mRNA can be regulated by interactions with miRNAs and in turn the availability of these miRNAs can be regulated by their interactions with alternate mRNAs. The concept of regulation of a given mRNA by alternate mRNA (competing endogenous mRNA) by virtue of interactions with miRNAs through shared miRNA response elements is poised to become a fundamental genetic regulatory mechanism. The molecular basis of the mRNA-mRNA cross talks is via miRNA response elements, which can be predicted based on both molecular interaction and evolutionary conservation. By examining the co-occurrence of miRNA response elements in the mRNAs on a genome-wide basis we predict competing endogenous RNA for specific mRNAs targeted by miRNAs. Comparison of the mRNAs predicted to regulate PTEN with recently published work, indicate that the results presented within the competing endogenous RNA database (ceRDB) have biological relevance.

Availability

http://www.oncomir.umn.edu/cefinder/     

Building a cellular switch: more lessons from a good egg.

Xenopus oocytes mature in response to the steroid hormone progesterone. At the level of a population of oocytes, the response is graded-the higher the concentration of progesterone, the larger the fraction of oocytes that will mature-but at the level of the individual oocyte, the response is all-or-none. The all-or-none character of this cell fate switch is hypothesized to arise out of two properties of the signal transduction machinery that mediates maturation, positive feedback, and ultrasensitivity. This combination of positive feedback plus ultrasensitivity crops up again and again in cellular switches, from the lysis-lysogeny switch in phage-infected bacteria to the action potential in neurons.     

Effects of sequestration on signal transduction cascades

The building blocks of most signal transduction pathways are pairs of enzymes, such as kinases and phosphatases, that control the activity of protein targets by covalent modification. It has previously been shown [Goldbeter A & Koshland DE (1981) Proc Natl Acad Sci USA 78, 6840-6844] that these systems can be highly sensitive to changes in stimuli if their catalysing enzymes are saturated with their target protein substrates. This mechanism, termed zero-order ultrasensitivity, may set thresholds that filter out subthreshold stimuli. Experimental data on protein abundance suggest that the enzymes and their target proteins are present in comparable concentrations. Under these conditions a large fraction of the target protein may be sequestrated by the enzymes. This causes a reduction in ultrasensitivity so that the proposed mechanism is unlikely to account for ultrasensitivity under the conditions present in most in vivo signalling cascades. Furthermore, we show that sequestration changes the dynamics of a covalent modification cycle and may account for signal termination and a sign-sensitive delay. Finally, we analyse the effect of sequestration on the dynamics of a complex signal transduction cascade: the mitogen-activated protein kinase (MAPK) cascade with negative feedback. We show that sequestration limits ultrasensitivity in this cascade and may thereby abolish the potential for oscillations induced by negative feedback.     

Effects of altered resource consumption rates by one consumer species on a competitor

Non‐mechanistic models of competition suggest that harming one of two competing species will increase the population density of the other. These models also suggest that any change in a fitness component of one competitor will make the densities of the two competitors change in opposite directions. However, models of competition that incorporate resource dynamics show that neither conclusion holds generally. Reducing the consumption abilities of one competitor may decrease the population size of the other by decreasing resource overexploitation by the first and thereby increasing its density. It is also possible for decreased consumption abilities of one species to increase the population densities of both species, when the increased density of the focal species is offset by its decreased ability to consume the main resources of its competitor. Finally, decreases in consumption may have the effects predicted by phenomenological models; a decrease in the focal species and an increase in its competitor. Unstable systems may exhibit more complicated patterns of changes in densities with changes in consumption rates. These counterintuitive effects depend on the presence of overexploitation of biotic resources, about which little is known. More generally, there have been few theoretical or empirical studies examining the indirect effects of changes in consumption rates of a focal species in a food web; these are termed ‘trait‐initiated indirect effects’. A better understanding of the potential consequences of altered consumption rates will be important for understanding biotic shifts in communities undergoing environmental change, and in using simple community modules to understand larger food webs.     

