Molecular Mechanism of DNA Topoisomerase I-Dependent rDNA Silencing: Sir2p Recruitment at Ribosomal Genes

Saccharomyces cerevisiae sir2Δ or top1Δ mutants exhibit similar phenotypes involving ribosomal DNA, including (i) loss of transcriptional silencing, resulting in non-coding RNA hyperproduction from cryptic RNA polymerase II promoters; (ii) alterations in recombination; and (iii) a general increase in histone acetylation. Given the distinct enzymatic activities of Sir2 and Top1 proteins, a histone deacetylase and a DNA topoisomerase, respectively, we investigated whether genetic and/or physical interactions between the two proteins could explain the shared ribosomal RNA genes (rDNA) phenotypes. We employed an approach of complementing top1Δ cells with yeast, human, truncated, and chimeric yeast/human TOP1 constructs and of assessing the extent of non-coding RNA silencing and histone H4K16 deacetylation. Our findings demonstrate that residues 115–125 within the yeast Top1p N-terminal domain are required for the complementation of the top1Δ rDNA phenotypes. In chromatin immunoprecipitation and co-immunoprecipitation experiments, we further demonstrate the physical interaction between Top1p and Sir2p. Our genetic and biochemical studies support a model whereby Top1p recruits Sir2p to the rDNA and clarifies a structural role of DNA topoisomerase I in the epigenetic regulation of rDNA, independent of its known catalytic activity

Characterization of glycan determinants that mediate recognition of the major Wuchereria bancrofti circulating antigen by diagnostic antibodies

The Global Program to Eliminate Lymphatic Filariasis (GPELF) relies heavily on a rapid diagnostic test (RDT) to a Wuchereria bancrofti circulating filarial antigen (Wb-CFA) to identify endemic areas and for determining when mass drug administration can stop. The antigen contains a carbohydrate epitope that is recognized by monoclonal antibody AD12. Og4C3, a monoclonal antibody that is used in a commercial ELISA for Wb-CFA recognizes the same moiety. Despite its diagnostic importance, little is known about the structure and function of this AD12 epitope . It is also present on other W. bancrofti glycoproteins and on glycoproteins of other filarial worms, but such antigens are not detected in the sera of individuals with most other filarial infections. We report here functional and biochemical analyses that shed light on the interaction between filarial glycoproteins and AD12 and/or Og4C3. Binding of these monoclonal antibodies to a mammalian glycan array suggests the reactive moiety has structural similarity to terminal β-d-glucuronic acid in a 1-3 linkage to other hexoses. However, sera collected from individuals with patent W. bancrofti infection had very low or undetectable serum antibodies to the GlcA-containing array glycans. Unlike other filarial glycoproteins, the Wb-CFA is relatively resistant to protease digestion by pronase and trypsin and completely resistant to the mucinase O-sialoglycoprotein endopeptidase (OSGE). The protease resistance of the Wb-CFA may contribute to its consistent detection in Wb-infected sera

Systems analysis-based assessment of post-treatment adverse events in lymphatic filariasis

BACKGROUND:Lymphatic filariasis (LF) is a neglected tropical disease, and the Global Program to Eliminate LF delivers mass drug administration (MDA) to 500 million people every year. Adverse events (AEs) are common after LF treatment. METHODOLOGY/PRINCIPAL FINDINGS:To better understand the pathogenesis of AEs, we studied LF-patients from a treatment trial. Plasma levels of many filarial antigens increased post-treatment in individuals with AEs, and this is consistent with parasite death. Circulating immune complexes were not elevated in these participants, and the classical complement cascade was not activated. Multiple cytokines increased after treatment in persons with AEs. A transcriptomic analysis was performed for nine individuals with moderate systemic AEs and nine matched controls. Differential gene expression analysis identified a significant transcriptional signature associated with post-treatment AEs; 744 genes were upregulated. The transcriptional signature was enriched for TLR and NF-κB signaling. Increased expression of seven out of the top eight genes upregulated in persons with AEs were validated by qRT-PCR, including TLR2. CONCLUSIONS/SIGNIFICANCE:This is the first global study of changes in gene expression associated with AEs after treatment of lymphatic filariasis. Changes in cytokines were consistent with prior studies and with the RNAseq data. These results suggest that Wolbachia lipoprotein is involved in AE development, because it activates TLR2-TLR6 and downstream NF-κB. Additionally, LPS Binding Protein (LBP, which shuttles lipoproteins to TLR2) increased post-treatment in individuals with AEs. Improved understanding of the pathogenesis of AEs may lead to improved management, increased MDA compliance, and accelerated LF elimination

