Multiple mRNA Decapping Enzymes in Mammalian Cells Man-Gen Song, You Li, Megerditch Kiledjian  Molecular Cell  Volume 40, Issue 3, Pages 423-432 (November 2010) DOI: 10.1016/j.molcel.2010.10.010 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 40, 423-432DOI: (10.1016/j.molcel.2010.10.010) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Dcp2 Protein Is Differentially Expressed in Mammalian Organs (A) Dcp2 protein is not detectable in a subset of organ extracts. Protein extract (100 μg) from the indicated organs were resolved by SDS-polyacrylamide gel electrophoresis and Dcp2 protein was detected by western blot analysis using GAPDH as a loading control. Fifty micrograms K562 extract was used as a positive control and migration of each protein is denoted on the right. (B) Extract from the indicated human organs were resolved and analyzed as in A except eIF4E was used as a loading control. The brain extract is from brain cortex. Fifty micrograms HeLa S15 extract and 293T cell total extract were used as positive controls. (C) The presence of Dcp2 was detected as in A from the indicated mouse extracts derived from embryonic day 14.5 (E14.5), embryonic day 16.5 (E16.5), postnatal day 0 (P0), and 8-week-old adults (Adult). Molecular Cell 2010 40, 423-432DOI: (10.1016/j.molcel.2010.10.010) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Dcp2 Protein and RNA Expression in WT and Dcp2β/β Mouse Tissues and MEF Cells (A–C) Dcp2 protein levels were detected by western blot analysis of 100 μg extract from the indicated organs obtained from wild-type (WT) and Dcp2β/β mice (A) or from 50 μg of the corresponding MEF cells (B). Human K562 erythroleukemia and mouse erythroleukemia (MEL) cell extracts were used as positive controls. Dcp2 mRNA levels in the indicated MEF cells as determined by quantitative real-time RT-PCR is shown in (C) and presented relative to β-actin mRNA levels. Results were obtained from three independent experiments and error bars represent ±SD. Molecular Cell 2010 40, 423-432DOI: (10.1016/j.molcel.2010.10.010) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 RNA Can Be Decapped Efficiently in Wild-Type and Dcp2β/β MEF Cells 32P-cap-labeled pcP-G16 RNA was electroporated into wild-type (WT) and Dcp2β/β MEF cells and RNAs isolated at the indicated time points and resolved by 7 M urea/6% polyacrylamide denaturing gel electrophoresis. The transfected RNA is indicated and I.C. denotes a uniformly labeled RNA internal control spiked into the reaction prior to RNA isolation for quantitation. Quantitation of three independent experiments are plotted on the right with ±SD denoted by error bars. Molecular Cell 2010 40, 423-432DOI: (10.1016/j.molcel.2010.10.010) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Nudt16 Is a Cytoplasm mRNA Decapping Enzyme (A) Decapping activity of the recombinant His-tagged human Dcp2, Nudt16 and mouse Nudt1, 4, 5, 7, and Nudt16L1 proteins are shown. Decapping products resolve on PEI-TLC developed in 0.45 M (NH4)2SO4. (B) 293T cells cytoplasm and nuclear proteins were fractionated, extract from equal cell numbers were resolved on SDS PAGE and the distribution of Nudt16 protein was tested by Western Blot analysis. hnRNP C1/C2 and GAPDH were used as nuclear and cytoplasmic markers, respectively. (C) FLAG-hNudt16 (Top) or hNudt16-FLAG (Bottom) was overexpressed in U2OS cells. The epitope tagged protein was localized by indirect immunofluorescence with an anti-FLAG antibody by confocal microscopy. The presented images are representative of >95% of the transfected cells. The differential interference contrast (DIC) images of the same cells are shown as indicated. Molecular Cell 2010 40, 423-432DOI: (10.1016/j.molcel.2010.10.010) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Nudt16 mRNA and Protein Are Expressed in All Mouse Tissues Tested (A and B) Nudt16 mRNA (A) and protein (B) levels within the indicated organ extracts were detected by northern blot (20 μg) and western blot (100 μg) analysis, respectively. Lanes containing 50 μg extract from human erythroleukemia K562 cells and murine erythroleukemia MEL cells are denoted. GAPDH was used as loading control. (C) The Nudt16 protein levels in 60 μg extract from WT or Dcp2β/β MEF cells were tested by western blot and tubulin was used as a loading control. Molecular Cell 2010 40, 423-432DOI: (10.1016/j.molcel.2010.10.010) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Nudt16 Is Involved in the Decay of a Subset of mRNAs Amotl2 and Ankrd1 mRNAs are stabilized following a reduction in Nudt16 protein levels. (A) Nudt16 protein levels were detected following lentiviral expressed mNudt16-directed shRNA in MEF cells as determined by western blot analysis. (B) Stability of Amotl2 mRNA in control and Nudt16 knockdown MEF cells are shown in the left panel and the fate of the same mRNA in wild-type and Dcp2β/β MEF cells is shown on the right. The middle panel shows the decay of Amotl2 mRNA in cells with a lentiviral shRNA directed 65% reduction in Xrn1 protein. Transcription was arrested by the addition of Actinomycin D 48 hr postlentiviral infection and mRNA levels tested at the times indicated. Quantitation of three independent experiments shown with ±SD denoted by error bars. (C and D) mRNA levels of Ankrd1 (C) and c-Jun (D) following transcriptional arrest are shown as described in (B) above. Molecular Cell 2010 40, 423-432DOI: (10.1016/j.molcel.2010.10.010) Copyright © 2010 Elsevier Inc. Terms and Conditions