Substrate competition as a source of ultrasensitivity in the inactivation of Wee1

The mitotic regulators Wee1 and Cdk1 can inactivate each other through inhibitory phosphorylations. This double-negative feedback loop is part of a bistable trigger that makes the transition into mitosis abrupt and decisive. To generate a bistable response, some component of a double-negative feedback loop must exhibit an ultrasensitive response to its upstream regulator. Here, we experimentally demonstrate that Wee1 exhibits a highly ultrasensitive response to Cdk1. Several mechanisms can, in principle, give rise to ultrasensitivity, including zero-order effects, multisite phosphorylation, and competition mechanisms. We found that the ultrasensitivity in the inactivation of Wee1 arises mainly through two competition mechanisms: competition between two sets of phosphorylation sites in Wee1 and between Wee1 and other high-affinity Cdk1 targets. Based on these findings, we were able to reconstitute a highly ultrasensitive Wee1 response with purified components. Competition provides a simple way of generating the equivalent of a highly cooperative allosteric response.     

Messenger RNA competition in living Xenopus oocytes.

When calf lens crystallin mRNA and rabbit globin mRNA are competing for factors limiting protein synthesis in living Xenopus oocytes, no mRNA species is preferentially selected for translation. Differences in the intrinsic translational efficiency of the mRNA species exist, but the relative efficiencies are the same at high and low mRNA concentrations. mRNAs already being translated, in particular endogenous oocyte mRNAs, are less sensitive to competitive inhibition by injected mRNAs. As injected mRNAs gradually become incorporated into the protein-synthesizing machinery of the oocyte, they acquire the same status as the oocyte's own active mRNAs. Exogenous mRNAs this become endogenous mRNAs. These results, together with previous estmates of the translational efficiency of injected heterologous mRNA species, are compatible with the assumption that a large proportion of the endogenous mRNAs is not competing for the translational apparatus of the oocyte and, therefore, probably is present in the temporarily inactivated form.     

Spatial heterogeneity, source-sink dynamics, and the local coexistence of competing species

Patch occupancy theory predicts that a trade-off between competition and dispersal should lead to regional coexistence of competing species. Empirical investigations, however, find local coexistence of superior and inferior competitors, an outcome that cannot be explained within the patch occupancy framework because of the decoupling of local and spatial dynamics. We develop two-patch metapopulation models that explicitly consider the interaction between competition and dispersal. We show that a dispersal-competition trade-off can lead to local coexistence provided the inferior competitor is superior at colonizing empty patches as well as immigrating among occupied patches. Immigration from patches that the superior competitor cannot colonize rescues the inferior competitor from extinction in patches that both species colonize. Too much immigration, however, can be detrimental to coexistence. When competitive asymmetry between species is high, local coexistence is possible only if the dispersal rate of the inferior competitor occurs below a critical threshold. If competing species have comparable colonization abilities and the environment is otherwise spatially homogeneous, a superior ability to immigrate among occupied patches cannot prevent exclusion of the inferior competitor. If, however, biotic or abiotic factors create spatial heterogeneity in competitive rankings across the landscape, local coexistence can occur even in the absence of a dispersal-competition trade-off. In fact, coexistence requires that the dispersal rate of the overall inferior competitor not exceed a critical threshold. Explicit consideration of how dispersal modifies local competitive interactions shifts the focus from the patch occupancy approach with its emphasis on extinction-colonization dynamics to the realm of source-sink dynamics. The key to coexistence in this framework is spatial variance in fitness. Unlike in the patch occupancy framework, high rates of dispersal can undermine coexistence, and hence diversity, by reducing spatial variance in fitness.     

Morphological response to competition for light in the clonal Trifolium repens (Fabaceae)

? Premise of the study: Plant communities in temperate zones are dominated by clonal plants that can plastically modify their growth characteristics in response to competition. Given that plants compete with one another, and the implications this has for species coexistence, we conducted a study to assess how clonal species morphologically respond to competition for light depending on its intensity and heterogeneity, which are determined by the competitor species. ? Methods: We assessed the morphological response to competition for light of the clonal species Trifolium repens L. by measuring its growth performance, and vertical and horizontal growth traits. We used five competitive environments, i.e., one without competitor and four differing by their competitor species creating different conditions of competition intensity and heterogeneity. ? Key results: The morphological response of Trifolium repens to competition for light depended on the competitor identity. Competition intensity and heterogeneity, determined by competitor identity, had an interactive effect on most traits. The increase in petiole elongation and specific leaf area due to increased competition intensity was observed only at low to intermediate competition heterogeneity. Competition heterogeneity promoted the elongation of clone connections allowing space exploration. ? Conclusions: Our results demonstrated that the intensity and heterogeneity of competition, which depended on competitor identity, are of primary importance in determining the plastic response of Trifolium repens. This emphasizes that it is important to consider the fine-scale spatial distribution of individuals when studying their interactions within plant communities.     

