CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3′ Untranslated Region, Altering Polysome Association and Stress Tolerance in <italic toggle="yes">Cryptococcus neoformans</italic>

ABSTRACT In Cryptococcus neoformans, mRNAs encoding ribosomal proteins (RP) are rapidly and specifically repressed during cellular stress, and the bulk of this repression is mediated by deadenylation-dependent mRNA decay. A motif-finding approach was applied to the 3′ untranslated regions (UTRs) of...

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Autores principales:	Jay Leipheimer, Amanda L. M. Bloom, Tilman Baumstark, John C. Panepinto
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	CNBP G-quadruplex posttranscriptional gene regulation stress response translation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/16ff5085e546445da27b8661628ed90a
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spelling	oai:doaj.org-article:16ff5085e546445da27b8661628ed90a2021-11-15T15:25:50ZCNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3′ Untranslated Region, Altering Polysome Association and Stress Tolerance in <italic toggle="yes">Cryptococcus neoformans</italic>10.1128/mSphere.00201-182379-5042https://doaj.org/article/16ff5085e546445da27b8661628ed90a2018-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00201-18https://doaj.org/toc/2379-5042ABSTRACT In Cryptococcus neoformans, mRNAs encoding ribosomal proteins (RP) are rapidly and specifically repressed during cellular stress, and the bulk of this repression is mediated by deadenylation-dependent mRNA decay. A motif-finding approach was applied to the 3′ untranslated regions (UTRs) of RP transcripts regulated by mRNA decay, and a single, significant motif, GGAUG, was identified. Znf9, a small zinc knuckle RNA binding protein identified by mass spectrometry, was found to interact specifically with the RPL2 3′-UTR probe. A second, homologous protein, Gis2, was identified in the genome of C. neoformans and also bound the 3′-UTR probe, and deletion of both genes resulted in loss of binding in cell extracts. The RPL2 3′ UTR contains four G-triplets (GGG) that have the potential to form a G-quadruplex, and temperature gradient gel electrophoresis revealed a potassium-dependent structure consistent with a G-quadruplex that was abrogated by mutation of G-triplets. However, deletion of G-triplets did not abrogate the binding of either Znf9 or Gis2, suggesting that these proteins either bind irrespective of structure or act to prevent structure formation. Deletion of both GIS2 and ZNF9 resulted in a modest increase in basal stability of the RPL2 mRNA which resulted in an association with higher-molecular-weight polysomes under unstressed conditions. The gis2Δ mutant and gis2Δ znf9Δ double mutant exhibited sensitivity to cobalt chloride, fluconazole, and oxidative stress, and although transcriptional induction of ERG25 was similar to that of the wild type, analysis of sterol content revealed repressed levels of sterols in the gis2Δ and gis2Δ znf9Δ double mutant, suggesting a role in translational regulation of sterol biosynthesis. IMPORTANCE Stress adaptation is fundamental to the success of Cryptococcus neoformans as a human pathogen and requires a reprogramming of the translating pool of mRNA. This reprogramming begins with the regulated degradation of mRNAs encoding the translational machinery. The mechanism by which these mRNAs are specified has not been determined. This study has identified a cis element within a G-quadruplex structure that binds two C. neoformans homologues of cellular nucleic acid binding protein (CNBP). These proteins regulate the polysome association of the target mRNA but perform functions related to sterol homeostasis which appear independent of ribosomal protein mRNAs. The presence of two CNBP homologues in C. neoformans suggests a diversification of function of these proteins, one of which appears to regulate sterol biosynthesis and fluconazole sensitivity.Jay LeipheimerAmanda L. M. BloomTilman BaumstarkJohn C. PanepintoAmerican Society for MicrobiologyarticleCNBPG-quadruplexposttranscriptional gene regulationstress responsetranslationMicrobiologyQR1-502ENmSphere, Vol 3, Iss 4 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	CNBP G-quadruplex posttranscriptional gene regulation stress response translation Microbiology QR1-502
spellingShingle	CNBP G-quadruplex posttranscriptional gene regulation stress response translation Microbiology QR1-502 Jay Leipheimer Amanda L. M. Bloom Tilman Baumstark John C. Panepinto CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3′ Untranslated Region, Altering Polysome Association and Stress Tolerance in <italic toggle="yes">Cryptococcus neoformans</italic>
