Cargando…

Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) a...

Descripción completa

Detalles Bibliográficos
Autores principales: Eskandari, Rosa, Ratnayake, Lalanthi, Lakin-Thomas, Patricia L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: SAGE Publications 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8276340/
https://www.ncbi.nlm.nih.gov/pubmed/33825541
http://dx.doi.org/10.1177/0748730421999948
_version_ 1783721885748428800
author Eskandari, Rosa
Ratnayake, Lalanthi
Lakin-Thomas, Patricia L.
author_facet Eskandari, Rosa
Ratnayake, Lalanthi
Lakin-Thomas, Patricia L.
author_sort Eskandari, Rosa
collection PubMed
description Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes (frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation (vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δgtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δgtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δgtr2 is defective in entrainment. In all of these assays, Δgtr2 is similar to Δvta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.
format Online
Article
Text
id pubmed-8276340
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher SAGE Publications
record_format MEDLINE/PubMed
spelling pubmed-82763402021-08-03 Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora Eskandari, Rosa Ratnayake, Lalanthi Lakin-Thomas, Patricia L. J Biol Rhythms Original Articles Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes (frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation (vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δgtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δgtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δgtr2 is defective in entrainment. In all of these assays, Δgtr2 is similar to Δvta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway. SAGE Publications 2021-04-07 2021-08 /pmc/articles/PMC8276340/ /pubmed/33825541 http://dx.doi.org/10.1177/0748730421999948 Text en © 2021 The Author(s) https://creativecommons.org/licenses/by-nc/4.0/This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage).
spellingShingle Original Articles
Eskandari, Rosa
Ratnayake, Lalanthi
Lakin-Thomas, Patricia L.
Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora
title Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora
title_full Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora
title_fullStr Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora
title_full_unstemmed Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora
title_short Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora
title_sort shared components of the frq-less oscillator and tor pathway maintain rhythmicity in neurospora
topic Original Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8276340/
https://www.ncbi.nlm.nih.gov/pubmed/33825541
http://dx.doi.org/10.1177/0748730421999948
work_keys_str_mv AT eskandarirosa sharedcomponentsofthefrqlessoscillatorandtorpathwaymaintainrhythmicityinneurospora
AT ratnayakelalanthi sharedcomponentsofthefrqlessoscillatorandtorpathwaymaintainrhythmicityinneurospora
AT lakinthomaspatricial sharedcomponentsofthefrqlessoscillatorandtorpathwaymaintainrhythmicityinneurospora