Fig. 5

MDA5 suppresses the promoter activity of LINE-1 5′-UTR. A Schematic representation of the amplicon regions on JM111 in the PCR and qRT-PCR assay detecting LINE-1 RNA. Primers L1–3F and EGFP-2F target cDNA derived from JM111 RNA, and the amplified fragment represent full-length JM111 RNA, while L1–1F and L1–1R target cDNA derived from all LINE-1 mRNA (including endogenous LINE-1 mRNA and exogenous JM111 RNA). B Electrophoresis image showing that MDA5-myc reduces levels of LINE-1 RNA generated from JM111. HEK293T cells seeded in a 24-well plate were co-transfected with 1 μg of JM111 and 225 ng of control vector VR1012 or 25, 75, or 225 ng of MDA5-myc-expressing vector, and subjected to the PCR assay at 12 h post-transfection to detect the levels of LINE-1 RNA from JM111. The western blotting results show the MDA5-myc protein levels in the transfected cells. C qRT-PCR results based on samples from panel B showing that exogenous MDA5-myc reduces levels of total LINE-1 mRNA. D Electrophoresis image showing that knocking down endogenous MDA5 expression increases levels of LINE-1 RNA generated from JM111. HEK293T cells seeded in a 24-well plate were first transfected with 100 nM siNC, siIFIH1–1, or siIFIH1–2, and then transfected with 1 μg of JM111 after 24 h. Transfected cells were subjected to the PCR assay at 12 h post-transfection to detect the levels of LINE-1 RNA from JM111. E qRT-PCR results based on samples from panel D showing that knocking down endogenous MDA5 expression enhances levels of total LINE-1 mRNA. F and G Electrophoresis image showing the potency of MDA5-myc mutants in reducing the levels of LINE-1 RNA generated from JM111. HEK293T cells seeded in a 24-well plate were co-transfected with 1 μg of JM111 and control vector VR1012 (225 ng) or one of MDA5-myc-expressing vectors (225 ng) and subjected to the PCR assay at 12 h post-transfection to detect the levels of LINE-1 RNA from JM111. The western blotting results show the MDA5 protein levels in the transfected cells. H Schematic representation of the 5UTR-Luc cassette, which was generated with the backbone vector pGL3-Basic. I and J Luciferase activity data indicating the potency of wild type MDA5-myc or its mutants in LINE-1 5′-UTR regulation. HEK293T cells seeded in a 24-well plate were co-transfected with 200 ng of 5UTR-Luc and control vector VR1012 (225 ng) or one of MDA5-myc-expressing vectors (225 ng). Luciferase activity was tested at 48 h post-transfection. The western blotting results show the MDA5-myc protein levels in the transfected cells