RNA-binding protein Rbpms is represented in human retinas by isoforms A and C and its transcriptional regulation involves Sp1-binding site.

ABSTRACT

Rbpms (RNA-binding protein with multiple splicing) is a member of the RRM (RNA Recognition Motif) family of RNA-binding proteins, which is expressed as multiple alternatively spliced transcripts encoding different protein isoforms. We have shown earlier that Rbpms expression in the retina is restricted to retinal ganglion cells (RGCs), and have characterized this gene as a marker for RGCs. The aim of this study was to identify isoforms representing Rbpms in human retinas and to analyze its transcriptional regulation. We found that Rbpms is expressed as transcription variants 1 and 3 encoding isoforms A and C, respectively. These isoforms are encoded by the same first 6 exons but have different C-terminal ends encoded by exon 8 in variant 1 and exon 7 in variant 3. Computational analysis of the Rbpms 5' untranslated and flanking regions reveals the presence of three CpG islands and four predicted promoter regions (PPRs). The effect of PPR 1 (- 1672/- 1420) and PPR2 (- 330/- 79) on transcriptional activation was minimal, whereas PPR 3 (- 73/+ 177) and PPR4 (+ 274/+ 524) induced the expression by ~ 7 and ninefold compared to control, respectively. The maximum activity, a 30-fold increase above the control level, was obtained from the construct containing both PPRs 3 and 4. Site-directed mutagenesis of several cis-elements within PPR3 and PPR4 including five for Sp1, one for AP1, and two for NF-kB showed that mutation of the first three and especially the first GC box resulted in a threefold downregulation of gene expression. AP1, NF-kB, and two downstream Sp1 sites had no significant effect on expression level. The possible involvement of the GC box 1 at position - 54 in transcriptional regulation of Rbpms was corroborated by EMSA, which showed formation of a DNA-protein complex in the presence of the oligonucleotide corresponding to this Sp1-binding site.