A recently evolved transcriptional network controls biofilm development in Candida albicans.

A biofilm is an organized, resilient group of microbes in which individual cells acquire properties, such as drug resistance, that are distinct from those observed in suspension cultures. Here, we describe and analyze the transcriptional network controlling biofilm formation in the pathogenic yeast Candida albicans, whose biofilms are a major source of medical device-associated infections. We have combined genetic screens, genome-wide approaches, and two in vivo animal models to describe a master circuit controlling biofilm formation, composed of six transcription regulators that form a tightly woven network with ∼1,000 target genes. Evolutionary analysis indicates that the biofilm network has rapidly evolved: genes in the biofilm circuit are significantly weighted toward genes that arose relatively recently with ancient genes being underrepresented. This circuit provides a framework for understanding many aspects of biofilm formation by C. albicans in a mammalian host. It also provides insights into how complex cell behaviors can arise from the evolution of transcription circuits.

Evolution of yeast noncoding RNAs reveals an alternative mechanism for widespread intron loss.

The evolutionary forces responsible for intron loss are unresolved. Whereas research has focused on protein-coding genes, here we analyze noncoding small nucleolar RNA (snoRNA) genes in which introns, rather than exons, are typically the functional elements. Within the yeast lineage exemplified by the human pathogen Candida albicans, we find--through deep RNA sequencing and genome-wide annotation of splice junctions--extreme compaction and loss of associated exons, but retention of snoRNAs within introns. In the Saccharomyces yeast lineage, however, we find it is the introns that have been lost through widespread degeneration of splicing signals. This intron loss, perhaps facilitated by innovations in snoRNA processing, is distinct from that observed in protein-coding genes with respect to both mechanism and evolutionary timing.

Genetics and molecular biology in Candida albicans.

Candida albicans is an opportunistic fungal pathogen of humans. Although a normal part of our gastrointestinal flora, C. albicans has the ability to colonize nearly every human tissue and organ, causing serious, invasive infections. In this chapter we describe current methodologies used in molecular genetic studies of this organism. These techniques include rapid sequential gene disruption, DNA transformation, RNA isolation, epitope tagging, and chromatin immunoprecipitation. The ease of these techniques, combined with the high-quality C. albicans genome sequences now available, have greatly facilitated research into this important pathogen. Candida albicans is a normal resident of the human gastrointestinal tract; it is also the most common fungal pathogen of humans, causing both mucosal and systemic infections, particularly in immune compromised patients. C. albicans and Saccharomyces cerevisiae last shared a common ancestor more than 900 million years ago; in terms of conserved coding sequences, the two species are approximately as divergent as fish and humans. Although C. albicans and S. cerevisiae share certain core features, they also exhibit many significant differences. This is not surprising as C. albicans has the ability to survive in nearly every niche of a mammalian host, a property not shared by S. cerevisiae. Research into C. albicans is important in its own right, particularly with regards to its ability to cause disease in humans; in addition, comparison with S. cerevisiae can reveal important insights into evolutionary processes. Many of the methodologies developed for use in S. cerevisiae have been adapted for C. albicans, and we describe some of the most common. Although alternative procedures are described in the literature, we have found those described below to be the most convenient. Because the C. albicans parasexual cycle is cumbersome to use in the laboratory, genetics in this organism has been based almost entirely on directed mutations. Because the organism is diploid, creating a deletion mutant requires two rounds of gene disruption. We describe a rapid method for creating sequential disruptions, one which can be scaled up to create large collections of C. albicans deletion mutants. We also describe a series of additional techniques including DNA transformation, mRNA isolation, epitope tagging, and chromatin immunoprecipitation (ChIP). The ease of these techniques, combined with the high-quality C. albicans genome sequences now available, has greatly increased the quality and pace of research into this important pathogen.

The transcriptomes of two heritable cell types illuminate the circuit governing their differentiation.

The differentiation of cells into distinct cell types, each of which is heritable for many generations, underlies many biological phenomena. White and opaque cells of the fungal pathogen Candida albicans are two such heritable cell types, each thought to be adapted to unique niches within their human host. To systematically investigate their differences, we performed strand-specific, massively-parallel sequencing of RNA from C. albicans white and opaque cells. With these data we first annotated the C. albicans transcriptome, finding hundreds of novel differentially-expressed transcripts. Using the new annotation, we compared differences in transcript abundance between the two cell types with the genomic regions bound by a master regulator of the white-opaque switch (Wor1). We found that the revised transcriptional landscape considerably alters our understanding of the circuit governing differentiation. In particular, we can now resolve the poor concordance between binding of a master regulator and the differential expression of adjacent genes, a discrepancy observed in several other studies of cell differentiation. More than one third of the Wor1-bound differentially-expressed transcripts were previously unannotated, which explains the formerly puzzling presence of Wor1 at these positions along the genome. Many of these newly identified Wor1-regulated genes are non-coding and transcribed antisense to coding transcripts. We also find that 5' and 3' UTRs of mRNAs in the circuit are unusually long and that 5' UTRs often differ in length between cell-types, suggesting UTRs encode important regulatory information and that use of alternative promoters is widespread. Further analysis revealed that the revised Wor1 circuit bears several striking similarities to the Oct4 circuit that specifies the pluripotency of mammalian embryonic stem cells. Additional characteristics shared with the Oct4 circuit suggest a set of general hallmarks characteristic of heritable differentiation states in eukaryotes.

