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Felix L Struebing, Ying Li, Eldon E Geisert; The Sox11 locus and its complex bidirectional transcription in retinal injury. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4794. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The transcription factor Sox11 plays an important role in retinal development and axon regeneration. Our previously published experimental data from GeneNetwork.org following optic nerve crush (ONC) indicate a 6-fold range difference between 2 probes designed to anneal to different parts of the Sox11 3’UTR as well as the occurence of antisense transcripts. The present study examines the complex expression patterns of Sox11 and its non-coding antisense RNA (Sox11 opposite strand, Sox11OS) under normal and ONC conditions in the mouse.
RNA was isolated and pooled from adult mouse C57BL/6 retinas for normal controls (n=4) and 5 days following ONC (n=4). Strand-specific reverse transcription PCR (RT-PCR) was performed using 14 different primers spanning the entire Sox11 locus to screen for various sense and antisense transcripts. Rapid amplification of cDNA ends (5’/3’ RACE) was carried out to identify the 5’ and 3’ ends of Sox11 and Sox11OS in control and ONC. Quantitative RT-PCR (qRT-PCR) was used in conjunction with the ΔΔCt-method to quantify differential expression of Sox11 and Sox11OS following ONC.
Strand-specific RT-PCR demonstrated the expression of multiple transcripts from the sense and antisense strands of Sox11. Originally, this region was annotated as a single 8.5kb long transcript; however, we were unable to detect this long isoform in the retina. At least 3 transcripts are present on the sense strand: a distal transcript excluding the Sox11 protein-coding region and two proximal transcripts including the Sox11 protein-coding region with different 3’ UTR lengths (transcript length 1.6kb and 2kb, respectively). In addition, we detected Sox11OS transcripts with multiple transcription start and polyadenylation sites. qRT-PCR revealed a 6.5-fold up-regulation of the Sox11 single exon message following ONC (SD = 1.15), whereas an antisense-specific primer pair was upregulated 13.2-fold (SD = 1.19).
Our data demonstrate the complexity of the Sox11 locus, which is considerably more elaborate than previously thought, featuring bidirectional transcription and alternative polyadenylation sites. The complexity of this locus reveals the need for careful analysis of microarray data, especially following experimental manipulation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
The original mouse Sox11 locus showing microarray probe and primer binding sites
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