Candida albicans repetitive elements display epigenetic diversity and plasticity

Transcriptionally silent heterochromatin is associated with repetitive DNA. It is poorly understood whether and how heterochromatin differs between different organisms and whether its structure can be remodelled in response to environmental signals. Here, we address this question by analysing the chromatin state associated with DNA repeats in the human fungal pathogen Candida albicans. Our analyses indicate that, contrary to model systems, each type of repetitive element is assembled into a distinct chromatin state. Classical Sir2-dependent hypoacetylated and hypomethylated chromatin is associated with the rDNA locus while telomeric regions are assembled into a weak heterochromatin that is only mildly hypoacetylated and hypomethylated. Major Repeat Sequences, a class of tandem repeats, are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Marker gene silencing assays and genome-wide RNA sequencing reveals that C. albicans heterochromatin represses expression of repeat-associated coding and non-coding RNAs. We find that telomeric heterochromatin is dynamic and remodelled upon an environmental change. Weak heterochromatin is associated with telomeres at 30?°C, while robust heterochromatin is assembled over these regions at 39?°C, a temperature mimicking moderate fever in the host. Thus in C. albicans, differential chromatin states controls gene expression and epigenetic plasticity is linked to adaptation

Characterization of the four genes encoding cytoplasmic ribosomal protein S15a in Arabidopsis thaliana

Eukaryotic cytosolic ribosomes are composed of two distinct subunits consisting of four individual ribosomal RNAs and, in Arabidopsis thaliana, 81 ribosomal proteins. Functional subunit assembly is dependent on the production of each ribosomal component. Arabidopsis thaliana r-protein genes exist in multi-gene families ranging in size from two to seven transcriptionally active members. The cytosolic RPS15a gene family consists of four members (RPS15aA, -C, -D and -F) that, at the amino acid level, share 87-100% identity. Using semi-quantitative RT-PCR I have shown that RPS15aC is not expressed and that transcript abundance differs both spatially and temporally among the remaining RPS15a genes in non-treated Arabidopsis tissues and in seedlings following a variety of abiotic stresses. A comprehensive analysis of the RPS15a 5' regulatory regions (RRs) using a series of deletion constructs was used to determine the minimal region required for gene expression and identify putative cis-regulatory elements. Transcription start site mapping using 5' RACE indicated multiple sites of initiation for RPS15aA and -F and only a single site for RPS15aD while all three genes contain a leader intron upstream of the start codon. Analysis of reporter gene activity in transgenic Arabidopsis containing a series of 5' RR deletion::GUS fusions showed that, similar to previous RT-PCR results, there was a trend for mitotically active tissues to stain for GUS activity. Putative cis-elements including the TELO box, PCNA Site II motif and pollen specific elements were identified. However, there was not always a clear correlation between the presence of a putative element and RPS15a transcript abundance or GUS activity. Although variation in transcriptional activity of each RPS15a gene has been observed, subcellular localization of both RPS15aA and -D in the nucleolus has been confirmed in planta by confocal microscopy. The results of this thesis research suggest while all three active RPS15a genes are transcriptionally regulated, additional post-transcriptional and/or translational regulation may be responsible for final RPS15a levels while differential isoform incorporation into ribosomal subunits may be the final point of r-protein regulation

Polyadenylation of ribosomal RNA in human cells

The addition of poly(A)-tails to RNA is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3′ ends of most nuclear-encoded mRNAs, but not to rRNAs. Contrarily, in prokaryotes and organelles, polyadenylation stimulates RNA degradation. Recently, polyadenylation of nuclear-encoded transcripts in yeast was reported to promote RNA degradation, demonstrating that polyadenylation can play a double-edged role for RNA of nuclear origin. Here we asked whether in human cells ribosomal RNA can undergo polyadenylation. Using both molecular and bioinformatic approaches, we detected non-abundant polyadenylated transcripts of the 18S and 28S rRNAs. Interestingly, many of the post-transcriptionally added tails were composed of heteropolymeric poly(A)-rich sequences containing the other nucleotides in addition to adenosine. These polyadenylated RNA fragments are most likely degradation intermediates, as primer extension (PE) analysis revealed the presence of distal fragmented molecules, some of which matched the polyadenylation sites of the proximal cleavage products revealed by oligo(dT) and circled RT–PCR. These results suggest the presence of a mechanism to degrade ribosomal RNAs in human cells, that possibly initiates with endonucleolytic cleavages and involves the addition of poly(A) or poly(A)-rich tails to truncated transcripts, similar to that which operates in prokaryotes and organelles

