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.

Novel 199 base pair NEFH promoter drives expression in retinal ganglion cells

“…Some promoters known to express specifically in RGCs have either not been expressed highly or are too large to be included in AAV vectors, which have a maximum cargo capacity of approximately 4.7 kb, e.g. Brn3a, Thy1 and Rbpms [8][9][10][11][12][13] . Notably, the size of both the human NEFH promoter (2.5 kb) and, particularly, the truncated hA-P promoter (199 bp) are ideal for application within AAV vectors and thereby represents a significant advantage over the larger RGC promoters published to date 6,[14][15][16] .…”

“…However, many promoter sequences of genes known to be RGC-specific, such as Brn3a , Thy1 and Rbpms , are too large to be incorporated into AAV vectors, the vector of choice for many ocular gene therapies. AAV has a maximum cargo capacity of approximately 4.7 kb 8 – 13 . One study demonstrated that the DCX promoter (3310 bp; doublecortin) can drive strong and specific RGC expression following AAV-QuadYF-mediated delivery 14 , 15 .…”

RBPMS promotes contractile smooth muscle splicing and alters phenotypic behaviour of human embryonic stem cell derived vascular smooth muscle cells

“…Third, the hES-VSMC model explores the splicing activity of a single isoform – RBPMS-A, overexpressed at non-physiological levels (Fig 4 associated supplementary C). RBPMS, (by virtue of its own splicing), is physiologically expressed as multiple transcript isoforms with at least 2 main (A and B) protein isoforms that we have observed to possess differential activity [7, 60]. To compound this, RBPMS activity is also regulated by post-translational modifications [61].…”

Single Cell Transcriptomic Analyses Reveal the Impact of bHLH Factors on Human Retinal Organoid Development

“…The following primary antibodies were used: mouse anti-GFP (1:200; Millipore, MAB#3580); goat anti-GFP (1:200; Rockland Inc., 600-101-215); rabbit anti-GFP (1:200; Rockland Inc., 400-401-215); mouse anti-FLAG (Clone M2) (1:500; Sigma-Aldrich, F3165); rabbit anti-ATOH7 (1:100; NovusBio, NBP1-88639); goat anti-Ngn2 (1:200; Santa Cruz Biotechnology, sc-19233); mouse anti-BrdU (1:1; GE Healthcare, RPN202); mouse anti-PCNA (1:500; Sigma-Aldrich, P3825); rabbit anti-phospho-histone 3 (ser10) (1:3,000; Upstate Biotechnology, 06-570); rabbit anti-Pax6 (1:200; Chemicon, ab5409); goat anti-CHX10/VSX2 (N-18) (1:50; Santa Cruz Biotechnology, sc-21690); goat anti-BRN3a (1:100; Santa Cruz Biotechnology, sc-31984); goat anti-pan BRN3 (1:50; Santa Cruz Biotechnology, sc-6026); mouse anti-Iselt1 (1:10; Developmental Study Hybridoma Bank, 39.4D5); mouse anti-doublecortin (E-6) (1:50; Santa Cruz Biotechnology, sc-271390); rabbit anti-NeuN (1:200; Abcam, ab177487); rabbit anti-NF145 (1:750; Millipore, AB1987); rabbit anti-RBPMS (1:200; Ye et al, 2018 ). Secondary antibodies used were: Alexa Fluor 488 donkey anti-mouse (1:500; Thermo Fisher, A32766); Alexa Fluor 488 donkey anti-rabbit (1:500; Thermo Fisher, A32790); Alexa Fluor 488 donkey anti-goat (1:500; Thermo Fisher, A32814); Alexa Fluor 594 donkey anti-mouse (1:500; Thermo Fisher, A32744); Alexa Fluor 594 donkey anti-rabbit (1:500; Thermo Fisher, A32754); Alexa Fluor 594 donkey anti-goat (1:500; Thermo Fisher, A32758); Alexa Fluor 647 donkey anti-mouse (1:500; Thermo Fisher, A32787); Alexa Fluor 647 donkey anti-rabbit (1:500; Thermo Fisher, A32795); Alexa Fluor 647 donkey anti-goat (1:500; Thermo Fisher, A32849).…”