Strain-dependent differences in coordination of yeast signalling networks.

The yeast mitogen-activated protein kinase pathways serve as a model system for understanding how network interactions affect the way in which cells coordinate the response to multiple signals. We have quantitatively compared two yeast strain backgrounds YPH499 and ∑1278b (both of which have previously been used to study these pathways) and found several important differences in how they coordinate the interaction between the high osmolarity glycerol (HOG) and mating pathways. In the ∑1278b background, in response to simultaneous stimulus, mating pathway activation is dampened and delayed in a dose-dependent manner. In the YPH499 background, only dampening is dose-dependent. Furthermore, leakage from the HOG pathway into the mating pathway (crosstalk) occurs during osmostress alone in the ∑1278b background only. The mitogen-activated protein kinase Hog1p suppresses crosstalk late in an induction time course in both strains but does not affect the early crosstalk seen in the ∑1278b background. Finally, the kinase Rck2p plays a greater role suppressing late crosstalk in the ∑1278b background than in the YPH499 background. Our results demonstrate that comparisons between laboratory yeast strains provide an important resource for understanding how signalling network interactions are tuned by genetic variation without significant alteration to network structure.

Biological signal generators: integrating synthetic biology tools and in silico control.

Biological networks sense extracellular stimuli and generate appropriate outputs within the cell that determine cellular response. Biological signal generators are becoming an important tool for understanding how information is transmitted in these networks and controlling network behavior. Signal generators produce well-defined, dynamic, intracellular signals of important network components, such as kinase activity or the concentration of a specific transcription factor. Synthetic biology tools coupled with in silico control have enabled the construction of these sophisticated biological signal generators. Here we review recent advances in biological signal generator construction and their use in systems biology studies. Challenges for constructing signal generators for a wider range of biological networks and generalizing their use are discussed.

Nonsense-mediated mRNA decay of the ferric and cupric reductase mRNAs FRE1 and FRE2 in Saccharomyces cerevisiae.

The nonsense-mediated mRNA decay (NMD) pathway regulates mRNAs that aberrantly terminate translation. This includes aberrant mRNAs and functional natural mRNAs. Natural mRNA degradation by NMD is triggered by mRNA features and environmental cues. Saccharomyces cerevisiae encodes multiple proteins with ferric and cupric reductase activity. Here, we examined the regulation by NMD of two mRNAs, FRE1 and FRE2, encoding ferric and cupric reductases in S. cerevisiae. We found that FRE2 mRNAs are regulated by NMD under noninducing conditions and that the FRE2 3'-UTR contributes to the degradation of the mRNAs by NMD. Conversely, FRE1 mRNAs are not regulated by NMD under comparable conditions. These findings suggest that regulation of functionally related mRNAs by NMD can be differential and conditional.

The nonsense-mediated mRNA decay (NMD) pathway differentially regulates COX17, COX19 and COX23 mRNAs.

The differential regulation of COX17, COX19 and COX23 mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was investigated. The NMD pathway regulates mRNAs that aberrantly terminate translation. This includes mRNAs harboring premature translation termination codons and natural mRNAs. Most natural mRNAs regulated by NMD encode fully functional proteins involved in various cellular processes. However, the cause and targeting of most of these mRNAs by the pathway is not understood. Analysis of a set of mRNAs involved in copper homeostasis showed that a subset of these mRNAs function in mitochondrial copper homeostasis. Here, we examined the regulation of COX17, COX19 and COX23 mRNAs by NMD. These mRNAs encode homologous mitochondrial proteins involved in metallation of cytochrome c oxidase. We found that COX17, COX19 and COX23 mRNAs are differentially regulated by NMD depending on environmental copper levels. A long 3'-UTR contributes to the direct regulation of COX19 mRNA by the pathway. Alternatively, COX23 mRNA contains a long 3'-UTR, but is indirectly regulated by the pathway under two conditions tested here. Analysis of the functionality of the NMD targeting features in COX23 mRNA showed that the COX23 3'-UTR is sufficient to trigger NMD. The regulation of mRNAs involved in mitochondrial copper metabolism by NMD is physiologically significant because excess copper enhances growth of NMD mutants on a non-fermentable carbon source. These findings suggest that regulation of mRNAs encoding homologous proteins by NMD can be differential depending on environmental copper levels. Furthermore, these findings suggest copper ion homeostatic mechanisms in the mitochondria occur at the mRNA level via the NMD pathway.

mRNAs involved in copper homeostasis are regulated by the nonsense-mediated mRNA decay pathway depending on environmental conditions.

