Multiple mRNA decapping enzymes in mammalian cells

Abstract

Regulation of RNA degradation plays an important role in the control of gene expression. One mechanism of eukaryotic mRNA decay proceeds through an initial deadenylation followed by 5' end decapping and exonucleolytic decay. Dcp2 is currently believed to be the only cytoplasmic decapping enzyme responsible for decapping of all mRNAs. Here we report that Dcp2 protein modestly contributes to bulk mRNA decay and surprisingly is not detectable in a subset of mouse and human tissues. Consistent with these findings, a hypomorphic knockout of Dcp2 had no adverse consequences in mice. In contrast, the previously reported Xenopus nucleolar decapping enzyme, Nudt16, is an ubiquitous cytoplasmic decapping enzyme in mammalian cells. Like Dcp2, Nudt16 also regulates the stability of a subset of mRNAs including a member of the motin family of proteins involved in angiogenesis, Angiomotin-like 2. These data demonstrate mammalian cells possess multiple mRNA decapping enzymes, including Nudt16 to regulate mRNA turnover.

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 weeks old adults (Adult).

Figure 2. Dcp2 Protein and RNA Expression in WT and Dcp2^β/β Mouse Tissues and MEF Cells

Dcp2 protein levels were detected by Western blot analysis of 100μg extract from the indicated organs obtained from wild type (WT) and Dcp2^β/β mice (A) or from 50μg 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 +/- Standard Deviation (SD).

Figure 3. RNA can be Decapped Efficiently in Wild Type and Dcp2^β/β MEF Cells

³²P-cap- RNA was electroporated into wild type (WT) and Dcp2^β/β MEF cells and RNAs labeled pcP-G₁₆ 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 +/- standard deviation denoted by error bars.

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.

Figure 5. Nudt16 mRNA and Protein are Expressed in All Mouse Tissues Tested

Nudt16 mRNA (A) and protein (B) levels within the indicated organ extracts were detected by Northern blot (20μg) and Western blot (100μg) analysis respectively. Lanes containing 50μg 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 60μg extract from WT or Dcp2^β/β MEF cells were tested by Western Blot and Tubulin was used as a loading control.

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 knock down MEF cells are shown in the left panel and the fate of the same mRNA in wild type and Dcp2^β/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 hours post-lentiviral infection and mRNA levels tested at the times indicated. Quantitation of three independent experiments are shown with +/- standard deviation denoted by error bars. mRNA levels of Ankrd1 (C) and c-Jun (D) following transcriptional arrest are shown as described in B above.