RNA Exosome & Chromatin: The Yin & Yang of Transcription: A Dissertation

Eukaryotic genomes can produce two types of transcripts: protein-coding and non-coding RNAs (ncRNAs). Cryptic ncRNA transcripts are bona fide RNA Pol II products that originate from bidirectional promoters, yet they are degraded by the RNA exosome. Such pervasive transcription is prevalent across eukaryotes, yet its regulation and function is poorly understood. We hypothesized that chromatin architecture at cryptic promoters may regulate ncRNA transcription. Nucleosomes that flank promoters are highly enriched in two histone marks: H3-K56Ac and the variant H2A.Z, which make nucleosomes highly dynamic. These histone modifications are present at a majority of promoters and their stereotypic pattern is conserved from yeast to mammals, suggesting their evolutionary importance. Although required for inducing a handful of genes, their contribution to steady-state transcription has remained elusive. In this work, we set out to understand if dynamic nucleosomes regulate cryptic transcription and how this is coordinated with the RNA exosome. Remarkably, we find that H3-K56Ac promotes RNA polymerase II occupancy at a large number of protein coding and noncoding loci, yet neither histone mark has a significant impact on steady state mRNA levels in budding yeast. Instead, broad effects of H3-K56Ac or H2A.Z on levels of both coding and ncRNAs are only revealed in the absence of the nuclear RNA exosome. We show that H2A.Z functions with H3-K56Ac in chromosome folding, facilitating formation of Chromosomal Interaction Domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA levels, perhaps in part by regulating higher order chromatin structures. Together, these chromatin factors achieve a balance of RNA exosome activity (yin; negative) and Pol II (yang; positive) to maintain transcriptional homeostasis

Alterations in urine, serum and brain metabolomic profiles exhibit sexual dimorphism during malaria disease progression

<p>Abstract</p> <p>Background</p> <p>Metabolic changes in the host in response to <it>Plasmodium </it>infection play a crucial role in the pathogenesis of malaria. Alterations in metabolism of male and female mice infected with <it>Plasmodium berghei </it>ANKA are reported here.</p> <p>Methods</p> <p>¹H NMR spectra of urine, sera and brain extracts of these mice were analysed over disease progression using Principle Component Analysis and Orthogonal Partial Least Square Discriminant Analysis.</p> <p>Results</p> <p>Analyses of overall changes in urinary profiles during disease progression demonstrate that females show a significant early post-infection shift in metabolism as compared to males. In contrast, serum profiles of female mice remain unaltered in the early infection stages; whereas that of the male mice changed. Brain metabolite profiles do not show global changes in the early stages of infection in either sex. By the late stages urine, serum and brain profiles of both sexes are severely affected. Analyses of individual metabolites show significant increase in lactate, alanine and lysine, kynurenic acid and quinolinic acid in sera of both males and females at this stage. Early changes in female urine are marked by an increase of ureidopropionate, lowering of carnitine and transient enhancement of asparagine and dimethylglycine. Several metabolites when analysed individually in sera and brain reveal significant changes in their levels in the early phase of infection mainly in female mice. Asparagine and dimethylglycine levels decrease and quinolinic acid increases early in sera of infected females. In brain extracts of females, an early rise in levels is also observed for lactate, alanine and glycerol, kynurenic acid, ureidopropionate and 2-hydroxy-2-methylbutyrate.</p> <p>Conclusions</p> <p>These results suggest that <it>P. berghei </it>infection leads to impairment of glycolysis, lipid metabolism, metabolism of tryptophan and degradation of uracil. Characterization of early changes along these pathways may be crucial for prognosis and better disease management. Additionally, the distinct sexual dimorphism exhibited in these responses has a bearing on the understanding of the pathophysiology of malaria.</p

RNA polymerase II depletion promotes transcription of alternative mRNA species

BACKGROUND: Cells respond to numerous internal and external stresses, such as heat, cold, oxidative stress, DNA damage, and osmotic pressure changes. In most cases, the primary response to stress is transcriptional induction of genes that assist the cells in tolerating the stress and facilitate the repair of the cellular damage. However, when the transcription machinery itself is stressed, responding by such standard mechanisms may not be possible. RESULTS: In this study, we demonstrate that depletion or inactivation of RNA polymerase II (RNAPII) changes the preferred polyadenylation site usage for several transcripts, and leads to increased transcription of a specific subset of genes. Surprisingly, depletion of RNA polymerase I (RNAPI) also promotes altered polyadenylation site usage, while depletion of RNA polymerase III (RNAPIII) does not appear to have an impact. CONCLUSIONS: Our results demonstrate that stressing the transcription machinery by depleting either RNAPI or RNAPII leads to a novel transcriptional response that results in induction of specific mRNAs and altered polyadenylation of many of the induced transcripts

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

MOESM1 of RNA polymerase II depletion promotes transcription of alternative mRNA species

