Search

The 7-methylguanosine cap structure at the 5' end of eukaryotic messenger RNAs is a critical determinant of their stability and translational efficiency (1-3). It is generally believed that 5'-end capping is a constitutive process that occurs during mRNA maturation and lacks the need for a quality-control mechanism to ensure its fidelity. We recently reported that the yeast Rail protein has pyrophosphohydrolase activity towards mRNAs lacking a 5'-end cap (4). Here we show that, in vitro as well as in yeast cells, Rail possesses a novel decapping endonuclease activity that can also remove the entire cap structure dinucleotide from an mRNA. This activity is targeted preferentially towards mRNAs with unmethylated caps in contrast to the canonical decapping enzyme, Dcp2, which targets mRNAs with a methylated cap. Capped but unmethylated mRNAs generated in yeast cells with a defect in the methyltransferase gene are more stable in a rail-gene-disrupted background. Moreover, railΔ yeast cells with wild-type capping enzymes show significant accumulation of mRNAs with 5' -end capping defects under nutritional stress conditions of glucose starvation or amino acid starvation. These findings provide evidence that 5' -end capping is not a constitutive process that necessarily always proceeds to completion and demonstrates that Rail has an essential role in clearing mRNAs with aberrant 5'-end caps. We propose that Rail is involved in an as yet uncharacterized quality control process that ensures mRNA 5' -end integrity by an aberrant-cap-mediated mRNA decay mechanism., The stability and translational efficiency of eukaryotic mRNAs are both influenced by the 5'-end cap (1-3). The cap is co-transcriptionally added and consists of a guanine nucleoside methylated at the [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.07.033 Byline: Ming-Der Lin (a), Xinfu Jiao (b), Dominic Grima (c), Sarah F. Newbury (c), Megerditch Kiledjian (b), Tze-Bin Chou (a) Keywords: Processing bodies; Drosophila decapping protein 1; Drosophila decapping protein 2; