Analysis of exonic splicing enhancers in the mouse gonadotropin-releasing hormone (GnRH) gene.
We have previously reported that efficient removal of the first intron (intron A) of gonadotropin-releasing hormone (GnRH) pre-mRNA is the prerequisite event for post-transcriptional regulation. In the present study, using an in vitro HeLa splicing system, we examined the enhancing activities of exonic elements for GnRH pre-mRNA splicing. While not excised by exon 2 alone, intron A was efficiently excised when exon 3 and/or exon 4 was combined with exon 2, suggesting the presence of exonic splicing enhancers (ESEs) in exons 3 and 4. Purine-rich sequences located in the border of exons 2 and 3 (denoted ESE3) and in exon 4 (ESE4) revealed strong splicing enhancing activities. Mutation in ESE3 decreased pre-mRNA splicing, while mutation in purine-rich sequences in exon 2 did not. We further analyzed the functional activity of ESE4 by mutations or deletions of the ESE4 sequence that consists of three purine-repeats separated by two spacers and a putative hairpin constructing sequence. An UV cross-linking assay using the RNA sequence of ESE4 examined the presence of ESE4-specific binding proteins in the nuclear extracts from GT1 hypothalamic GnRH neurons. Collectively, this study indicates that a sequence context of ESE4 and its binding proteins may be crucially involved in enhanced GnRH pre-mRNA splicing. However, it should be further clarified as to which splicing factor(s) is responsible for ESE4-dependent GnRH pre-mRNA splicing.
Han
J
J
School of Biological Sciences, Seoul National University, 151-742, Seoul, South Korea.
Seong
J Y
JY
Kim
K
K
Wuttke
W
W
Jarry
H
H
D000818:Animals; D001483:Base Sequence; D005091:Exons / Q000235:genetics*; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D051379:Mice; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011685:Purine Nucleotides / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D014466:Ultraviolet Rays
D011685:Purine Nucleotides; D012322:RNA Precursors; D007987:Gonadotropin-Releasing Hormone