Polyadenylation of <i>Dnmt1</i> in the mouse ovary.

<p>(<b>A</b>) Phylogenetic analysis of the <i>Dnmt1</i> 3′UTR in Clustal X shows that the CPE and MBE sites form part of a larger conserved element (CON) which is identical across diverse species. Grey shading denotes areas of 100% homology between species. The CPEB binding site (CPE: UUUUAU in mRNA) is boxed by a thick black line; the consensus RNA binding sequence for mammalian Musashi (MBE; G/AU_1–3AGU), is present as GTAGT (fine black line). The Hex sequence is also indicated. Species identity and RefSeq accession numbers are indicated at left; Hex- polyA hexanucleotide; CON- conserved region. (<b>B</b>) Rapid Amplification of cDNA End-Poly(A) Test (RACE-PAT) to assess polyadenylation levels. <i>Top panel:</i> Synthesis of cDNA was carried out with oligo dT primer, and subsequent PCR used a <i>Dnmt1</i> gene-specific primer and anchored oligo dT to prevent artificial shortening of the PCR products in subsequent cycles. <i>Bottom left panel:</i> Endogenous <i>Dnmt1</i> mRNA undergoes polyadenylation in ovaries where the first wave of oocytes are in the growth phase (6–24 days post partum-dpp). <i>Bottom right panel</i>: Southern blotting and hybridization with the DNMT1-specific probe indicated above. (<b>C</b>) RNA ligation-coupled RT-PCR was used to confirm polyadenylation. Following ligation of an oligo to the poly(A) tails of all mRNA species present in oocytes harvested from ovaries of the indicated age, RT-PCR was carried out using the gene-specific forward primers indicated at right and a primer anti-sense to the ligated oligo. For the <i>Gdf9</i> positive control, more of the signal is dispersed upwards on the gel (direction of arrow) as mRNA tails become longer in growing oocytes. <i>Dnmt1</i> mRNA can be seen to follow a similar pattern. Control PCR using gene-specific primers for β-actin demonstrates RNA integrity. (<b>D</b>) RT-PCR shows that transcripts of <i>Cpeb1</i> are present at high levels in ovaries throughout postnatal development, while <i>Msi1</i> is expressed in growing oocytes only (10–24 dpp). (<b>E</b>) Western blotting shows that only DNMT1^s is found in ES cells, whereas DNMT1^o begins to appear in growing oocytes and accumulates by 10 dpp. MSI1 shows strongest expression at 10 dpp. Signal for DNMT1^o is visible in 3 month old (3 m) adult ovaries on longer exposure. Knockout ES cells lacking DNMT1 (1KO) and GAPDH are shown as controls.