Rutledge CE , Lau HT , Mangan H , Hardy LL , Sunnotel O , Guo F , MacNicol AM , Walsh CP , Lees-Murdock DJ .

082595 Wellcome Trust , MR/J007773/1 Medical Research Council , R01 HD035688 NICHD NIH HHS , MRC_MR/J007773/1 Medical Research Council , Wellcome Trust

	Figure 2. Msi plays a key role in polyadenylation in the Xenopus oocyte.(A) Polyadenylation occurs at GVBD in Xenopus oocytes. RNA ligation-coupled RT-PCR indicates that the endogenous Xenopus Dnmt1 mRNA (xDnmt1) has a short poly(A) tail in immature oocytes, but becomes elongated during progesterone stimulated maturation (arrow indicates extent of polyadenylation). Oocytes were collected when 50% had undergone germinal vesicle breakdown (GVBD) and grouped into those which had (+) or had not (−) completed GVBD. The maximum size of poly(A) tail confirmed by sequencing is indicated in nucleotides (nt) beside the arrow. (B) Polyadenylation of mouse Dnmt1 (mDnmt1) in Xenopus oocytes requires the MBE but not CPE. In vitro transcribed wildtype (WT), MBE mutant (MBE mut), or CPE mutant (CPE mut) Dnmt1 3′UTR constructs were injected into immature Xenopus oocytes, which were then stimulated with progesterone treatment and assayed as above. When the MBE is mutated, polyadenylation does not occur, while the pattern of polyadenylation of the CPE mutant is similar to wildtype. Max confirmed poly(A) tail size by cloning indicated as for (A).
	Figure 3. Redundant control involving MSI and CPEin mouse embryonic stem cells.(A) Schematic to show the mutations used to test functional requirements in the Dnmt1 3′UTR after cloning it downstream of a luciferase reporter. As well as the CPE and MBE, the region contains a potential Pumilio binding element (PBE): all three were mutated (dark grey letters) using point mutation or insertions (MBE). The ΔCON construct contains a deletion of the entire conserved block indicated in Fig. 1. (B) Mutations in the CPE (ΔCPE) MBE (ΔMBE), or PBE significantly decrease translation of the luciferase reporter in mouse ES cells, but combined mutations (ΔCΔM; or triple mutant, TM) do not have an additive effect. Deletion of the conserved region reduces translation to below 40% of the wildtype Dnmt1 3′UTR. Readings in triplicate from more than three independent experiments are presented as mean +/−SEM; ***p<0.001 (by 2-tailed unpaired Students T-test) compared to expression of wildtype, which was arbitrarily set to 100%. (C) MSI1 interacts with endogenous Dnmt1 mRNA. RNA-immunoprecipitation was performed in R63 mouse ES cells with anti-MSI1 antibody or normal rabbit IgG. Immunoprecipitated RNA was analysed by RT-PCR (top panel). The known MSI1 target Numb could be amplified, while Actb which is not a target, was absent, confirming the technique was working. Dnmt1 could be amplified from MSI1 immunoprecipitates, but not negative controls, indicating MSI1 also binds to this mRNA. A no template control (NTC) was also included. Quantification by RT-qPCR (bottom panel) indicated significant enrichment of Dnmt1, to levels approx. half that of Numb (bottom right). Results are the average of three independent experiments carried out in triplicate. (D) Transient knockdown of RNA binding proteins in ESC. Levels of depletion of the respective target mRNAs following transfection by the indicated siRNA were assessed by RT-qPCR. A scrambled siRNA (mock) was used as a control. An example multiplex experiment (dark bars) is shown at right. All target mRNA were significantly reduced (p<0.05) in single or multiplex experiments. (E) Western blotting showing redundant control ensures DNMT1 translation in ESC. Transient depletion of any one of the RNA binding proteins MSI1, MSI2, CPEB or PUM2, or of combinations thereof such as MSI1 and MSI (MSi1&2) and MSI1, CPEB1 and PUM2 (TKD), had no detectable effect on DNMT1 protein levels in ESC. DNMT1 siRNA are shown as a positive control, mock and WT are negative controls.
	Figure 4. Cells lacking MSI1 show greater CPEB dependence.(A) HeLa cells lack MSI1. Western blotting shows that while DNMT1 is expressed in HeLa, HCT116 and R63 ES cells, the expression of MSI1 is limited and detected only in HCT116 and R63 ES cells. Sizes are indicated in kilodaltons (kDa). Similar results were found for PUM2 (B) CPE is one important component of translational control in HeLa. Luciferase constructs as indicated in Fig. 3 were transfected into HeLa cells. Mutation of the CPE reduced expression of the reporter gene to a similar extent as observed in ESC cells. Significance **p<0.01; ***p<0.001 (C) CPEB1 was targeted for knockdown in HeLa cells by stable shRNA expression. Levels of CPEB1 mRNA in two clonally-derived cell lines B11 and B12 carrying the shRNA are shown. (D) Dnmt1 mRNA levels remain stable in CPEB1 knockdown cell lines. (E) Stable CPEB1 knockdown prevents efficient DNMT1 translation. Protein levels of CCNB1, which is known to be translationally repressed by CPEB1, show upregulation in both stable knockdown cell lines as expected. Reprobing the same membrane shows DNMT1 protein levels are reduced in these samples. Molecular weights are indicated to the left in kiloDaltons (KDa). (F) CPEB4 transient knockdown in HeLa. RT-qPCR shows mRNA levels relative to mock-transfected cells following transient knockdown of CPEB4 and DNMT1 in HeLa. (G) Transient depletion of CPEB4 reduces DNMT1 protein levels to an extent comparable to that achieved by DNMT1 siRNA.
	Figure 1. Polyadenylation of Dnmt1 in the mouse ovary.(A) Phylogenetic analysis of the Dnmt1 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/AU1–3AGU), 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) Rapid Amplification of cDNA End-Poly(A) Test (RACE-PAT) to assess polyadenylation levels. Top panel: Synthesis of cDNA was carried out with oligo dT primer, and subsequent PCR used a Dnmt1 gene-specific primer and anchored oligo dT to prevent artificial shortening of the PCR products in subsequent cycles. Bottom left panel: Endogenous Dnmt1 mRNA undergoes polyadenylation in ovaries where the first wave of oocytes are in the growth phase (6–24 days post partum-dpp). Bottom right panel: Southern blotting and hybridization with the DNMT1-specific probe indicated above. (C) 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 Gdf9 positive control, more of the signal is dispersed upwards on the gel (direction of arrow) as mRNA tails become longer in growing oocytes. Dnmt1 mRNA can be seen to follow a similar pattern. Control PCR using gene-specific primers for β-actin demonstrates RNA integrity. (D) RT-PCR shows that transcripts of Cpeb1 are present at high levels in ovaries throughout postnatal development, while Msi1 is expressed in growing oocytes only (10–24 dpp). (E) Western blotting shows that only DNMT1s is found in ES cells, whereas DNMT1o begins to appear in growing oocytes and accumulates by 10 dpp. MSI1 shows strongest expression at 10 dpp. Signal for DNMT1o 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.

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