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Global protein conjugation by ubiquitin-like-modifiers during ischemic stress is regulated by microRNAs and confers robust tolerance to ischemia.

Lee YJ, Johnson KR, Hallenbeck JM - PLoS ONE (2012)

Bottom Line: We found that not only SUMO conjugation, but also global protein conjugation by other ULMs including NEDD8, ISG15, UFM1 and FUB1 were significantly increased in the brains of hibernating ground squirrels during torpor.By means of miRNA microarrays of ground squirrel brain samples (from active and torpor phase) we found that the miR-200 family (miR-200a,b,c/miR-141/miR-429) and the miR-182 family (miR-182/miR-183/miR-96) were among the most consistently depressed miRNAs in the brain during the torpor phase as compared to active animals.This is the first report to describe that the natural tolerance to brain ischemia in hibernators is linked to regulation by microRNAs of a broad range of ubiquitin-like modifiers.

View Article: PubMed Central - PubMed

Affiliation: Stroke Branch, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.

ABSTRACT
Hibernation torpor provides an excellent model of natural tolerance to ischemia. We have previously shown that massive global SUMOylation occurs during hibernation torpor in ground squirrels. We have also shown that overexpression of Ubc9, SUMO-1, or SUMO-2/3 provides protection against ischemic damage in cell lines and cortical neurons exposed to oxygen/glucose deprivation, and in mice exposed to middle cerebral artery occlusion. We have now extended our study to other Ubiquitin-Like-Modifiers (ULMs), which have multiple cellular functions during stress, in order to assess the possibility that they also have roles in tolerance to ischemia. We found that not only SUMO conjugation, but also global protein conjugation by other ULMs including NEDD8, ISG15, UFM1 and FUB1 were significantly increased in the brains of hibernating ground squirrels during torpor. By means of miRNA microarrays of ground squirrel brain samples (from active and torpor phase) we found that the miR-200 family (miR-200a,b,c/miR-141/miR-429) and the miR-182 family (miR-182/miR-183/miR-96) were among the most consistently depressed miRNAs in the brain during the torpor phase as compared to active animals. In addition, we showed that these miRNAs are involved in the expression of various ULM proteins and their global conjugation to proteins. We observed that inhibition of the miR-200 family and/or miR-182 family miRNA activities in SHSY5Y cells increases global protein conjugation by the above ULMs and makes these cells more tolerant to OGD-induced cell death. This is the first report to describe that the natural tolerance to brain ischemia in hibernators is linked to regulation by microRNAs of a broad range of ubiquitin-like modifiers.

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miR-200 family and miR-182 family members of miRNAs indeed target various ULM and/or ULM related genes.SHSY5Y cells were co-transfected with the pmirGLO Dual-Luciferase miRNA Target Expression (Promega) constructs into which putative target sites for the various ULMs had been inserted, and with mimics of miRNAs as indicated. Two days later, cells were lysed, luciferase acivities (both firefly and Renilla) were measured, firefly luciferase activities were normalized with Renilla firefly activity, and expressed as relative to control (transfected with empty vector and negative control mimic). Data are shown as the mean±SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05 compared to control.
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pone-0047787-g005: miR-200 family and miR-182 family members of miRNAs indeed target various ULM and/or ULM related genes.SHSY5Y cells were co-transfected with the pmirGLO Dual-Luciferase miRNA Target Expression (Promega) constructs into which putative target sites for the various ULMs had been inserted, and with mimics of miRNAs as indicated. Two days later, cells were lysed, luciferase acivities (both firefly and Renilla) were measured, firefly luciferase activities were normalized with Renilla firefly activity, and expressed as relative to control (transfected with empty vector and negative control mimic). Data are shown as the mean±SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05 compared to control.

Mentions: In order to confirm that miR-200 family miRNAs and miR-182/96/183 family miRNAs are involved in ULM and/or ULM-related gene expression, we compiled a list of potential targets of these miRNAs in various ULM mRNAs using three computational target prediction programs: TargetScan (www.targetscan.org); MicroInspector (http://bioinfo.uni-plovdiv.bg/microinspector/); RegRNA tool (http://regrna.mbc.nctu.edu.tw/html/prediction.html). We found quite a few potential target sites for miR-200 and miR-141, and some for miR-182/183/96 in SUMO-related genes and other ULMs as shown in Table 2. miRNA::mRNA target prediction is based on assumptions of certain values for temperature, salt and other conditions and the results are all too often dependent on the algorithm employed by each tool. For example, some of the potential target sites were predicted at 32°C but not at 37°C using the MicroInspector which is the only program you can run at different temperature settings. The DNA sequences of target sites for miR-200 family and/or miR-182 family miRNAs in each gene are shown in the supplemental materials (Table S2). In order to examine whether some (if not all) of these predicted target sites are really recognized by either miR-200 family or miR-182/183/96 family miRNAs, we made firefly luciferase reporter constructs containing individual predicted target sequences for the miRNAs of interest. The pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) was designed to quantitatively evaluate miRNA activity by the insertion of putative target sites 3′ of the firefly luciferase gene. The oligonucleotides used for these reporter plasmids are shown in Table 1 with the predicted miRNA target sequences in each gene shown in bold letters. Firefly luciferase is the primary reporter gene, and reduced firefly luciferase expression indicates the endogenous or introduced miRNA level of binding to the cloned miRNA target sequence. SHSY5Y human neuroblastoma cells were transfected with these designed constructs along with a corresponding miRNA mimic, and luciferase activities were measured. The firefly luciferase activities were normalized by Renilla luciferase activities, and shown as the percentage of control (i.e. transfected with empty vector and miRNA mimic negative control) activity (Fig. 5). As shown in the figure, each of these transfectants, except one that was transfected with the SUMO-1 miR-141 target site incorporated construct and miR-141 mimic, showed significantly reduced activities. The results strongly suggest that theses predicted target sites are real and SUMO and other ULM mRNAs are indeed targeted and controlled by the miR-200 family and the miR-182 family miRNAs.


Global protein conjugation by ubiquitin-like-modifiers during ischemic stress is regulated by microRNAs and confers robust tolerance to ischemia.

Lee YJ, Johnson KR, Hallenbeck JM - PLoS ONE (2012)

miR-200 family and miR-182 family members of miRNAs indeed target various ULM and/or ULM related genes.SHSY5Y cells were co-transfected with the pmirGLO Dual-Luciferase miRNA Target Expression (Promega) constructs into which putative target sites for the various ULMs had been inserted, and with mimics of miRNAs as indicated. Two days later, cells were lysed, luciferase acivities (both firefly and Renilla) were measured, firefly luciferase activities were normalized with Renilla firefly activity, and expressed as relative to control (transfected with empty vector and negative control mimic). Data are shown as the mean±SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05 compared to control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475703&req=5

