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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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2018-08-01
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| Series: | mSphere |
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| Online Access: | https://journals.asm.org/doi/10.1128/mSphere.00201-18 |
| _version_ | 1818652007921090560 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-17T02:15:10Z |
| format | Article |
| id | doaj.art-16ff5085e546445da27b8661628ed90a |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-12-17T02:15:10Z |
| publishDate | 2018-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSphere |
| spelling | doaj.art-16ff5085e546445da27b8661628ed90a2022-12-21T22:07:26ZengAmerican Society for MicrobiologymSphere2379-50422018-08-013410.1128/mSphere.00201-18CNBP 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>Jay Leipheimer0Amanda L. M. Bloom1Tilman Baumstark2John C. Panepinto3Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York, USADepartment of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York, USADepartment of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York, USADepartment of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York, USAABSTRACT 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.https://journals.asm.org/doi/10.1128/mSphere.00201-18CNBPG-quadruplexposttranscriptional gene regulationstress responsetranslation |
| spellingShingle | 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> mSphere CNBP G-quadruplex posttranscriptional gene regulation stress response translation |
| 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_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_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_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 |
| topic | CNBP G-quadruplex posttranscriptional gene regulation stress response translation |
| url | https://journals.asm.org/doi/10.1128/mSphere.00201-18 |
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