Figure 5

IRES-GFP fusion constructs mislocalize RNCR2 to the cytoplasm. (A, B) Model for a mechanism by which overexpression of IRES-GFP fusion-constructs can result in dominant-negative phenotypes in transfected cells. Fusion to the IRES-GFP sequence mislocalizes lncRNAs and associated effector proteins to the ribosome, and mislocalizes proteins that bind RNCR2 and mediate the biochemical effectors of lncRNA function away from endogenous nuclear-retained lncRNAs, effectively inhibiting their function. (C) RNCR2-IRES-GFP RNA is mislocalized from the nucleus to the cytoplasm. HeLa cells were transfected with RNCR2 or RNCR2-IRES-GFP fusion constructs and RNA location analyzed by fISH followed by immunohistochemistry against the cytoplasmic S6 ribosomal protein. White arrows indicate regions of prominent RNCR2 localization. The cell nucleus, defined by the region encompassed by DAPI-stained chromatin, is delineated by the white dashed line. The relative fraction of nuclear RNCR2 signal found within in cells transfected with CAG-RNCR2 and CAG-RNCR2-IRES-GFP is shown in the bar graph. *p < 1.5^-07.