The Drosophila Neuroblasts: A Model System For Human Ribosomopathies

This dissertation describes the use of Drosophila neuroblasts (NBs) to model human ribosomopathies; the overall goal is to understand why specific stem cell and progenitor cell populations are the primary targets in nucleolar stress as seen in the ribosomopathies. Chapter 1 provides an overview of relevant literature. Chapter 2 describes nucleolar stress in Drosophila neuroblasts as a model for human ribosomopathies. For this, we induce nucleolar stress by using the UAS-GAL4 system to express RNAi that depletes Nopp140 transcripts, and we also employ homozygous, CRISPR-Cas9-generated Nopp140 gene disruptions with a systemic null phenotype (Nopp140-/-). Embryonic lethality was observed under RNAi depletion of Nopp140 as well as homozygous and heterozygous Nopp140 disruption. Larval lethality occurred at the second instar stage in Nopp140-/- line, similar to the previously generated complete Nopp140 deletion line, KO121. Larval brain development was severely impaired in Nopp140-/- larvae and in larvae that expressed neuron-specific RNAi that depletes Nopp140. The hypoplastic brain phenotype was due to reduction in NB populations as well as the proliferative capacity of the dividing NBs. While the majority of NB lineages in wild-type brains are at S-phase and proliferative at day 3 after larval hatching as indicated by EdU labeling assay, only the Mushroom Body (MB) NBs are at S-phase and proliferative in the Nopp140-/- larval brain. Furthermore, these MB NBs retained fibrillarin within their nucleoli, while fibrillarin redistributed to the nucleoplasm in the surrounding cells. Hence, we conclude that MB NBs are more resilient to nucleolar stress induced by the loss of Nopp140 compared to other neuroblast lineages. This finding strengthens the use of Drosophila neuroblasts as a model for the human ribosomopathies, and we hypothesize that different neuroblast lineages respond variably to nucleolar stress. Chapter 3 describes repeat polymorphisms in the Nopp140 gene that result in two Nopp140 alleles, Nopp140-Long and Nopp140-Short, that differ by exactly 96 bps within the central domain. We provide evidence showing preferential amplification of the Nopp140-Long allele compared to that of the Nopp140-Short allele, which we determined to be a PCR artefact, for reasons that remain unknown. Chapter 4 closes with conclusions and future studies

The post-translational methylation of arginine in the glycine arginine rich region of CHO nucleoin

Nucleolin is a nucleolar protein important for ribosome biogenesis. Nucleolin contains a conserved glycine arginine rich (GAR) domain near its carboxy terminus. GAR domains are defined by repeating tri-peptide, (arginine -glycine-glycine (RGG)) motifs. The arginine in the RGG motif is post-translationally modified by dimethylation on one of the two guanido nitrogens. Although arginine methylation was identified over 30 years ago, the function of this modification remains unknown. The GAR domain of nucleolin is important for nucleic acid binding and for nucleolar localization of nucleolin. This dissertation describes investigates possible in vivo interactions between nucleolin and other nucleolar proteins involved in ribosome biogenesis. This dissertation also addresses the possible roles that arginine dimethylation may play in the function of the GAR domain. A GAR truncated hamster nucleolin (DGAR) localizes to the nucleoplasm and not to the nucleoli of CHO cells. This version of nucleolin was subsequently used to probe the in vivo interactions of nucleolin with other non-ribosomal nucleolar proteins. In support of previous work, DGAR caused endogenous B23 to redistribute to the nucleoplasm, suggesting an in vivo interaction. Endogenous Nopp140 shows no redistribution in the presence of DGAR. The effect of DGAR on the nucleolar protein, fibrillarin, remains unclear. A redistribution of exogenously expressed wild type nucleolin from nucleoli to the nucleoplasm is observed when CHO cells are treated with the methyltransferase inhibitors 5-methyl 5¢ deoxythioadenosine (MTA) and 3-deaza-adenosine (DAA). This redistribution of nucleolin is reversible and independent of protein synthesis. The arginines in the RGG motifs of the GAR domain of hamster nucleolin were changed to lysine, and the fully substituted protein was not a substrate for the RGG specific protein arginine methyltransferase, Hmt1p. The lysine substituted protein bound nucleic acids and behaved in vivo in a manner indistinguishable from wild type nucleolin. These results indicate that methylation is not necessary for in vitro nucleic acid binding or for in vivo localization of nucleolin, and that the redistribution of nucleolin observed following MTA treatment is likely due to inhibiting the methylation of another nucleolar substrate

Relative expression of rRNA transcripts and 45S rDNA promoter methylation status are dysregulated in tumors in comparison with matched-normal tissues in breast cancer

