Cryptic Transcription Mediates Repression of Subtelomeric Metal Homeostasis Genes

Nonsense-mediated mRNA decay (NMD) prevents the accumulation of transcripts bearing premature termination codons. Here we show that Saccharomyces cerevisiae NMD mutants accumulate 5′–extended RNAs (CD-CUTs) of many subtelomeric genes. Using the subtelomeric ZRT1 and FIT3 genes activated in response to zinc and iron deficiency, respectively, we show that transcription of these CD-CUTs mediates repression at the bona fide promoters, by preventing premature binding of RNA polymerase II in conditions of metal repletion. Expression of the main ZRT1 CD-CUT is controlled by the histone deacetylase Rpd3p, showing that histone deacetylases can regulate expression of genes through modulation of the level of CD-CUTs. Analysis of binding of the transcriptional activator Zap1p and insertion of transcriptional terminators upstream from the Zap1p binding sites show that CD-CUT transcription or accumulation also interferes with binding of the transcriptional activator Zap1p. Consistent with this model, overexpressing Zap1p or using a constitutively active version of the Aft1p transcriptional activator rescues the induction defect of ZRT1 and FIT3 in NMD mutants. These results show that cryptic upstream sense transcription resulting in unstable transcripts degraded by NMD controls repression of a large number of genes located in subtelomeric regions, and in particular of many metal homeostasis genes

Etude fonctionnelle du dégradosome d'Escherichia coli et régulation de la RNase E par phosphorylation

Dr Gutierrez, Dr Putzer, Dr Hajnsdorf, Dr FeldenThe RNA degradosome of E. coli is a multienzymatic complex involved in mRNA degradation. It is composed of four major proteins: RNase E, PNPase, RhlB and enolase. In our first study, we examined the interaction between RNase E and RhlB, and other DEAD-box RNA helicases. We showed the existence of two distinct helicase binding sites: one that is RhlB specific and another that binds SrmB, RhlE or CsdA. A second study was aimed at elucidating the role of enolase in the degadosome. Considering these results, the hypothesis that enolase has a general role in mRNA degradation seems unlikely. Studies on inhibition of the RNase E due to phosphorylation of its non-catalytic domain by a protein kinase from bacteriophage T7 led to mechanistic models for the control of RNase E activity. In addition, these studies showed a close link between the catalytic and non-catalytic domains of RNase E. Furthermore, there apparently is an endogenous kinase in E. coli that phosphorylates the non-catalytic domain of the RNase E, but only in the absence of the catalytic domain.Le dégradosome d'E. coli est un complexe protéique impliqué dans la dégradation des ARNm constitué de quatre protéines majeures : RNase E, PNPase, RhlB et l'Enolase. Dans le but de mieux comprendre l'action du dégradosome, des études de l'interaction de RhlB et des autres hélicases DEAD-box de la cellule avec la RNase E ont été menées. Deux sites distincts d'interaction ont été mis en évidence dont l'un spécifique de RhlB. Ceci suggère l'existence de dégradosome alternatif différents selon les conditions de croissance. Des travaux menés sur l'Enolase afin de déterminer son rôle au sein du complexe semblent écarter un rôle structural du complexe ou global de la dégradation des ARNm. L'hypothèse d'un effet plus spécifique est privilégiée. Enfin, des travaux sur la régulation de la RNase E par phosphorylation ont conduit à plusieurs modèles de mécanismes son inhibition par ce processus. Une relation étroite entre le domaine NTH et CTH de la RNase E a ainsi été mise en évidence. De plus, il semble qu'une kinase endogène de E. coli puisse phosphoryler le CTH de la RNase E, uniquement en absence du NTH

Étude fonctionnelle du dégradosome d'Escherichia coli et régulation de la RNase E par phosphorylation

Le dégradosome d'E. coli est un complexe protéique impliqué dans la dégradation des ARNm constitué de quatre protéines majeures : RNase E, PNPase, RhlB et l'Enolase. Dans le but de mieux comprendre l'action du dégradosome, des études de l'interaction de RhlB et des autres hélicases DEAD-box de la cellule avec la RNase E ont été menées. Deux sites distincts d'interaction ont été mis en évidence dont l'un spécifique de RhlB. Ceci suggère l'existence de dégradosome alternatif différents selon les conditions de croissance. Des travaux menés sur l'Enolase afin de déterminer son rôle au sein du complexe semblent écarter un rôle structural du complexe ou global de la dégradation des ARNm. L'hypothèse d'un effet plus spécifique est privilégiée. Enfin, des travaux sur la régulation de la RNase E par phosphorylation ont conduit à plusieurs modèles de mécanismes son inhibition par ce processus. Une relation étroite entre le domaine NTH et CTH de la RNase E a ainsi été mise en évidence. De plus, il semble qu'une kinase endogène de E. coli puisse phosphoryler le CTH de la RNase E, uniquement en absence du NTH.The RNA degradosome of E. coli is a multienzymatic complex involved in mRNA degradation. It is composed of four major proteins: RNase E, PNPase, RhlB and enolase. In our first study, we examined the interaction between RNase E and RhlB, and other DEAD-box RNA helicases. We showed the existence of two distinct helicase binding sites: one that is RhlB specific and another that binds SrmB, RhlE or CsdA. A second study was aimed at elucidating the role of enolase in the degadosome. Considering these results, the hypothesis that enolase has a general role in mRNA degradation seems unlikely. Studies on inhibition of the RNase E due to phosphorylation of its non-catalytic domain by a protein kinase from bacteriophage T7 led to mechanistic models for the control of RNase E activity. In addition, these studies showed a close link between the catalytic and non-catalytic domains of RNase E. Furthermore, there apparently is an endogenous kinase in E. coli that phosphorylates the non-catalytic domain of the RNase E, but only in the absence of the catalytic domain.TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

