Automated high-throughput RNAi screening in human cells combined with reporter mRNA transfection to identify novel regulators of translation.
Standard
Automated high-throughput RNAi screening in human cells combined with reporter mRNA transfection to identify novel regulators of translation. / Casanova, Claudia M; Sehr, Peter; Putzker, Kerstin; Hentze, Matthias W; Neumann, Beate; Duncan, Kent E.; Thoma, Christian.
in: PLOS ONE, Jahrgang 7, Nr. 9, 9, 2012, S. 45943.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
Harvard
APA
Vancouver
Bibtex
}
RIS
TY - JOUR
T1 - Automated high-throughput RNAi screening in human cells combined with reporter mRNA transfection to identify novel regulators of translation.
AU - Casanova, Claudia M
AU - Sehr, Peter
AU - Putzker, Kerstin
AU - Hentze, Matthias W
AU - Neumann, Beate
AU - Duncan, Kent E.
AU - Thoma, Christian
PY - 2012
Y1 - 2012
N2 - Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.
AB - Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.
KW - Humans
KW - Hela Cells
KW - Protein Biosynthesis
KW - Phosphoric Monoester Hydrolases/metabolism
KW - High-Throughput Screening Assays
KW - RNA, Messenger/genetics/metabolism
KW - RNA Interference
KW - Mitogen-Activated Protein Kinase 3/metabolism
KW - Phosphotransferases/metabolism
KW - Transfection
KW - Vascular Endothelial Growth Factor A/genetics/metabolism
KW - Humans
KW - Hela Cells
KW - Protein Biosynthesis
KW - Phosphoric Monoester Hydrolases/metabolism
KW - High-Throughput Screening Assays
KW - RNA, Messenger/genetics/metabolism
KW - RNA Interference
KW - Mitogen-Activated Protein Kinase 3/metabolism
KW - Phosphotransferases/metabolism
KW - Transfection
KW - Vascular Endothelial Growth Factor A/genetics/metabolism
U2 - 10.1371/journal.pone.0045943
DO - 10.1371/journal.pone.0045943
M3 - SCORING: Journal article
VL - 7
SP - 45943
JO - PLOS ONE
JF - PLOS ONE
SN - 1932-6203
IS - 9
M1 - 9
ER -
