Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling.

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interacts with the conserved 5' splice-site (5'SS) GU dinucleotide and makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. We further show that Prp28's ATPase activity is potentiated by the phosphorylated Npl3, but not the unphosphorylated Npl3, thus suggesting a strategy for regulating DExD/H-box ATPases. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal region, which can be phosphorylated and serves as an anchor to human spliceosome.

Sex alters molecular evolution in diploid experimental populations of S. cerevisiae.

Sex is common among eukaryotes, but entails considerable costs. The selective conditions that drive the evolutionary maintenance of sexual reproduction remain an open question. One long-standing explanation is that sex and recombination facilitate adaptation to fluctuating environmental conditions, although the genetic mechanisms that underlie such a benefit have not been empirically observed. In this study, we compare the dynamics and fitness effects of mutations in sexual and asexual diploid populations of the yeast Saccharomyces cerevisiae during adaptation to a fluctuating environment. While we find no detectable difference in the rate of adaptation between sexual and asexual populations, only the former evolve high fitness mutations in parallel, a genetic signature of adaptation. Using genetic reconstructions and fitness assays, we demonstrate that evolved, overdominant mutations can be beneficial in asexual populations, but maintained at lower frequencies in sexual populations due to segregation load. Overall these data show that sex alters the molecular basis of adaptation in diploids, and confers both costs and benefits.

Abundant and diverse Tetrahymena species living in the bladder traps of aquatic carnivorous Utricularia plants.

Ciliates are unicellular eukaryotes known for their cellular complexity and wide range of natural habitats. How they adapt to their niches and what roles they play in ecology remain largely unknown. The genus Tetrahymena is among the best-studied groups of ciliates and one particular species, Tetrahymena thermophila, is a well-known laboratory model organism in cell and molecular biology, making it an excellent candidate for study in protist ecology. Here, based on cytochrome c oxidase subunit I (COX1) gene barcoding, we identify a total of 19 different putative Tetrahymena species and two closely related Glaucoma lineages isolated from distinct natural habitats, of which 13 are new species. These latter include 11 Tetrahymena species found in the bladder traps of Utricularia plants, the most species-rich and widely distributed aquatic carnivorous plant, thus revealing a previously unknown but significant symbiosis of Tetrahymena species living among the microbial community of Utricularia bladder traps. Additional species were collected using an artificial trap method we have developed. We show that diverse Tetrahymena species may live even within the same habitat and that their populations are highly dynamic, suggesting that the diversity and biomass of species worldwide is far greater than currently appreciated.

Adaptive transcription-splicing resynchronization upon losing an essential splicing factor.

Essential genes form the core of a genome and are therefore thought to be indispensable for cellular viability. However, recent findings have challenged this notion in that cells may survive in the absence of some essential genes provided that relevant genetic modifiers are in existence. We therefore hypothesized that the loss of an essential gene may not always be fatefully detrimental; instead, it may pave the way towards genome evolution. We experimentally tested this hypothesis in the context of pre-messenger RNA splicing by evolving yeast cells harbouring a permanent loss of the essential splicing factor Prp28 in the presence of a genetic modifier. Here, we show that cellular fitness can be restored by compensatory mutations that alter either the splicing machinery per se or the Spt-Ada-Gcn5 acetyltransferase transcription co-activator complex in the cells with no Prp28. Biochemical and genetic analysis revealed that slowing down transcription compensates for splicing deficiency, which in turn boosts cellular fitness. In addition, we found that inefficient splicing also conversely decreases nascent RNA production. Taken together, our data suggest that transcription-splicing synchronization contributes to robustness in the gene-expression pathway and argue that the intrinsic interconnectivity within a biological system can be exploited for compensatory evolution and system re-optimization.

The conserved AU dinucleotide at the 5' end of nascent U1 snRNA is optimized for the interaction with nuclear cap-binding-complex.

Splicing is initiated by a productive interaction between the pre-mRNA and the U1 snRNP, in which a short RNA duplex is established between the 5' splice site of a pre-mRNA and the 5' end of the U1 snRNA. A long-standing puzzle has been why the AU dincucleotide at the 5'-end of the U1 snRNA is highly conserved, despite the absence of an apparent role in the formation of the duplex. To explore this conundrum, we varied this AU dinucleotide into all possible permutations and analyzed the resulting molecular consequences. This led to the unexpected findings that the AU dinucleotide dictates the optimal binding of cap-binding complex (CBC) to the 5' end of the nascent U1 snRNA, which ultimately influences the utilization of U1 snRNP in splicing. Our data also provide a structural interpretation as to why the AU dinucleotide is conserved during evolution.

