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Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism

BACKGROUND: Glutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and...

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Autores principales: Martín-Rufián, Mercedes, Tosina, Marta, Campos-Sandoval, José A., Manzanares, Elisa, Lobo, Carolina, Segura, J. A., Alonso, Francisco J., Matés, José M., Márquez, Javier
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367983/
https://www.ncbi.nlm.nih.gov/pubmed/22679499
http://dx.doi.org/10.1371/journal.pone.0038380
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author Martín-Rufián, Mercedes
Tosina, Marta
Campos-Sandoval, José A.
Manzanares, Elisa
Lobo, Carolina
Segura, J. A.
Alonso, Francisco J.
Matés, José M.
Márquez, Javier
author_facet Martín-Rufián, Mercedes
Tosina, Marta
Campos-Sandoval, José A.
Manzanares, Elisa
Lobo, Carolina
Segura, J. A.
Alonso, Francisco J.
Matés, José M.
Márquez, Javier
author_sort Martín-Rufián, Mercedes
collection PubMed
description BACKGROUND: Glutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and characterize transcript variants of the mammalian glutaminase Gls2 gene. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate for the first time simultaneous expression of two transcript variants from the Gls2 gene in human, rat and mouse. A combination of RT-PCR, primer-extension analysis, bioinformatics, real-time PCR, in vitro transcription and translation and immunoblot analysis was applied to investigate GLS2 transcripts in mammalian tissues. Short (LGA) and long (GAB) transcript forms were isolated in brain and liver tissue of human, rat and mouse. The short LGA transcript arises by a combination of two mechanisms of transcriptional modulation: alternative transcription initiation and alternative promoter. The LGA variant contains both the transcription start site (TSS) and the alternative promoter in the first intron of the Gls2 gene. The full human LGA transcript has two in-frame ATGs in the first exon, which are missing in orthologous rat and mouse transcripts. In vitro transcription and translation of human LGA yielded two polypeptides of the predicted size, but only the canonical full-length protein displayed catalytic activity. Relative abundance of GAB and LGA transcripts showed marked variations depending on species and tissues analyzed. CONCLUSIONS/SIGNIFICANCE: This is the first report demonstrating expression of alternative transcripts of the mammalian Gls2 gene. Transcriptional mechanisms giving rise to GLS2 variants and isolation of novel GLS2 transcripts in human, rat and mouse are presented. Results were also confirmed at the protein level, where catalytic activity was demonstrated for the human LGA protein. Relative abundance of GAB and LGA transcripts was species- and tissue-specific providing evidence of a differential regulation of GLS2 transcripts in mammals.
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spelling pubmed-33679832012-06-07 Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism Martín-Rufián, Mercedes Tosina, Marta Campos-Sandoval, José A. Manzanares, Elisa Lobo, Carolina Segura, J. A. Alonso, Francisco J. Matés, José M. Márquez, Javier PLoS One Research Article BACKGROUND: Glutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and characterize transcript variants of the mammalian glutaminase Gls2 gene. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate for the first time simultaneous expression of two transcript variants from the Gls2 gene in human, rat and mouse. A combination of RT-PCR, primer-extension analysis, bioinformatics, real-time PCR, in vitro transcription and translation and immunoblot analysis was applied to investigate GLS2 transcripts in mammalian tissues. Short (LGA) and long (GAB) transcript forms were isolated in brain and liver tissue of human, rat and mouse. The short LGA transcript arises by a combination of two mechanisms of transcriptional modulation: alternative transcription initiation and alternative promoter. The LGA variant contains both the transcription start site (TSS) and the alternative promoter in the first intron of the Gls2 gene. The full human LGA transcript has two in-frame ATGs in the first exon, which are missing in orthologous rat and mouse transcripts. In vitro transcription and translation of human LGA yielded two polypeptides of the predicted size, but only the canonical full-length protein displayed catalytic activity. Relative abundance of GAB and LGA transcripts showed marked variations depending on species and tissues analyzed. CONCLUSIONS/SIGNIFICANCE: This is the first report demonstrating expression of alternative transcripts of the mammalian Gls2 gene. Transcriptional mechanisms giving rise to GLS2 variants and isolation of novel GLS2 transcripts in human, rat and mouse are presented. Results were also confirmed at the protein level, where catalytic activity was demonstrated for the human LGA protein. Relative abundance of GAB and LGA transcripts was species- and tissue-specific providing evidence of a differential regulation of GLS2 transcripts in mammals. Public Library of Science 2012-06-05 /pmc/articles/PMC3367983/ /pubmed/22679499 http://dx.doi.org/10.1371/journal.pone.0038380 Text en Martín-Rufián et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Martín-Rufián, Mercedes
Tosina, Marta
Campos-Sandoval, José A.
Manzanares, Elisa
Lobo, Carolina
Segura, J. A.
Alonso, Francisco J.
Matés, José M.
Márquez, Javier
Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism
title Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism
title_full Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism
title_fullStr Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism
title_full_unstemmed Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism
title_short Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism
title_sort mammalian glutaminase gls2 gene encodes two functional alternative transcripts by a surrogate promoter usage mechanism
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367983/
https://www.ncbi.nlm.nih.gov/pubmed/22679499
http://dx.doi.org/10.1371/journal.pone.0038380
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