Evolution of small nuclear RNAs in S. cerevisiae, C. albicans, and other hemiascomycetous yeasts.

The spliceosome is a large, dynamic ribonuclear protein complex, required for the removal of intron sequences from newly synthesized eukaryotic RNAs. The spliceosome contains five essential small nuclear RNAs (snRNAs): U1, U2, U4, U5, and U6. Phylogenetic comparisons of snRNAs from protists to mammals have long demonstrated remarkable conservation in both primary sequence and secondary structure. In contrast, the snRNAs of the hemiascomycetous yeast Saccharomyces cerevisiae have highly unusual features that set them apart from the snRNAs of other eukaryotes. With an emphasis on the pathogenic yeast Candida albicans, we have now identified and compared snRNAs from newly sequenced yeast genomes, providing a perspective on spliceosome evolution within the hemiascomycetes. In addition to tracing the origins of previously identified snRNA variations present in Saccharomyces cerevisiae, we have found numerous unexpected changes occurring throughout the hemiascomycetous lineages. Our observations reveal interesting examples of RNA and protein coevolution, giving rise to altered interaction domains, losses of deeply conserved snRNA-binding proteins, and unique snRNA sequence changes within the catalytic center of the spliceosome. These same yeast lineages have experienced exceptionally high rates of intron loss, such that modern hemiascomycetous genomes contain introns in only approximately 5% of their genes. Also, the splice site sequences of those introns that remain adhere to an unusually strict consensus. Some of the snRNA variations we observe may thus reflect the altered intron landscape with which the hemiascomycetous spliceosome must contend.

Computational and experimental approaches double the number of known introns in the pathogenic yeast Candida albicans.

Candida albicans is the most common fungal pathogen of humans. Frequently found as a commensal within the digestive tracts of healthy individuals, C. albicans is an opportunistic pathogen that causes a wide variety of clinical syndromes in immuno-compromised individuals. A comprehensive annotation of the C. albicans genome sequence was recently published. Because many C. albicans coding sequences are interrupted by introns, proper intron annotation is essential for the accurate definition of genes in this pathogen. Intron annotation is also important for identifying potential targets of splicing regulation, a common mechanism of gene control in eukaryotes. In this study, we report an improved annotation of C. albicans introns. In addition to correcting the existing intron annotations, 25% of which were incorrect, we have used novel computational and experimental approaches to identify new introns, bringing the total to 415, almost double the number previously known. Our identification methods focus primarily on intron features rather than protein-coding features, overcoming biases of traditional intron annotation methods. Introns are not randomly distributed in C. albicans, and are over-represented in genes involved in specific cellular processes, such as splicing, translation, and mitochondrial respiration. This nonrandom distribution suggests functional roles for these introns, and we demonstrate that splicing of two transcripts whose introns have unusual sequence features is responsive to environmental factors.

mRNA surveillance of expressed pseudogenes in C. elegans.

Messenger RNAs (mRNAs) that contain premature translation termination codons (PTCs) are targeted for rapid degradation in all eukaryotes tested. The mechanisms of nonsense-mediated mRNA decay (NMD) have been described in considerable detail, but the biological roles of NMD in wild-type organisms are poorly understood. mRNAs of wild-type organisms known to be degraded by NMD ("natural targets" of NMD) include by-products of regulated alternative splicing, out-of-frame mRNAs derived from unproductive gene rearrangements, cytoplasmic pre-mRNAs, endogenous retroviral and transposon RNAs, and mRNAs having upstream open reading frames or other unusual sequence features. NMD may function to eliminate aberrant PTC-containing mRNAs in order to protect cells from expression of potentially deleterious truncated proteins. Pseudogenes are nonfunctional genes or gene fragments that accumulate mutations through genetic drift. Such mutations will often introduce shifts of reading frame and/or PTCs, and mRNAs of expressed pseudogenes may thus be substrates of NMD. We demonstrate that mRNAs expressed from C. elegans pseudogenes are degraded by NMD and discuss possible implications for both mRNA surveillance and protein evolution. We describe an expressed pseudogene that encodes a small nucleolar RNA (snoRNA) within an intron and suggest this represents an evolutionary intermediate between snoRNA-encoding host genes that do or do not encode proteins.