In vivo functional analysis of the Dicistroviridae intergenic region internal ribosome entry sites

Some viral and cellular messages use an alternative mechanism to initiate protein synthesis that involves internal recruitment of the ribosome to an internal ribosome entry site (IRES). The Dicistroviridae intergenic regions (IGR) have been studied as model IRESs to understand the mechanism of IRES-mediated translation. In this study, the in vivo activity of IGR IRESs were compared. Our analysis demonstrates that Class I and II IGR IRESs have comparable translation efficiency in yeast and that Class II is significantly more active in mammalian cells. Furthermore, while Class II IGR IRES activity was enhanced in yeast grown at a higher temperature, temperature did not affect IGR IRES activity in mammalian cells. This suggests that Class II IRESs may not function optimally with yeast ribosomes. Examination of chimeric IGR IRESs, established that the IRES strength and temperature sensitivity are mediated by the ribosome binding domain. In addition, the sequence of the first translated codon is also an important determinant of IRES activity. Our findings provide us with a comprehensive overview of IGR IRES activities and allow us to begin to understand the differences between Classes I and II IGR IRESs

RPS25 is essential for translation initiation by the Dicistroviridae and hepatitis C viral IRESs

Most eukaryotic mRNAs are translated using a cap-dependent mechanism of translation. However, ∼10% of mammalian mRNAs initiate translation using a cap-independent mechanism that is not well understood. These mRNAs contain an internal ribosome entry site (IRES) located in the 5′ untranslated region. The cricket paralysis virus (CrPV) intergenic region IRES (IGR IRES) functions in yeast, mammals, and plants, and does not require any translation initiation factors. We used yeast genetics to understand how ribosomes are recruited directly to the mRNA by an IRES. We found that Rps25p has an essential role in CrPV IGR IRES activity in yeast and mammalian cells but not in cap-dependent translation. Purified 40S ribosomal subunits lacking Rps25 are unable to bind to the IGR IRES in vitro. The hepatitis C virus (HCV) IRES also requires Rps25, demonstrating the function of Rps25 is conserved across IRES types. Yeast strains lacking Rps25 exhibit only slight defects in global translation, readthrough, ribosome biogenesis, and programmed ribosomal frameshifting. This work is the first demonstration of a ribosomal protein that is specifically required for IRES-mediated translation initiation. Our findings provide us with the beginnings of a model for the molecular interactions of an IRES with the ribosome

Identification and characterization of Loa loa antigens responsible for cross-reactivity with rapid diagnostic tests for lymphatic filariasis.

The Global Program to Eliminate Lymphatic Filariasis (LF) relies on rapid diagnostic tests (RDTs) to determine where annual mass drug administration for LF is required and when it can be stopped. These tests detect a Wuchereria bancrofti glycoprotein in the blood of infected persons via a carbohydrate moiety recognized by the monoclonal antibodies AD12 and DH6.5. Loiasis cross-reactivity with LF RDTs has recently been recognized as a serious obstacle to LF elimination in loiasis-endemic areas. To better understand the nature of this cross-reactivity, we used the DH6.5 antibody to immunoaffinity purify Loa loa antigens from the sera of individuals with a positive RDT due to loiasis. Immunoblot analysis revealed many circulating AD12/DH6.5-reactive antigens, and proteomic analysis identified multiple L. loa proteins in LF RDT-positive loiasis sera. These included both secreted and somatic proteins, suggesting that they may be released by dying L. loa adult worms and/or microfilariae. Unlike the single high molecular weight W. bancrofti circulating filarial antigen that is reliably present in the blood of persons with bancroftian filariasis, reactive L. loa antigens appeared to be only transiently present in the blood of a subset of persons with loiasis. These key differences between the circulating antigens of W. bancrofti and L. loa can be used to differentiate positive results generated by both species and may lead to improved diagnostic tests for LF and loiasis