A population study of killer viruses reveals different evolutionary histories of two closely related Saccharomyces sensu stricto yeasts.

Microbes have evolved ways of interference competition to gain advantage over their ecological competitors. The use of secreted killer toxins by yeast cells through acquiring double-stranded RNA viruses is one such prominent example. Although the killer behaviour has been well studied in laboratory yeast strains, our knowledge regarding how killer viruses are spread and maintained in nature and how yeast cells co-evolve with viruses remains limited. We investigated these issues using a panel of 81 yeast populations belonging to three Saccharomyces sensu stricto species isolated from diverse ecological niches and geographic locations. We found that killer strains are rare among all three species. In contrast, killer toxin resistance is widespread in Saccharomyces paradoxus populations, but not in Saccharomyces cerevisiae or Saccharomyces eubayanus populations. Genetic analyses revealed that toxin resistance in S. paradoxus is often caused by dominant alleles that have independently evolved in different populations. Molecular typing identified one M28 and two types of M1 killer viruses in those killer strains. We further showed that killer viruses of the same type could lead to distinct killer phenotypes under different host backgrounds, suggesting co-evolution between the viruses and hosts in different populations. Taken together, our data suggest that killer viruses vary in their evolutionary histories even within closely related yeast species.

Functions of the DExD/H-box proteins in nuclear pre-mRNA splicing.

In eukaryotes, many genes are transcribed as precursor messenger RNAs (pre-mRNAs) that contain exons and introns, the latter of which must be removed and exons ligated to form the mature mRNAs. This process is called pre-mRNA splicing, which occurs in the nucleus. Although the chemistry of pre-mRNA splicing is identical to that of the self-splicing Group II introns, hundreds of proteins and five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6, are essential for executing pre-mRNA splicing. Spliceosome, arguably the most complex cellular machine made up of all those proteins and snRNAs, is responsible for carrying out pre-mRNA splicing. In contrast to the transcription and the translation machineries, spliceosome is formed anew onto each pre-mRNA and undergoes a series of highly coordinated reconfigurations to form the catalytic center. This amazing process is orchestrated by a number of DExD/H-proteins that are the focus of this article, which aims to review the field in general and to project the exciting challenges and opportunities ahead. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.

Dynamic large-scale chromosomal rearrangements fuel rapid adaptation in yeast populations.

Large-scale genome rearrangements have been observed in cells adapting to various selective conditions during laboratory evolution experiments. However, it remains unclear whether these types of mutations can be stably maintained in populations and how they impact the evolutionary trajectories. Here we show that chromosomal rearrangements contribute to extremely high copper tolerance in a set of natural yeast strains isolated from Evolution Canyon (EC), Israel. The chromosomal rearrangements in EC strains result in segmental duplications in chromosomes 7 and 8, which increase the copy number of genes involved in copper regulation, including the crucial transcriptional activator CUP2 and the metallothionein CUP1. The copy number of CUP2 is correlated with the level of copper tolerance, indicating that increasing dosages of a single transcriptional activator by chromosomal rearrangements has a profound effect on a regulatory pathway. By gene expression analysis and functional assays, we identified three previously unknown downstream targets of CUP2: PHO84, SCM4, and CIN2, all of which contributed to copper tolerance in EC strains. Finally, we conducted an evolution experiment to examine how cells maintained these changes in a fluctuating environment. Interestingly, the rearranged chromosomes were reverted back to the wild-type configuration at a high frequency and the recovered chromosome became fixed in less selective conditions. Our results suggest that transposon-mediated chromosomal rearrangements can be highly dynamic and can serve as a reversible mechanism during early stages of adaptive evolution.

The evolution of low mutation rates in experimental mutator populations of Saccharomyces cerevisiae.

Mutation is the source of both beneficial adaptive variation and deleterious genetic load, fueling the opposing selective forces than shape mutation rate evolution. This dichotomy is well illustrated by the evolution of the mutator phenotype, a genome-wide 10- to 100-fold increase in mutation rate. This phenotype has often been observed in clonally expanding populations exposed to novel or frequently changing conditions. Although studies of both experimental and natural populations have shed light on the evolutionary forces that lead to the spread of the mutator allele through a population, significant gaps in our understanding of mutator evolution remain. Here we use an experimental evolution approach to investigate the conditions required for the evolution of a reduction in mutation rate and the mechanisms by which populations tolerate the accumulation of deleterious mutations. We find that after ∼6,700 generations, four out of eight experimental mutator lines had evolved a decreased mutation rate. We provide evidence that the accumulation of deleterious mutations leads to selection for reduced mutation rate clones in populations of mutators. Finally, we test the long-term consequences of the mutator phenotype, finding that mutator lines follow different evolutionary trajectories, some of which lead to drug resistance.

