Comparative Genomics Reveals Two Novel RNAi Factors in Trypanosoma brucei and Provides Insight into the Core Machinery

The introduction ten years ago of RNA interference (RNAi) as a tool for molecular exploration in Trypanosoma brucei has led to a surge in our understanding of the pathogenesis and biology of this human parasite. In particular, a genome-wide RNAi screen has recently been combined with next-generation Illumina sequencing to expose catalogues of genes associated with loss of fitness in distinct developmental stages. At present, this technology is restricted to RNAi-positive protozoan parasites, which excludes T. cruzi, Leishmania major, and Plasmodium falciparum. Therefore, elucidating the mechanism of RNAi and identifying the essential components of the pathway is fundamental for improving RNAi efficiency in T. brucei and for transferring the RNAi tool to RNAi-deficient pathogens. Here we used comparative genomics of RNAi-positive and -negative trypanosomatid protozoans to identify the repertoire of factors in T. brucei. In addition to the previously characterized Argonaute 1 (AGO1) protein and the cytoplasmic and nuclear Dicers, TbDCL1 and TbDCL2, respectively, we identified the RNA Interference Factors 4 and 5 (TbRIF4 and TbRIF5). TbRIF4 is a 3′-5′ exonuclease of the DnaQ superfamily and plays a critical role in the conversion of duplex siRNAs to the single-stranded form, thus generating a TbAGO1-siRNA complex required for target-specific cleavage. TbRIF5 is essential for cytoplasmic RNAi and appears to act as a TbDCL1 cofactor. The availability of the core RNAi machinery in T. brucei provides a platform to gain mechanistic insights in this ancient eukaryote and to identify the minimal set of components required to reconstitute RNAi in RNAi-deficient parasites

Genome and Transcriptome Analysis of the Food-Yeast Candida utilis

The industrially important food-yeast Candida utilis is a Crabtree effect-negative yeast used to produce valuable chemicals and recombinant proteins. In the present study, we conducted whole genome sequencing and phylogenetic analysis of C. utilis, which showed that this yeast diverged long before the formation of the CUG and Saccharomyces/Kluyveromyces clades. In addition, we performed comparative genome and transcriptome analyses using next-generation sequencing, which resulted in the identification of genes important for characteristic phenotypes of C. utilis such as those involved in nitrate assimilation, in addition to the gene encoding the functional hexose transporter. We also found that an antisense transcript of the alcohol dehydrogenase gene, which in silico analysis did not predict to be a functional gene, was transcribed in the stationary-phase, suggesting a novel system of repression of ethanol production. These findings should facilitate the development of more sophisticated systems for the production of useful reagents using C. utilis

Tinkering Evolution of Post-Transcriptional RNA Regulons: Puf3p in Fungi as an Example

Genome-wide studies of post-transcriptional mRNA regulation in model organisms indicate a “post-transcriptional RNA regulon” model, in which a set of functionally related genes is regulated by mRNA–binding RNAs or proteins. One well-studied post-transcriptional regulon by Puf3p functions in mitochondrial biogenesis in budding yeast. The evolution of the Puf3p regulon remains unclear because previous studies have shown functional divergence of Puf3p regulon targets among yeast, fruit fly, and humans. By analyzing evolutionary patterns of Puf3p and its targeted genes in forty-two sequenced fungi, we demonstrated that, although the Puf3p regulon is conserved among all of the studied fungi, the dedicated regulation of mitochondrial biogenesis by Puf3p emerged only in the Saccharomycotina clade. Moreover, the evolution of the Puf3p regulon was coupled with evolution of codon usage bias in down-regulating expression of genes that function in mitochondria in yeast species after genome duplication. Our results provide a scenario for how evolution like a tinker exploits pre-existing materials of a conserved post-transcriptional regulon to regulate gene expression for novel functional roles

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

Quantitative Microscopy Reveals Centromeric Chromatin Stability, Size, and Cell Cycle Mechanisms to Maintain Centromere Homeostasis

The deposited item is a book chapter and is part of the series "Centromeres and Kinetochores" published by the publisher Springer Verlag. The deposited book chapter is a post-print version and has been submitted to peer reviewing. There is no public supplementary material available for this publication. This publication hasn't any creative commons license associated.Centromeres are chromatin domains specified by nucleosomes containing the histone H3 variant, CENP-A. This unique centromeric structure is at the heart of a strong self-templating epigenetic mechanism that renders centromeres heritable. We review how specific quantitative microscopy approaches have contributed to the determination of the copy number, architecture, size, and dynamics of centromeric chromatin and its associated centromere complex and kinetochore. These efforts revealed that the key to long-term centromere maintenance is the slow turnover of CENP-A nucleosomes, a critical size of the chromatin domain and its cell cycle-coupled replication. These features come together to maintain homeostasis of a chromatin locus that directs its own epigenetic inheritance and facilitates the assembly of the mitotic kinetochore.There are no funders and sponsors indicated explicitly in the document.info:eu-repo/semantics/publishedVersio

Evolutionary Lessons from Species with Unique Kinetochores

The kinetochore is the multi-protein complex that drives chromosome segregation in eukaryotes. It assembles onto centromeric DNA and mediates attachment to spindle microtubules. Kinetochore research over the last several decades has been focused on a few animal and fungal model organisms, which revealed a detailed understanding of the composition and organization of their kinetochores. Yet, these traditional model organisms represent only a small fraction of all eukaryotes. To gain insights into the actual degree of kinetochore diversity, it is critical to extend these studies to nontraditional model organisms from evolutionarily distant lineages. In this chapter, we review the current knowledge of kinetochores across diverse eukaryotes with an emphasis on variations that arose in nontraditional model organisms. In addition, we also review the literature on species, in which the subcellular localization of kinetochores has changed from the nucleoplasm to the nuclear membrane. Finally, we speculate on the organization of the chromosome segregation machinery in an early eukaryotic ancestor to gain insights into fundamental principles of the chromosome segregation machinery, which are common to all eukaryotes

Evolutionary Lessons from Species with Unique Kinetochores

The kinetochore is the multi-protein complex that drives chromosome segregation in eukaryotes. It assembles onto centromeric DNA and mediates attachment to spindle microtubules. Kinetochore research over the last several decades has been focused on a few animal and fungal model organisms, which revealed a detailed understanding of the composition and organization of their kinetochores. Yet, these traditional model organisms represent only a small fraction of all eukaryotes. To gain insights into the actual degree of kinetochore diversity, it is critical to extend these studies to nontraditional model organisms from evolutionarily distant lineages. In this chapter, we review the current knowledge of kinetochores across diverse eukaryotes with an emphasis on variations that arose in nontraditional model organisms. In addition, we also review the literature on species, in which the subcellular localization of kinetochores has changed from the nucleoplasm to the nuclear membrane. Finally, we speculate on the organization of the chromosome segregation machinery in an early eukaryotic ancestor to gain insights into fundamental principles of the chromosome segregation machinery, which are common to all eukaryotes