184 ALTERNATIVE SPLICING OF DNA METHYLTRANSFERASE 1 IN PORCINE OOCYTES

Abstract

In human, mouse, and some marsupials, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (Dnmt1) enzyme. The identification and characterization of a similar oocyte transcript variant in farm animals would greatly contribute to the understanding of the methylation processes that occur during nuclear remodeling of in vivo embryos as well as of cloned embryos. The objective of this study was to identify and sequence isoforms of Dnmt1 expressed in porcine oocytes. Total RNA was isolated from pools of 50 denuded mature oocytes as well as from fibroblast cells using the Trizol method. RNA was co-precipitated with glycogen, and residual genomic DNA was removed with DNase I. A RACE System (Invitrogen, Carlsbad, CA, USA) was used to amplify the 5′ cDNA end of Dnmt1. Briefly, the first strand of cDNA was synthesized using SuperScript II and a Dnmt1-specific primer followed by degradation of the RNA strands and incorporation of TdT and dCTP tails to the 3′ ends of the cDNA. Tailed cDNA was amplified by PCR using a forward anchor primer and a reverse Dnmt1-specific primer. PCR products were separated by electrophoresis on an agarose gel. Resulting PCR products were subcloned into a cloning vector, and the cDNA inserts were sequenced. PCR primers capable of amplifying all possible alternatively spliced isoforms of Dnmt1 were used to identify the presence of the RNA sequences found in fibroblasts and oocytes of pigs. Two distinctive bands (290 and 390 bp) were observed after 5′RACE, PCR, and electrophoresis of oocyte Dnmt1 cDNA. Only 1 band of 290 bp was observed after amplification of fibroblast cDNA. The location of exons and introns of every transcript variant was determined by aligning the 5′RACE-derived sequences to the Sus scrofa Dnmt1 genomic sequence located on chromosome 2 (CU462940). The smaller 290-bp band, amplified from both oocytes and fibroblasts, had an identical DNA sequence (EU908731). Structure analysis of the larger 390-bp band indicates that this oocyte Dnmt1 isoform has an additional exon located between exon 1 and 2 of the somatic form of Dnmt1 (EU908730). PCR products amplified using primers specific for the oocyte or somatic transcript verified the presence of the additional exon in the oocyte Dnmt1 splicing variant. In conclusion, this study shows that porcine oocytes express an alternative isoform of Dnmt1 in addition to the somatic transcript. The 2 identified isoforms are produced by alternative splicing of the Dnmt1 gene. Analysis of the oocyte and somatic Dnmt1 isoforms in pre-implantation embryos will determine the expression pattern of this transcript during genomic methylation and its involvement during nuclear reprogramming and cellular differentiation.