pone-0047787-g005: miR-200 family and miR-182 family members of miRNAs indeed target various ULM and/or ULM related genes.SHSY5Y cells were co-transfected with the pmirGLO Dual-Luciferase miRNA Target Expression (Promega) constructs into which putative target sites for the various ULMs had been inserted, and with mimics of miRNAs as indicated. Two days later, cells were lysed, luciferase acivities (both firefly and Renilla) were measured, firefly luciferase activities were normalized with Renilla firefly activity, and expressed as relative to control (transfected with empty vector and negative control mimic). Data are shown as the mean±SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05 compared to control.
Mentions: In order to confirm that miR-200 family miRNAs and miR-182/96/183 family miRNAs are involved in ULM and/or ULM-related gene expression, we compiled a list of potential targets of these miRNAs in various ULM mRNAs using three computational target prediction programs: TargetScan (www.targetscan.org); MicroInspector (http://bioinfo.uni-plovdiv.bg/microinspector/); RegRNA tool (http://regrna.mbc.nctu.edu.tw/html/prediction.html). We found quite a few potential target sites for miR-200 and miR-141, and some for miR-182/183/96 in SUMO-related genes and other ULMs as shown in Table 2. miRNA::mRNA target prediction is based on assumptions of certain values for temperature, salt and other conditions and the results are all too often dependent on the algorithm employed by each tool. For example, some of the potential target sites were predicted at 32°C but not at 37°C using the MicroInspector which is the only program you can run at different temperature settings. The DNA sequences of target sites for miR-200 family and/or miR-182 family miRNAs in each gene are shown in the supplemental materials (Table S2). In order to examine whether some (if not all) of these predicted target sites are really recognized by either miR-200 family or miR-182/183/96 family miRNAs, we made firefly luciferase reporter constructs containing individual predicted target sequences for the miRNAs of interest. The pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) was designed to quantitatively evaluate miRNA activity by the insertion of putative target sites 3′ of the firefly luciferase gene. The oligonucleotides used for these reporter plasmids are shown in Table 1 with the predicted miRNA target sequences in each gene shown in bold letters. Firefly luciferase is the primary reporter gene, and reduced firefly luciferase expression indicates the endogenous or introduced miRNA level of binding to the cloned miRNA target sequence. SHSY5Y human neuroblastoma cells were transfected with these designed constructs along with a corresponding miRNA mimic, and luciferase activities were measured. The firefly luciferase activities were normalized by Renilla luciferase activities, and shown as the percentage of control (i.e. transfected with empty vector and miRNA mimic negative control) activity (Fig. 5). As shown in the figure, each of these transfectants, except one that was transfected with the SUMO-1 miR-141 target site incorporated construct and miR-141 mimic, showed significantly reduced activities. The results strongly suggest that theses predicted target sites are real and SUMO and other ULM mRNAs are indeed targeted and controlled by the miR-200 family and the miR-182 family miRNAs.

Bottom Line: We found that not only SUMO conjugation, but also global protein conjugation by other ULMs including NEDD8, ISG15, UFM1 and FUB1 were significantly increased in the brains of hibernating ground squirrels during torpor.By means of miRNA microarrays of ground squirrel brain samples (from active and torpor phase) we found that the miR-200 family (miR-200a,b,c/miR-141/miR-429) and the miR-182 family (miR-182/miR-183/miR-96) were among the most consistently depressed miRNAs in the brain during the torpor phase as compared to active animals.This is the first report to describe that the natural tolerance to brain ischemia in hibernators is linked to regulation by microRNAs of a broad range of ubiquitin-like modifiers.

View Article: PubMed Central - PubMed

Affiliation: Stroke Branch, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.

ABSTRACT
Hibernation torpor provides an excellent model of natural tolerance to ischemia. We have previously shown that massive global SUMOylation occurs during hibernation torpor in ground squirrels. We have also shown that overexpression of Ubc9, SUMO-1, or SUMO-2/3 provides protection against ischemic damage in cell lines and cortical neurons exposed to oxygen/glucose deprivation, and in mice exposed to middle cerebral artery occlusion. We have now extended our study to other Ubiquitin-Like-Modifiers (ULMs), which have multiple cellular functions during stress, in order to assess the possibility that they also have roles in tolerance to ischemia. We found that not only SUMO conjugation, but also global protein conjugation by other ULMs including NEDD8, ISG15, UFM1 and FUB1 were significantly increased in the brains of hibernating ground squirrels during torpor. By means of miRNA microarrays of ground squirrel brain samples (from active and torpor phase) we found that the miR-200 family (miR-200a,b,c/miR-141/miR-429) and the miR-182 family (miR-182/miR-183/miR-96) were among the most consistently depressed miRNAs in the brain during the torpor phase as compared to active animals. In addition, we showed that these miRNAs are involved in the expression of various ULM proteins and their global conjugation to proteins. We observed that inhibition of the miR-200 family and/or miR-182 family miRNA activities in SHSY5Y cells increases global protein conjugation by the above ULMs and makes these cells more tolerant to OGD-induced cell death. This is the first report to describe that the natural tolerance to brain ischemia in hibernators is linked to regulation by microRNAs of a broad range of ubiquitin-like modifiers.

Show MeSH
Related in: MedlinePlus