The Type VI Secretion System Spike Protein VgrG5 Mediates Membrane Fusion during Intercellular Spread by Pseudomallei Group Burkholderia Species

ABSTRACT Pseudomallei group Burkholderia species are facultative intracellular parasites that spread efficiently from cell to cell by a mechanism involving the fusion of adjacent cell membranes. Intercellular fusion requires the function of the cluster 5 type VI secretion system (T6SS-5) and its associated valine-glycine repeat protein, VgrG5. Here we show that VgrG5 alleles are conserved and functionally interchangeable between Burkholderia pseudomallei and its relatives B. mallei , B. oklahomensis , and B. thailandensis . We also demonstrate that the integrity of the VgrG5 C-terminal domain is required for fusogenic activity, and we identify sequence motifs, including two hydrophobic segments, that are important for fusion. Mutagenesis and secretion experiments using B. pseudomallei strains engineered to express T6SS-5 in vitro show that the VgrG5 C-terminal domain is dispensable for T6SS-mediated secretion of Hcp5, demonstrating that the ability of VgrG5 to mediate membrane fusion can be uncoupled from its essential role in type VI secretion. We propose a model in which a unique fusogenic activity at the C terminus of VgrG5 facilitates intercellular spread by B. pseudomallei and related species following injection across the plasma membranes of infected cells. </jats:p

Evidence in vivo that the DEAD-box RNA helicase RhlB facilitates the degradation of ribosome-free mRNA by RNase E

The RNA degradosome of Escherichia coli is a ribonucleolytic multienzyme complex containing RNase E, polynucleotide phosphorylase, RhlB, and enolase. Previous in vitro and in vivo work has shown that RhlB facilitates the exonucleolytic degradation of structured mRNA decay intermediates by polynucleotide phosphorylase in an ATPase-dependent reaction. Here, we show that deleting the gene encoding RhlB stabilizes a lacZ mRNA transcribed by bacteriophage T7 RNA polymerase. Deleting the gene encoding enolase has little if any effect. Other messages transcribed by T7 polymerase are also stabilized by ΔrhlB. The effect of point mutations inactivating RhlB is comparable with the effect of deleting the gene. Primer extension analysis of the lacZ message indicates that RhlB facilitates endoribonucleolytic cleavage by RNase E, demonstrating a functional interaction between the RNA helicase and the endoribonuclease. The possible physiological role of an RhlB-RNase E pathway and the mechanisms by which RhlB could facilitate RNase E cleavage are discussed

The human macrophage mannose receptor is not a professional phagocytic receptor

International audienceThe macrophage mannose receptor (MR) appears to play an important role in the binding and phagocytosis of several human pathogens, but its phagocytic property and signaling pathways have been poorly defined. The general strategy to explore such topics is to express the protein of interest in nonphagocytic cells, but in the case of MR, there are few reports using the full-length MR cDNA. When we searched to clone de novo the human MR (hMR) cDNA, problems were encountered, and full-length hMR cDNA was only obtained after devising a complex cloning strategy. Chinese hamster ovary cells, which have a fully functional phagocytic machinery when expressing professional phagocytic receptors, were stably transfected, and cell clones expressing hMR at quantitatively comparable levels than human macrophages or J774E cells were obtained. They exhibited a functional hMR-mediated endocytic capacity of a soluble ligand but failed to ingest classical particulate ligands of MR such as zymosan, Mycobacterium kansasii, or trimannoside bovine serum albumin-coated latex beads. Transient expression of hMR in two human cell lines did not provide a phagocytic capacity either. In conclusion, we show that MR is not a professional phagocytic receptor, as it does not possess the ability to promote particle ingestion in nonphagocytic cells on its own. We propose that MR is a binding receptor, which requires a partner to trigger phagocytosis in some specialized cells such as macrophages. Our new expression vector could represent a useful tool to study the receptor and its partnership further