Thiamine Pyrophosphate Riboswitches Are Targets for the Antimicrobial Compound Pyrithiamine

SummaryThiamine metabolism genes are regulated in numerous bacteria by a riboswitch class that binds the coenzyme thiamine pyrophosphate (TPP). We demonstrate that the antimicrobial action of the thiamine analog pyrithiamine (PT) is mediated by interaction with TPP riboswitches in bacteria and fungi. For example, pyrithiamine pyrophosphate (PTPP) binds the TPP riboswitch controlling the tenA operon in Bacillus subtilis. Expression of a TPP riboswitch-regulated reporter gene is reduced in transgenic B. subtilis or Escherichia coli when grown in the presence of thiamine or PT, while mutant riboswitches in these organisms are unresponsive to these ligands. Bacteria selected for PT resistance bear specific mutations that disrupt ligand binding to TPP riboswitches and derepress certain TPP metabolic genes. Our findings demonstrate that riboswitches can serve as antimicrobial drug targets and expand our understanding of thiamine metabolism in bacteria

Control of mRNA Splicing by Intragenic RNA Activators of Stress Signaling: Potential Implications for Human Disease

A critical step in the cellular stress response is transient activation of the RNA-dependent protein kinase PKR by double-helical RNA, resulting in down-regulation of protein synthesis through phosphorylation of the α chain of translation initiation factor eIF2, a major PKR substrate. However, intragenic elements of 100–200 nucleotides in length within primary transcripts of cellular genes, exemplified by the tumor necrosis factor (TNF)-α gene and fetal and adult globin genes, are capable of forming RNA structures that potently activate PKR and thereby strongly enhance mRNA splicing efficiency. By inducing nuclear eIF2α phosphorylation, these PKR activator elements enable highly efficient early spliceosome assembly yet do not impair translation of the mature spliced mRNA. The TNF-α RNA activator of PKR folds into a compact pseudoknot that is highly conserved within the phylogeny. Upon excision of β-globin first intron, the RNA activator of PKR, located in exon 1, is silenced through strand displacement by a short sequence within exon 2, restricting thereby the ability to activate PKR to the splicing process without impeding subsequent synthesis of β-globin essential for survival. This activator/silencer mechanism likewise controls splicing of α-globin pre-mRNA, but the exonic locations of PKR activator and silencer sequences are reversed, demonstrating evolutionary flexibility. Impaired splicing efficiency may underlie numerous human β-thalassemia mutations that map to the β-globin RNA activator of PKR or its silencer. Even where such mutations change the encoded amino acid sequence during subsequent translation, they carry the potential of first impairing PKR-dependent mRNA splicing or shutoff of PKR activation needed for optimal translation

A long noncoding RNA promotes parasite differentiation in African trypanosomes

Copyright © 2022 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC)The parasite Trypanosoma brucei causes African sleeping sickness that is fatal to patients if untreated. Parasite differentiation from a replicative slender form into a quiescent stumpy form promotes host survival and parasite transmission. Long noncoding RNAs (lncRNAs) are known to regulate cell differentiation in other eukaryotes. To determine whether lncRNAs are also involved in parasite differentiation, we used RNA sequencing to survey the T. brucei genome, identifying 1428 previously uncharacterized lncRNA genes. We find that grumpy lncRNA is a key regulator that promotes parasite differentiation into the quiescent stumpy form. This function is promoted by a small nucleolar RNA encoded within the grumpy lncRNA. snoGRUMPY binds to messenger RNAs of at least two stumpy regulatory genes, promoting their expression. grumpy overexpression reduces parasitemia in infected mice. Our analyses suggest that T. brucei lncRNAs modulate parasite-host interactions and provide a mechanism by which grumpy regulates cell differentiation in trypanosomes.This work was supported in part by Fundação para a Ciência e Tecnologia (FCT) grant, awarded to F.G. and entitled “Long noncoding RNAs as new diagnostic biomarkers for African Sleeping sickness” (PTDC/DTPEPI/7099/2014, start date: 1 January 2016, end date: 31 December 2018); also by Howard Hughes Medical Institute International Early Career Scientist Program (project title: “How parasites use epigenetics to evade host defenses,” project no. 55007419, start date: 1 February 2012, end date: 31 January 2017); and by the European Research Council (project title: “Exploring the hidden life of African trypanosomes: parasite fat tropism and implications for the disease,” project no. 771714, start date: 1 August 2018, end date: 31 January 2024), both awarded to L.M.F. The project leading to these results have received funding from “la Caixa” Foundation under the agreement LCF/PR/HR20/52400019 [project title: “Mechanism and function of epitranscriptomic poly(A) tail modifications in African trypanosomes,” project no. HR20-00361, start date: 1 March 2021, end date: 29 February 2024]. L.M.F. is supported by FCT (IF/01050/2014, project title: “Molecular basis for the efficient biology of trypanossome parasitism,” start date: 1 January 2015, end date: 31 December 2019) and by CEEC institutional program (CEECINST/00110/2018, start date: 1 January 2020, end date: 14 December 2020). C.N. acknowledges the support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya. S. Michaeli acknowledges the support of the Israel Science Foundation (ref. 1959/20) from October 2020 to October 2025, entitled “Functional analysis of rRNA processing and the role of rRNA modification for specialized translation in the two life stages of trypanosomes” and U.S. Binational Science Foundation (ref. 2015/219) from October 2015 to October 2019, entitled “The role and mechanism of RNA pseudo-uridylation and sugar methylation (Nm) during the developmental cycle of trypanosomes.” The work done in A.D.’s laboratory was supported by National Science Center SONATA BIS grant, entitled “Non-canonical RNA tailing and other post-transcriptional regulatory mechanisms in T cell-mediated adaptive immunity” (proposal ID: 492777, agreement no: UMO-2020/38/E/NZ2/00372, start date: 22 March 2021, end date: 21 March 2026); National Science Center OPUS grant, entitled “Analysis of the role of cytoplasmic polyadenylation in the regulation of the innate immune response” (proposal ID: 443521, agreement no.: UMO-2019/33/B/NZ2/01773, start date: 2 March 2020, end date: 1 March 2023); and European Union’s Horizon 2020 (H2020-WIDESPREAD-03-2017)–ERAChair, entitled “MOlecular Signaling in Health and Disease - Interdisciplinary Centre of Excellence” (acronym: MOSaIC, agreement no.: 810425, implementation period: start date: 1 November 2018, end date: 31 October 2023).info:eu-repo/semantics/publishedVersio