The nonsense-mediated mRNA decay pathway (NMD) is an mRNA degradation pathway that degrades mRNAs that prematurely terminate translation. These mRNAs include mRNAs with premature termination codons as well as many natural mRNAs. In Saccharomyces cerevisiae a number of features have been shown to target natural mRNAs to NMD. However, the extent to which natural mRNAs from the same functional group are regulated by NMD and how environmental conditions influence this regulation is not known. Here, we examined mRNAs involved in copper homeostasis and are predicted to be sensitive to NMD. We found that the majority of these mRNAs have long 3'-UTRs that could target them for degradation by NMD. Analysis of one of these mRNAs, COX19, found that the long 3'-UTR contributes to regulation of this mRNA by NMD. Furthermore, we examined an additional mRNA, MAC1 under low copper conditions. We found that low copper growth conditions affect NMD sensitivity of the MAC1 mRNA demonstrating that sensitivity to NMD can be altered by environmental conditions. MAC1 is a copper sensitive transcription factor that regulates genes involved with high affinity copper transport. Our results expand our understanding of how NMD regulates mRNAs from the same functional group and how the environment influences this regulation.

Genetic Subdivision and Variation in Selfing Rates Among Central American Populations of the Mangrove Rivulus, Kryptolebias marmoratus.

We used 32 polymorphic microsatellite loci to investigate how a mixed-mating system affects population genetic structure in Central American populations (N = 243 individuals) of the killifish Kryptolebias marmoratus (mangrove rivulus), 1 of 2 of the world's only known self-fertilizing vertebrates. Results were also compared with previous microsatellite surveys of Floridian populations of this species. For several populations in Belize and Honduras, population structure and genetic differentiation were pronounced and higher than in Florida, even though the opposite trend was expected because populations in the latter region were presumably smaller and highly selfing. The deduced frequency of selfing (s) ranged from s = 0.39-0.99 across geographic locales in Central America. This heterogeneity in selfing rates was in stark contrast to Florida, where s > 0.9. The frequency of outcrossing in a population (t = 1 - s) was tenuously correlated with local frequencies of males, suggesting that males are one of many factors influencing outcrossing. Observed distributions of individual heterozygosity showed good agreement with expected distributions under an equilibrium mixed-mating model, indicating that rates of selfing remained relatively constant over many generations. Overall, our results demonstrate the profound consequences of a mixed-mating system for the genetic architecture of a hermaphroditic vertebrate.

Regulation of CTR2 mRNA by the nonsense-mediated mRNA decay pathway.

The nonsense-mediated mRNA decay (NMD) pathway was originally identified as a pathway that degrades mRNAs with premature termination codons; however, NMD is now known to regulate natural mRNAs as well. Natural mRNAs are degraded by NMD due to the presence of specific NMD targeting features. An atypically long 3'-UTR is one of the features that has been shown to induce the rapid degradation of mRNAs by NMD in Saccharomyces cerevisiae and other organisms. S. cerevisiae CTR2 mRNAs have long 3'-UTRs and are sensitive to NMD, although the extent by which these long 3'-UTRs target the CTR2 mRNAs to the pathway is unknown. Here, we investigated the sequence elements that induce NMD of the CTR2 mRNAs and determined that the long CTR2 3'-UTR is sufficient to target an NMD-insensitive mRNA to the pathway. We also found that, although the CTR2 3'-UTR contributes to NMD-induced degradation, CTR2 mRNAs contain additional NMD-inducing features that function cooperatively with the atypically long 3'-UTR to trigger mRNA degradation. Lengthening the CTR2 ORF abrogates NMD and renders the mRNAs immune to the NMD pathway. Moreover, we found that transcription of CTR2 driven by the GPD promoter, which is not identical to the CTR2 promoter, affects degradation of the transcripts by NMD.