Local features determine Ty3 targeting frequency at RNA polymerase III transcription start sites.

Retroelement integration into host genomes affects chromosome structure and function. A goal of a considerable number of investigations is to elucidate features influencing insertion site selection. The Saccharomyces cerevisiae Ty3 retrotransposon inserts proximal to the transcription start sites (TSS) of genes transcribed by RNA polymerase III (RNAP3). In this study, differential patterns of insertion were profiled genome-wide using a random barcode-tagged Ty3. Saturation transposition showed that tRNA genes (tDNAs) are targeted at widely different frequencies even within isoacceptor families. Ectopic expression of Ty3 integrase (IN) showed that it localized to targets independent of other Ty3 proteins and cDNA. IN, RNAP3, and transcription factor Brf1 were enriched at tDNA targets with high frequencies of transposition. To examine potential effects of cis-acting DNA features on transposition, targeting was tested on high-copy plasmids with restricted amounts of 5' flanking sequence plus tDNA. Relative activity of targets was reconstituted in these constructions. Weighting of genomic insertions according to frequency identified an A/T-rich sequence followed by C as the dominant site of strand transfer. This site lies immediately adjacent to the adenines previously implicated in the RNAP3 TSS motif (CAA). In silico DNA structural analysis upstream of this motif showed that targets with elevated DNA curvature coincide with reduced integration. We propose that integration mediated by the Ty3 intasome complex (IN and cDNA) is subject to inputs from a combination of host factor occupancy and insertion site architecture, and that this results in the wide range of Ty3 targeting frequencies.

Functional genomics for the oleaginous yeast Yarrowia lipolytica.

Oleaginous yeasts are valuable systems for biosustainable production of hydrocarbon-based chemicals. Yarrowia lipolytica is one of the best characterized of these yeast with respect to genome annotation and flux analysis of metabolic processes. Nonetheless, progress is hampered by a dearth of genome-wide tools enabling functional genomics. In order to remedy this deficiency, we developed a library of Y. lipolytica insertion mutants via transposon mutagenesis. The Hermes DNA transposon was expressed to achieve saturation mutagenesis of the genome. Over 534,000 independent insertions were identified by next-generation sequencing. Poisson analysis of insertion density classified ~ 22% of genes as essential. As expected, most essential genes have homologs in Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the majority of those are also essential. As an obligate aerobe, Y. lipolytica has significantly more respiration - related genes that are classified as essential than do S. cerevisiae and S. pombe. Contributions of non-essential genes to growth in glucose and glycerol carbon sources were assessed and used to evaluate two recent genome-scale models of Y. lipolytica metabolism. Fluorescence-activated cell sorting identified mutants in which lipid accumulation is increased. Our findings provide insights into biosynthetic pathways, compartmentalization of enzymes, and distinct functions of paralogs. This functional genomic analysis of the oleaginous yeast Y. lipolytica provides an important resource for modeling, bioengineering, and design of synthetic minimalized strains of respiratory yeasts.

Control of yeast retrotransposons mediated through nucleoporin evolution.

Yeasts serve as hosts to several types of genetic parasites. Few studies have addressed the evolutionary trajectory of yeast genes that control the stable co-existence of these parasites with their host cell. In Saccharomyces yeasts, the retrovirus-like Ty retrotransposons must access the nucleus. We show that several genes encoding components of the yeast nuclear pore complex have experienced natural selection for substitutions that change the encoded protein sequence. By replacing these S. cerevisiae genes with orthologs from other Saccharomyces species, we discovered that natural sequence changes have affected the mobility of Ty retrotransposons. Specifically, changing the genetic sequence of NUP84 or NUP82 to match that of other Saccharomyces species alters the mobility of S. cerevisiae Ty1 and Ty3. Importantly, all tested housekeeping functions of NUP84 and NUP82 remained equivalent across species. Signatures of natural selection, resulting in altered interactions with viruses and parasitic genetic elements, are common in host defense proteins. Yet, few instances have been documented in essential housekeeping proteins. The nuclear pore complex is the gatekeeper of the nucleus. This study shows how the evolution of this large, ubiquitous eukaryotic complex can alter the replication of a molecular parasite, but concurrently maintain essential host functionalities regarding nucleocytoplasmic trafficking.

Nitrate-Regulated Glutaredoxins Control Arabidopsis Primary Root Growth.