Tunable signal processing in synthetic MAP kinase cascades

The flexibility of MAPK cascade responses enables regulation of a vast array of cell fate decisions, but elucidating the mechanisms underlying this plasticity is difficult in endogenous signaling networks. We constructed insulated mammalian MAPK cascades in yeast to explore how intrinsic and extrinsic perturbations affect the flexibility of these synthetic signaling modules. Contrary to biphasic dependence on scaffold concentration, we observe monotonic decreases in signal strength as scaffold concentration increases. We find that augmenting the concentration of sequential kinases can enhance ultrasensitivity and lower the activation threshold. Further, integrating negative regulation and concentration variation can decouple ultrasensitivity and threshold from the strength of the response. Computational analyses show that cascading can generate ultrasensitivity and that natural cascades with different kinase concentrations are innately biased toward their distinct activation profiles. This work demonstrates that tunable signal processing is inherent to minimal MAPK modules and elucidates principles for rational design of synthetic signaling systems.     

Analysing microarray data using modular regulation analysis

MOTIVATION: Microarray experiments measure complex changes in the abundance of many mRNAs under different conditions. Current analysis methods cannot distinguish between direct and indirect effects on expression, or calculate the relative importance of mRNAs in effecting responses. RESULTS: Application of modular regulation analysis to microarray data reveals and quantifies which mRNA changes are important for cellular responses. The mRNAs are clustered, and then we calculate how perturbations alter each cluster and how strongly those clusters affect an output response. The product of these values quantifies how an input changes a response through each cluster. Two published datasets are analysed. Two mRNA clusters transmit most of the response of yeast doubling time to galactose; one contains mainly galactose metabolic genes, and the other a regulatory gene. Analysis of the response of yeast relative fitness to 2-deoxy-D-glucose reveals that control is distributed between several mRNA clusters, but experimental error limits statistical significance.     

Construction and analysis of the abnormal lncRNA–miRNA–mRNA network in hypoxic pulmonary hypertension

The incidence of hypoxic pulmonary hypertension (HPH) is increasing. Accumulating evidence suggests that long noncoding RNAs (lncRNAs) play an important role in HPH, but the functions and mechanism have yet to be fully elucidated. In the present study, we established a HPH rat model with 8 h of hypoxia exposure (10% O₂) per day for 21 days. High-throughput sequencing identified 60 differentially expressed (DE) lncRNAs, 20 DE miRNAs and 695 DE mRNAs in rat lung tissue. qRT-PCR verified the accuracy of the results. The DE mRNAs were significantly enriched in immune response, inflammatory response, leukocyte migration, cell cycle, cellular response to interleukin-1, IL-17 signalling pathway, cytokine–cytokine receptor interaction and Toll-like receptor signalling pathway. According to the theory of competing endogenous RNA (ceRNA) networks, lncRNA–miRNA–mRNA network was constructed by Cytoscape software, 16 miRNAs and 144 mRNAs. The results suggested that seven DE lncRNAs (Ly6l, AABR07038849.2, AABR07069008.2, AABR07064873.1, AABR07001382.1, AABR07068161.1 and AABR07060341.2) may serve as molecular sponges of the corresponding miRNAs and play a major role in HPH.     

To construct a ceRNA regulatory network as prognostic biomarkers for bladder cancer

Emerging evidence demonstrates that competing endogenous RNA (ceRNA) hypothesis has played a role in molecular biological mechanisms of cancer occurrence and development. But the effect of ceRNA network in bladder cancer (BC), especially lncRNA‐miRNA‐mRNA regulatory network of BC, was not completely expounded. By means of The Cancer Genome Atlas (TCGA) database, we compared the expression of RNA sequencing (RNA‐Seq) data between 19 normal bladder tissue and 414 primary bladder tumours. Then, weighted gene co‐expression network analysis (WGCNA) was conducted to analyse the correlation between two sets of genes with traits. Interactions between miRNAs, lncRNAs and target mRNAs were predicted by MiRcode, miRDB, starBase, miRTarBase and TargetScan. Next, by univariate Cox regression and LASSO regression analysis, the 86 mRNAs obtained by prediction were used to construct a prognostic model which contained 4 mRNAs (ACTC1 + FAM129A + OSBPL10 + EPHA2). Then, by the 4 mRNAs in the prognostic model, a ceRNA regulatory network with 48 lncRNAs, 14 miRNAs and 4 mRNAs was constructed. To sum up, the ceRNA network can further explore gene regulation and predict the prognosis of BC patients.