A tradeoff drives the evolution of reduced metal resistance in natural populations of yeast.

Various types of genetic modification and selective forces have been implicated in the process of adaptation to novel or adverse environments. However, the underlying molecular mechanisms are not well understood in most natural populations. Here we report that a set of yeast strains collected from Evolution Canyon (EC), Israel, exhibit an extremely high tolerance to the heavy metal cadmium. We found that cadmium resistance is primarily caused by an enhanced function of a metal efflux pump, PCA1. Molecular analyses demonstrate that this enhancement can be largely attributed to mutations in the promoter sequence, while mutations in the coding region have a minor effect. Reconstruction experiments show that three single nucleotide substitutions in the PCA1 promoter quantitatively increase its activity and thus enhance the cells' cadmium resistance. Comparison among different yeast species shows that the critical nucleotides found in EC strains are conserved and functionally important for cadmium resistance in other species, suggesting that they represent an ancestral type. However, these nucleotides had diverged in most Saccharomyces cerevisiae populations, which gave cells growth advantages under conditions where cadmium is low or absent. Our results provide a rare example of a selective sweep in yeast populations driven by a tradeoff in metal resistance.

Heterothallism in Saccharomyces cerevisiae isolates from nature: effect of HO locus on the mode of reproduction.

Understanding the evolution of sex and recombination, key factors in the evolution of life, is a major challenge in biology. Studies of reproduction strategies of natural populations are important to complement the theoretical and experimental models. Fungi with both sexual and asexual life cycles are an interesting system for understanding the evolution of sex. In a study of natural populations of yeast Saccharomyces cerevisiae, we found that the isolates are heterothallic, meaning their mating type is stable, while the general belief is that natural S. cerevisiae strains are homothallic (can undergo mating-type switching). Mating-type switching is a gene-conversion process initiated by a site-specific endonuclease HO; this process can be followed by mother-daughter mating. Heterothallic yeast can mate with unrelated haploids (amphimixis), or undergo mating between spores from the same tetrad (intratetrad mating, or automixis), but cannot undergo mother-daughter mating as homothallic yeasts can. Sequence analysis of HO gene in a panel of natural S. cerevisiae isolates revealed multiple mutations. Good correspondence was found in the comparison of population structure characterized using 19 microsatellite markers spread over eight chromosomes and the HO sequence. Experiments that tested whether the mating-type switching pathway upstream and downstream of HO is functional, together with the detected HO mutations, strongly suggest that loss of function of HO is the cause of heterothallism. Furthermore, our results support the hypothesis that clonal reproduction and intratetrad mating may predominate in natural yeast populations, while mother-daughter mating might not be as significant as was considered.

Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates the expression of PACAP in cultured tilapia astrocytes.

Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic hormone that is involved in numerous physiologic functions. The present study examines the presence and the functional significance of PACAP and its receptor in the brain and astrocytes of tilapia (Oreochromis mossambicus). This is the first demonstration of the full-length nucleotide sequence of tPACAP gene in tilapia pituitary, brain, and cultured astrocytes. Two cDNA variants of the growth hormone-releasing hormone (GHRH)-PACAP gene were identified in tilapia pituitary, brain, and cultured astrocytes as a result of exon skipping with a long form (271 bp) encoding both tPACAP(38) and tGHRH and a short form (166 bp) encoding only tPACAP(38). The short form was found to be more abundant in astrocytes. Addition of ovine PACAP(38) (1 nM) to cultured astrocytes significantly stimulated the expression of tPACAP(38) at 4 hrs, but the effect dropped after 8 hrs of treatment. By contrast, the expression of PACAP type I receptor (PAC(1)-R) mRNA in the astrocytes was not responsive to PACAP(38) treatment. The tPACAP(38) expression also was activated by the cAMP analog, dibutyryl-cAMP, in a dose-dependent manner. Adding high salinity (170 mM NaCl, 500 mOsm/kg osmolarity) to cultured medium substantially increased astroglial tPACAP(38) expression over 4 hrs to a level that was maintained for 16 hrs. This observation was not found when mannitol (270 mM) was supplemented as an osmolarity-enhancing agent (500 mOsm/ kg). Taken together, tPACAP expression in tilapia astrocytes was well regulated by exogenous PACAP, cAMP, and salinity and might be involved in the adaptation to high salinity when the fish is in a seawater environment.