Related Experiment Videos
Complex expression pattern of RPGR reveals a role for purine-rich exonic splicing enhancers
1Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye Ear Infirmary, Boston, Massachusetts 02114, USA.
Purpose:
To examine the mechanism underlying transcript heterogeneity in the gene for the retinitis pigmentosa GTPase regulator (RPGR).
Methods:
Transcript heterogeneity was analyzed by reverse transcription-polymerase chain reactions (RT-PCR), rapid amplification of cDNA ends (RACE), and transient expression of minigene constructs. Protein variants were identified by immunoblot analysis and by immunocytochemistry.
Results:
RPGR transcripts terminated either uniformly at the end of exon 19, producing the constitutive transcript with few variants, or at variable sites downstream from exon 15. The latter transcripts resembled the previously described open reading frame (ORF)14/15 variant, but the ORF14/15 exon was not found in full length. Instead, various portions of a purine-rich region were removed as introns. Numerous splice site combinations were used, giving rise to innumerable variants. Analysis of the purine-rich region found multiple exonic splicing enhancers (ESEs) known to promote splicing through interaction with serine-arginine repeat (SR) proteins. Antibodies targeting different regions of RPGR detected a multitude of RPGR proteins in photoreceptors, concentrated in the connecting cilium. Predominant ORF14/15-encoded RPGR polypeptides migrated at approximately 200 kDa and were photoreceptor specific.
Conclusions:
The exceptional heterogeneity in RPGR transcript processing results primarily from a novel form of alternative RNA splicing mediated by multiple exonic splicing enhancers. RPGR is composed of a population of proteins with a constant N-terminal core encompassing the RCC1 homology domain followed by a C-terminal portion of variable lengths and sequences.