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Linda Ye, Haksu Kyung, Jacky Man Kwong Kwong, Joseph Caprioli, Natik Piri; Transcriptional Regulation Of The RNA Binding Protein Rbpms In RGC-5 And 293T Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):29.
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RNA binding protein with multiple splicing (Rbpms) has been characterized in the retina as a specific marker for ganglion cells (RGCs). The aim of the present study is to identify and analyze the basic promoter region of RBPMS and to understand the mechanisms controlling RBPMS transcription in RGCs.
Ten deletion constructs containing 5’-flanking regions of Rbpms were generated by PCR with sequence-specific primers and inserted into pGL3-Basic and pEGFP-N3 vectors. Transfection of human embryonic kidney 293T and RGC-5 cells was performed with lipofectamine. A pCMV-β-galactosidase expression plasmid was used as an internal normalization control for variations in transfection efficiency.
Three potential promoter regions located at -1603 to -1353 and -259 to -9 in the 5’-flanking region and at +344 to +594 in the 5’-untranslated region (5’-UTR) of the Rbpms gene were identified. In 293T cells, 5'-UTR deletion from constructs containing both -1603/-1353 and -259/-9, and -259/-9 only predicted promoter regions led to an approximately 9- and 11-fold decrease in reporter gene expression, respectively. Expression levels from these constructs were only 2-fold higher than that obtained from the promoter-less pGL3-Basic vector used as a negative control. In RGC-5 cells, the effect of the 5’-UTR was even more significant. Expression levels from the construct containing the 5’-UTR was 60- and 25-fold higher compared to that from the construct with both -1603/-1353 and -259/-9 only promoter regions, respectively.
We have determined that the 5’-UTR of Rbpms is critical for the induction of gene expression in both 293T and RGC-5 cells. The presence of the 5’ flanking region that is predicted to have two promoter regions has a negative effect on the level of reporter gene expression in these cell lines. Further detailed analysis of these regions in vivo will help us to identify elements required for Rbpms transcription in RGCs.
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