Lytic Reactivation of the Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) Is Accompanied by Major Nucleolar Alterations

The nucleolus is a subnuclear compartment whose primary function is the biogenesis of ribosomal subunits. Certain viral infections affect the morphology and composition of the nucleolar compartment and influence ribosomal RNA (rRNA) transcription and maturation. However, no description of nucleolar morphology and function during infection with Kaposi’s sarcoma-associated herpesvirus (KSHV) is available to date. Using immunofluorescence microscopy, we documented extensive destruction of the nuclear and nucleolar architecture during the lytic reactivation of KSHV. This was manifested by the redistribution of key nucleolar proteins, including the rRNA transcription factor UBF. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Significantly, the redistribution of UBF was dependent on viral DNA replication or late viral gene expression. No significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found by RT-qPCR and Northern blot analysis. Furthermore, fluorescent in situ hybridization (FISH), combined with immunofluorescence, revealed an overlap between Fibrillarin and internal transcribed spacer 1 (ITS1), which represents the primary product of the pre-rRNA, suggesting that the processing of rRNA proceeds during lytic reactivation. Finally, small changes in the levels of pseudouridylation (Ψ) and 2′-O-methylation (Nm) were documented across the rRNA; however, none were localized to the functional domain. Taken together, our results suggest that despite dramatic changes in the nucleolar organization, rRNA transcription and processing persist during lytic reactivation of KSHV. Whether the observed nucleolar alterations favor productive infection or signify cellular anti-viral responses remains to be determined

Genome instability drives epistatic adaptation in the human pathogen Leishmania

International audienceHow genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania , which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2′- O -methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer

Exosome secretion affects social motility in <i>Trypanosoma brucei</i>

<div><p>Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite <i>Trypanosoma brucei</i>, exosome secretion is induced by stress that affects <i>trans</i>-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, <i>Vps36</i>, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites.</p></div

A single pseudouridine on rRNA regulates ribosome structure and function in the mammalian parasite Trypanosoma brucei

Abstract Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal. A single knock-out of a snoRNA guiding Ψ530 on H69 altered the composition of the 80S monosome. These changes specifically affected the translation of only a subset of proteins. This study correlates a single site Ψ modification with changes in ribosomal protein stoichiometry, supported by a high-resolution cryo-EM structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins

Exosome secretion affects social motility in <i>Trypanosoma brucei</i> - Fig 5

<p><b>(A) Immunogold TEM analysis demonstrating the location ZC3H41 in ILVs within MVBs.</b> Cells carrying the <i>SmD1</i> silencing construct, and the YFP-VPS36 construct were used. <b>(a, b)</b> un-induced (-Tet) or <b>(c-g)</b> silenced for 40 hrs. (+Tet) were used to prepare Cryo-EM sections, and were subjected to immunogold analysis. Imaging was performed using antibody to ZC3H41. <b>(B)</b> as in <b>(A)</b>, but using anti-GFP antibody to detect the VPS36. The scale bars are indicated.</p

Exosome secretion repels the migration of wild-type cells.

<p><b>Cells (~10</b>^<b>7</b><b>) were plated on semi solid agar containing tetracycline at a distance of 2.5 cm from each other.</b> The <i>SmD1 and SmD1/Vps36</i> silenced cells were plated on plates containing tetracycline. Pattern formation was analyzed 2 days after plating. The parasites from the plates were blotted, and the blot was stained with Ponceau and then reacted with anti-GPEET and anti-EP. The distance between the wild-type colony and the adjacent colony is presented. The statistical analysis represents the mean ± s.e.m **P< 0.01 comparing the distance between wild-type to wild-type, wild-type to <i>SmD1and</i> wild-type to <i>SmD1/Vps36</i> silenced cells based on three independent experiments.</p