Additional file 1: Figure S1. Confirmation of RPB2 Northern blot data by qRT-PCR. The schematic in panel A indicates locations of the primers that amplify the long RPB2 mRNA (purple; amplifies only the 4297 nt long form) and the short RPB2 mRNA (green; amplifies both 4010 nt and 4297 nt forms). Panel B presents the RT- PCR analysis of RNA isolated from the RPB1-FRB strain following nuclear depletion (+RAP). RNA levels were normalized to RNA from an intergenic region on chromosome V as done in Figure 5

Chromatin Dynamics and the RNA Exosome Function in Concert to Regulate Transcriptional Homeostasis

The histone variant H2A.Z is a hallmark of nucleosomes flanking promoters of protein-coding genes and is often found in nucleosomes that carry lysine 56-acetylated histone H3 (H3-K56Ac), a mark that promotes replication-independent nucleosome turnover. Here, we find that H3-K56Ac promotes RNA polymerase II occupancy at many protein-coding and noncoding loci, yet neither H3-K56Ac nor H2A.Z has a significant impact on steady-state mRNA levels in yeast. Instead, broad effects of H3-K56Ac or H2A.Z on RNA levels are revealed only in the absence of the nuclear RNA exosome. H2A.Z is also necessary for the expression of divergent, promoter-proximal noncoding RNAs (ncRNAs) in mouse embryonic stem cells. Finally, we show that H2A.Z functions with H3-K56Ac to facilitate formation of chromosome interaction domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA expression, perhaps in part by regulating higher-order chromatin structures

Age-Dependent Sex Bias in Clinical Malarial Disease in Hypoendemic Regions

<div><h3>Background and Objectives</h3><p>Experimental models show a male bias in murine malaria; however, extant literature on biases in human clinical malaria is inconclusive. Studies in hyperendemic areas document an absence of sexual dimorphism in clinical malaria. Data on sex bias in clinical malaria in hypoendemic areas is ambiguous—some reports note a male bias but do not investigate the role of differential mosquito exposure in that bias. Moreover, these studies do not examine whether the bias is age related. This study investigates whether clinical malaria in hypoendemic regions exhibits a sex bias and whether this bias is age-dependent. We also consider the role of vector exposure in this bias.</p> <h3>Methods</h3><p>Retrospective passive clinical malaria datasets (2002–2007) and active surveillance datasets (2000–2009) were captured for the hypoendemic Mumbai region in Western India. To validate findings, passive retrospective data was captured from a primary malaria clinic (2006–2007) in hypoendemic Rourkela (Eastern India). Data was normalized by determining percent slide-positivity rates (SPRs) for males and females, and parasite-positivity distributions were established across age groups. The Mann–Whitney test, Wilcoxon Signed Rank test, and Chi-square analysis were used to determine statistical significances.</p> <h3>Results</h3><p>In both the Mumbai and Rourkela regions, clinical malaria exhibited an adult male bias (p<0.01). A sex bias was not observed in children aged ≤10. Post-puberty, male SPRs were significantly greater than females SPRs (p<0.01). This adult male bias was observed for both vivax and falciparum clinical disease. Analysis of active surveillance data did not reveal an age or sex bias in the frequency of parasite positivity.</p> <h3>Conclusion</h3><p>This study demonstrates an age-dependent sex bias in clinical malaria in hypoendemic regions and enhanced incidence of clinical malaria in males following puberty. Possible roles of sex hormones, vector exposure, co-infections, and other factors in this enhanced susceptibility are discussed.</p> </div

Age distributions of SPRs of males and females in the clinical malaria datasets.

<p>A, B, Box plots showing the 25th and 75th percentiles, together with the median, with whiskers showing the minimum and maximum percent slide-positivity rates for males and females across age groups in the Mumbai region for clinical vivax (A) and falciparum (B) malaria. C,D, Box plots as above showing the minimum and maximum percent slide-positivity rates for males and females across age groups in Rourkela for clinical vivax (C) and falciparum (D) malaria. Statistically significant values obtained by the Mann–Whitney test are indicated.</p

Age distributions of differences between male and female SPRs in the clinical malaria and active surveillance datasets in the Mumbai region.

<p>A,C, Box plot showing the 25th and 75th percentiles, together with the median, with whiskers showing the minimum and maximum difference in the percent slide-positivity rates between males and females across age groups in the clinical malaria dataset for vivax (A) and falciparum (C) malaria. B,D, Box plots as in A showing the difference in the percent slide-positivity rates between males and females across age groups who tested positive for <i>P. vivax</i> (B) and <i>P. falciparum</i> (D) in the active surveillance programme. Data were compared with the difference of male/female SPRs expected under the hypothesis of neutrality (0, red line) and were analyzed with the Mann–Whitney test. Statistically significant values are shown in black. Numbers in red indicate statistically significant p values obtained by Wilcoxon Signed Rank test under the hypothesis that the median of the group did not differ significantly from zero. The test could not be applied to the falciparum data in the active surveillance dataset.</p