Intron retention as an alternative splice variant of the rat urocortin 1 gene.
Urocortin 1, highly conserved metazoan gene of the corticotropin-releasing hormone family, is a simple gene structured in two exons and the corresponding intron. The urocortin 1 prepropeptide is entirely coded in the second exon. Preliminary non-isotopic in situ hybridization experiments with an oligonucleotide complementary to an intron sequence of the urocortin 1 gene showed a significant cytoplasmic-like staining, suggesting the occurrence of an intron-retained urocortin 1 transcript. This observation prompted us to study whether the urocortin 1 gene presents alternative splicing by intron retention event. Confocal fluorescent in situ hybridization for urocortin 1 RNA and the use of the specific DNA dye TOPRO-3 allowed us to show significant expression of the intron-retained urocortin 1 transcript that did not colocalize with TOPRO-3 staining indicating a cytoplasmic localization for the intron-retained urocortin 1 transcript. The natural occurrence of a polyadenylated intron-retained urocortin 1 RNA was further documented by reverse transcriptase polymerase chain reaction (PCR), primed with oligo(dT), of total RNA extracted from three brain regions, a midbrain region containing the Edinger-Westphal nucleus, cerebellum and prefrontal cortex. In the three brain regions studied, it was possible to amplify both intron-less as well as intron-retained urocortin 1 transcripts. The use of PCR primers that simultaneously amplify both urocortin 1 transcripts allowed us to show that the expression of both urocortin 1 transcripts differs among the brain regions analyzed, suggesting a tissue specific regulation of this alternative splicing. In silico analysis of the five known mammalian urocortin 1 genomic sequences showed high conservation of the urocortin 1 intron sequence. Further studies should investigate the regulation of this intron retention event and its consequence for the functionality of the urocortin 1 gene.
Blanco
E
E
Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Catholic University of Chile, Alameda 340, 833-1010, Santiago, Chile.
Rojas
R
R
Haeger
P
P
Cuevas
R
R
Perez
C
C
Munita
R
R
Quiroz
G
G
Andrés
M Estela
ME
Forray
M I
MI
Gysling
K
K
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D002531:Cerebellum / Q000378:metabolism; D003346:Corticotropin-Releasing Hormone / Q000235:genetics* / Q000502:physiology; D014644:Genetic Variation; D007438:Introns / Q000235:genetics*; D008297:Male; D008636:Mesencephalon / Q000378:metabolism; D017397:Prefrontal Cortex / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D054832:Urocortins
D054832:Urocortins; D012313:RNA; D003346:Corticotropin-Releasing Hormone