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Over‐expression of FXRG and miR‐1 increases formation of RISC complexes in H9C2 cell line
Author(s) -
Walker Lisa,
Dockstader Karen,
Slavov Dobromir,
Sucharov Carmen
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1189.4
Subject(s) - microrna , rna binding protein , psychological repression , transfection , microbiology and biotechnology , immunoprecipitation , messenger rna , cell culture , gene isoform , argonaute , hek 293 cells , untranslated region , biology , translation (biology) , gene expression , chemistry , small interfering rna , gene , genetics
miRNAs regulate gene expression by binding to mRNAs and triggering the formation of the RISC complex, which can promote mRNA degradation or translation repression in association with RNA binding proteins. Previous studies have shown that the Fragile X Related Protein 1 (FXR1) can modulate miRNA function. Undifferentiated H9C2 cells do not express the cardiac isoform of FXR1, whereas these isoforms (G and E) are expressed in differentiated cells. In undifferentiated cells miR‐1‐mediasted repression of connexin 43 (Cx43) is dependent on expression of FXR1G or E. To determine if FXR1G was involved in the loading of Cx43 in the Risc complex, H9C2 cells were co‐transfected with a DNA construct containing the Cx43 3′UTR (contains the miR‐1 binding site) linked to 24 MS2 binding sites and a YFP‐MS2 binding protein construct. The cells were infected with a construct over‐expressing FXRG‐FLAG and miR‐1. To quantify the mRNA bound to the RISC complex, immunoprecipitation was performed with AGO2 (a component of the RISC complex), YFP and FLAG antibodies. RNA was extracted and analyzed by qRT‐PCR. In undifferentiated H9C2 cells, FXRG and miR‐1 over‐expression results in increased binding of Ago2 to the Cx43 3′UTR. These results suggest that FXR1G is necessary for loading of the target mRNA to the RISC complex. Supported by the American Physiological Society Undergraduate Summer Research Fellowship.