Nitrogen is an essential soil nutrient for plants, and lack of nitrogen commonly limits plant growth. Soil nitrogen is typically available to plants in two inorganic forms: nitrate and ammonium. To better understand how nitrate and ammonium differentially affect plant metabolism and development, we performed transcriptional profiling of the shoots of ammonium-supplied and nitrate-supplied Arabidopsis (Arabidopsis thaliana) plants. Seven genes encoding class III glutaredoxins were found to be strongly and specifically induced by nitrate. RNA silencing of four of these glutaredoxin genes (AtGRXS3/4/5/8) resulted in plants with increased primary root length (approximately 25% longer than the wild type) and decreased sensitivity to nitrate-mediated inhibition of primary root growth. Increased primary root growth is also a well-characterized phenotype of many cytokinin-deficient plant lines. We determined that nitrate induction of glutaredoxin gene expression was dependent upon cytokinin signaling and that cytokinins could activate glutaredoxin gene expression independent of plant nitrate status. In addition, crosses between "long-root" cytokinin-deficient plants and "long-root" glutaredoxin-silenced plants generated hybrids that displayed no further increase in primary root length (i.e. epistasis). Collectively, these findings suggest that AtGRXS3/4/5/8 operate downstream of cytokinins in a signal transduction pathway that negatively regulates plant primary root growth in response to nitrate. This pathway could allow Arabidopsis to actively discriminate between different nitrogen sources in the soil, with the preferred nitrogen source, nitrate, acting to suppress primary root growth (vertical dimension) in concert with its well-characterized stimulatory effect on lateral root growth (horizontal dimension).

Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes.

Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5' to 3' exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs.

Ty3, a Position-specific Retrotransposon in Budding Yeast.

Long terminal repeat (LTR) retrotransposons constitute significant fractions of many eukaryotic genomes. Two ancient families are Ty1/Copia (Pseudoviridae) and Ty3/Gypsy (Metaviridae). The Ty3/Gypsy family probably gave rise to retroviruses based on the domain order, similarity of sequences, and the envelopes encoded by some members. The Ty3 element of Saccharomyces cerevisiae is one of the most completely characterized elements at the molecular level. Ty3 is induced in mating cells by pheromone stimulation of the mitogen-activated protein kinase pathway as cells accumulate in G1. The two Ty3 open reading frames are translated into Gag3 and Gag3-Pol3 polyprotein precursors. In haploid mating cells Gag3 and Gag3-Pol3 are assembled together with Ty3 genomic RNA into immature virus-like particles in cellular foci containing RNA processing body proteins. Virus-like particle Gag3 is then processed by Ty3 protease into capsid, spacer, and nucleocapsid, and Gag3-Pol3 into those proteins and additionally, protease, reverse transcriptase, and integrase. After haploid cells mate and become diploid, genomic RNA is reverse transcribed into cDNA. Ty3 integration complexes interact with components of the RNA polymerase III transcription complex resulting in Ty3 integration precisely at the transcription start site. Ty3 activation during mating enables proliferation of Ty3 between genomes and has intriguing parallels with metazoan retrotransposon activation in germ cell lineages. Identification of nuclear pore, DNA replication, transcription, and repair host factors that affect retrotransposition has provided insights into how hosts and retrotransposons interact to balance genome stability and plasticity.

Distinct signalling pathways and transcriptome response signatures differentiate ammonium- and nitrate-supplied plants.

Nitrogen is the only macronutrient that is commonly available to plants in both oxidized and reduced forms, mainly nitrate and ammonium. The physiological and molecular effects of nitrate supply have been well studied, but comparatively little is known about ammonium nutrition and its differential effects on cell function and gene expression. We have used a physiologically realistic hydroponic growth system to compare the transcriptomes and redox status of the roots of ammonium- and nitrate-supplied Arabidopsis thaliana plants. While approximately 60% of nitrogen-regulated genes displayed common responses to both ammonium and nitrate, significant 'nitrate-specific' and 'ammonium-specific' gene sets were identified. Pathways involved in cytokinin response and reductant generation/distribution were specifically altered by nitrate, while a complex biotic stress response and changes in nodulin gene expression were characteristic of ammonium-supplied plants. Nitrate supply was associated with a rapid decrease in H(2)O(2) production, potentially because of an increased export of reductant from the mitochondrial matrix. The underlying basis of the nitrate- and ammonium-specific patterns of gene expression appears to be different signals elaborated from each nitrogen source, including alterations in extracellular pH that are associated with ammonium uptake, downstream metabolites in the ammonium assimilation pathway, and the presence or absence of the nitrate ion.