Ribosomal RNA (rRNA) expression, one of the most important factors regulating ribosome production, is primarily controlled by a CG-rich 45S rDNA promoter. However, the DNA methylation state of the 45S rDNA promoter, as well as its effect on rRNA gene expression in types of human cancers is controversial. In the present study we analyzed the methylation status of the rDNA promoter (-380 to +53 bp) as well as associated rRNA expression levels in breast cancer cell lines and breast tumor-normal tissue pairs. We found that the aforementioned regulatory region was extensively methylated (74-96%) in all cell lines and in 68% (13/19 tumor-normal pairs) of the tumors. Expression levels of rRNA transcripts 18S, 28S, 5.8S and 45S external transcribed spacer (45S ETS) greatly varied in the breast cancer cell lines regardless of their methylation status. Analyses of rRNA transcript expression levels in the breast tumor and normal matched tissues showed no significant difference when normalized with TBP. On the other hand, using the geometric mean of the rRNA expression values (GM-rRNA) as reference enabled us to identify significant changes in the relative expression of rRNAs in the tissue samples. We propose GM-rRNA normalization as a novel strategy to analyze expression differences between rRNA transcripts. Accordingly, the 18S rRNA/GM-rRNA ratio was significantly higher whereas the 5.8S rRNA/GM-rRNA ratio was significantly lower in breast tumor samples than this ratio in the matched normal samples. Moreover, the 18S rRNA/GM-rRNA ratio was negatively correlated with the 45S rDNA promoter methylation level in the normal breast tissue samples, yet not in the breast tumors. Significant correlations observed between the expression levels of rRNA transcripts in the normal samples were lost in the tumor samples. We showed that the expression of rRNA transcripts may not be based solely on promoter methylation. Carcinogenesis may cause dysregulation of the correlation between spliced rRNA expression levels, possibly due to changes in rRNA processing, which requires further investigation

Targets Identification and Characterization of Tramp Comples in Mouse

RNA surveillance and degradation play an important role in the development and growth of organisms by eliminating RNA that contains errors, or that is no longer needed by the cell. In some processes, RNAs designated to be degraded are first labeled and then specifically recognized by the exosome, which performs the final degradation. One of the key labeling factors in yeast is the TRAMP complex, a three-subunit complex composed of Air2, Trf4 and Mtr4. Air2 facilitates TRAMP binding of RNA, Trf4 appends a 3′ end polyA tail and Mtr4 regulates the rate of adenylation and modifies RNA structures for ease of degradation through its RNA helicase activity. Though TRAMP has been studied extensively in yeast and its biochemistry and RNA recognition functions well delineated, the recent identification of TRAMP in mammals has made it possible for work to characterize mammalian TRAMP function in tissue culture cells. The mammalian transcriptome is much more complex and diverse compared to yeast in that a large portion is consisted of non-coding RNAs such as lncRNA, snoRNA, miRNA and so on. To understand the role of TRAMP complex in gene expression regulation, we knockdown the SKIV2L2 (mouse Mtr4) subunit and performed a polyA sequencing. With bioinformatics tools such as Bowtie, F-Seqq, MEDIPS, miRCompare, we constructed a data pipeline and identified several categories of targets including snoRNA, rRNA, miRNA and long-noncoding RNA in mouse cells. These data suggests that the targets of TRAMP are widely spread along the genome, and these targets involve a myriad of regulatory pathways. Understanding the relationship between the targets will help reveal the function and effect of this complex. Also, a more accurate and comprehensive target identification method remains to be developed

RNAi knockdown of Nopp140 induces the Minute syndrome in Drosophila: a potential model for the human Treacher Collins syndorme

Nopp140 is believed to play a chaperone function in pre-rRNA processing and ribosome assembly. Alternative splicing in Drosophila may yield two isoforms: the first, Nopp140-True, shares a conserved carboxy terminus with human Nopp140. The second contains a distinctive glycine and arginine rich (RGG) carboxy terminus typically found in vertebrate nucleolin. To further characterize Nopp140 in Drosophila, we expressed interfering RNAs in transgenic flies using the GAL4 inducible system. RT-PCR and Western blot analyses showed a loss of Nopp140 mRNA and protein in transgenic larvae. Resulting phenotypes fall within the Minute syndrome of Drosophila; they include slow growth, larval and pupal lethality, or variably deformed wings, legs, and tergites in surviving adults. Analogous to the Drosophila Minute syndrome is the human Treacher Collins syndrome which displays craniofacial birth defects due to the haplo-insufficiency in treacle, a nucleolar protein structurally related to Nopp140. We further show that severe over-expression of GFP-Nopp140-True or GFP-Nopp140-RGG in a GAL4-dependent manner is also embryonic and larval lethal. We could mutually complement RNAi-induced lethality and lethality caused by over-expression in trans-heterozygous flies. Expressing either of the two Nopp140 isoforms rescued RNAi-induced lethality, suggesting functional overlap between the two proteins