5 research outputs found
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<sub>1–3</sub>AGU), 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<sup>s</sup> is found in ES cells, whereas DNMT1<sup>o</sup> begins to appear in growing oocytes and accumulates by 10 dpp. MSI1 shows strongest expression at 10 dpp. Signal for DNMT1<sup>o</sup> 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.</p
Msi plays a key role in polyadenylation in the <i>Xenopus</i> oocyte.
<p>(<b>A</b>) Polyadenylation occurs at GVBD in <i>Xenopus</i> oocytes. RNA ligation-coupled RT-PCR indicates that the endogenous <i>Xenopus Dnmt1</i> mRNA (<i>xDnmt1</i>) 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>B</b>) Polyadenylation of mouse <i>Dnmt1</i> (<i>mDnmt1</i>) in <i>Xenopus</i> oocytes requires the MBE but not CPE. In vitro transcribed wildtype (WT), MBE mutant (MBE mut), or CPE mutant (CPE mut) <i>Dnmt1</i> 3′UTR constructs were injected into immature <i>Xenopus</i> 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).</p
Efficient Translation of Dnmt1 Requires Cytoplasmic Polyadenylation and Musashi Binding Elements
<div><p>Regulation of DNMT1 is critical for epigenetic control of many genes and for genome stability. Using phylogenetic analysis we characterized a block of 27 nucleotides in the 3′UTR of <i>Dnmt1</i> mRNA identical between humans and <i>Xenopus</i> and investigated the role of the individual elements contained within it. This region contains a cytoplasmic polyadenylation element (CPE) and a Musashi binding element (MBE), with CPE binding protein 1 (CPEB1) known to bind to the former in mouse oocytes. The presence of these elements usually indicates translational control by elongation and shortening of the poly(A) tail in the cytoplasm of the oocyte and in some somatic cell types. We demonstrate for the first time cytoplasmic polyadenylation of <i>Dnmt1</i> during periods of oocyte growth in mouse and during oocyte activation in <i>Xenopus.</i> Furthermore we show by RNA immunoprecipitation that Musashi1 (MSI1) binds to the MBE and that this element is required for polyadenylation in oocytes. As well as a role in oocytes, site-directed mutagenesis and reporter assays confirm that mutation of either the MBE or CPE reduce DNMT1 translation in somatic cells, but likely act in the same pathway: deletion of the whole conserved region has more severe effects on translation in both ES and differentiated cells. In adult cells lacking MSI1 there is a greater dependency on the CPE, with depletion of CPEB1 or CPEB4 by RNAi resulting in substantially reduced levels of endogenous DNMT1 protein and concurrent upregulation of the well characterised CPEB target mRNA cyclin B1. Our findings demonstrate that CPE- and MBE-mediated translation regulate DNMT1 expression, representing a novel mechanism of post-transcriptional control for this gene.</p></div
Cells lacking MSI1 show greater CPEB dependence.
<p>(<b>A</b>) 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>B</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 (<b>C</b>) CPEB1 was targeted for knockdown in HeLa cells by stable shRNA expression. Levels of <i>CPEB1</i> mRNA in two clonally-derived cell lines B11 and B12 carrying the shRNA are shown. (<b>D</b>) <i>Dnmt1</i> mRNA levels remain stable in CPEB1 knockdown cell lines. (<b>E</b>) 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). (<b>F</b>) CPEB4 transient knockdown in HeLa. RT-qPCR shows mRNA levels relative to mock-transfected cells following transient knockdown of CPEB4 and DNMT1 in HeLa. (<b>G</b>) Transient depletion of CPEB4 reduces DNMT1 protein levels to an extent comparable to that achieved by <i>DNMT1</i> siRNA.</p
Redundant control involving MSI and CPEin mouse embryonic stem cells.
<p>(A) Schematic to show the mutations used to test functional requirements in the <i>Dnmt1</i> 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>B</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 <i>Dnmt1</i> 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%. (<b>C</b>) MSI1 interacts with endogenous <i>Dnmt1</i> 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 <i>Numb</i> could be amplified, while <i>Actb</i> which is not a target, was absent, confirming the technique was working. <i>Dnmt1</i> 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 <i>Dnmt1,</i> to levels approx. half that of <i>Numb</i> (bottom right). Results are the average of three independent experiments carried out in triplicate. (<b>D</b>) 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. (<b>E</b>) 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.</p