description	ABSTRACT In Cryptococcus neoformans, mRNAs encoding ribosomal proteins (RP) are rapidly and specifically repressed during cellular stress, and the bulk of this repression is mediated by deadenylation-dependent mRNA decay. A motif-finding approach was applied to the 3′ untranslated regions (UTRs) of RP transcripts regulated by mRNA decay, and a single, significant motif, GGAUG, was identified. Znf9, a small zinc knuckle RNA binding protein identified by mass spectrometry, was found to interact specifically with the RPL2 3′-UTR probe. A second, homologous protein, Gis2, was identified in the genome of C. neoformans and also bound the 3′-UTR probe, and deletion of both genes resulted in loss of binding in cell extracts. The RPL2 3′ UTR contains four G-triplets (GGG) that have the potential to form a G-quadruplex, and temperature gradient gel electrophoresis revealed a potassium-dependent structure consistent with a G-quadruplex that was abrogated by mutation of G-triplets. However, deletion of G-triplets did not abrogate the binding of either Znf9 or Gis2, suggesting that these proteins either bind irrespective of structure or act to prevent structure formation. Deletion of both GIS2 and ZNF9 resulted in a modest increase in basal stability of the RPL2 mRNA which resulted in an association with higher-molecular-weight polysomes under unstressed conditions. The gis2Δ mutant and gis2Δ znf9Δ double mutant exhibited sensitivity to cobalt chloride, fluconazole, and oxidative stress, and although transcriptional induction of ERG25 was similar to that of the wild type, analysis of sterol content revealed repressed levels of sterols in the gis2Δ and gis2Δ znf9Δ double mutant, suggesting a role in translational regulation of sterol biosynthesis. IMPORTANCE Stress adaptation is fundamental to the success of Cryptococcus neoformans as a human pathogen and requires a reprogramming of the translating pool of mRNA. This reprogramming begins with the regulated degradation of mRNAs encoding the translational machinery. The mechanism by which these mRNAs are specified has not been determined. This study has identified a cis element within a G-quadruplex structure that binds two C. neoformans homologues of cellular nucleic acid binding protein (CNBP). These proteins regulate the polysome association of the target mRNA but perform functions related to sterol homeostasis which appear independent of ribosomal protein mRNAs. The presence of two CNBP homologues in C. neoformans suggests a diversification of function of these proteins, one of which appears to regulate sterol biosynthesis and fluconazole sensitivity.
format	article
author	Jay Leipheimer Amanda L. M. Bloom Tilman Baumstark John C. Panepinto
author_facet	Jay Leipheimer Amanda L. M. Bloom Tilman Baumstark John C. Panepinto
author_sort	Jay Leipheimer
title	CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3′ Untranslated Region, Altering Polysome Association and Stress Tolerance in <italic toggle="yes">Cryptococcus neoformans</italic>
title_short	CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3′ Untranslated Region, Altering Polysome Association and Stress Tolerance in <italic toggle="yes">Cryptococcus neoformans</italic>
title_full	CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3′ Untranslated Region, Altering Polysome Association and Stress Tolerance in <italic toggle="yes">Cryptococcus neoformans</italic>
title_fullStr	CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3′ Untranslated Region, Altering Polysome Association and Stress Tolerance in <italic toggle="yes">Cryptococcus neoformans</italic>
title_full_unstemmed	CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3′ Untranslated Region, Altering Polysome Association and Stress Tolerance in <italic toggle="yes">Cryptococcus neoformans</italic>
title_sort	cnbp homologues gis2 and znf9 interact with a putative g-quadruplex-forming 3′ untranslated region, altering polysome association and stress tolerance in <italic toggle="yes">cryptococcus neoformans</italic>
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/16ff5085e546445da27b8661628ed90a
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CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3′ Untranslated Region, Altering Polysome Association and Stress Tolerance in <italic toggle="yes">Cryptococcus neoformans</italic>

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