Genomic Checkpoints for Exon 10 Usage in the Luteinizing Hormone Receptor Type 1 and Type 2

Alternative splicing is a hallmark of glycoprotein hormone receptor gene regulation, but its molecular mechanism is unknown. The LH receptor (LHR) gene possesses 11 exons, but exon 10 is constitutively skipped in the New World monkey lineage (LHR type 2), whereas it is constitutively spliced in the human (LHR type 1). This study identifies the regulatory elements of exon 10 usage. Sequencing of genomic marmoset DNA revealed that the cryptic LHR exon 10 is highly homologous to exon 10 from other species and displays intact splice sites. Functional studies using a minigene approach excluded the contribution of intronic, marmoset-specific long interspersed nucleotide-1 elements to exon 10 skipping. Sequencing of the genomic regions surrounding exon 10 from several primate lineages, sequence comparisons including the human and mouse LHR gene, revealed the presence of unique nucleotides at 3′-intronic position −19 and −10 and at position +26 within exon 10 of the marmoset LHR. Exon trap experiments and in vitro mutagenesis of these nucleotides resulted in the identification of a composite regulatory element of splicing consisting of cis-acting elements represented by two polypyrimidine tracts and a trans-acting element within exon 10, which affect the secondary RNA structure. Changes within this complex resulted either in constitutive exon inclusion, constitutive skipping, or alternative splicing of exon 10. This work delineates the molecular pathway leading to intronization of exon 10 in the LHR type 2 and reveals, for the first time, the essential function of regulatory and structural elements involved in glycoprotein hormone receptor splicing.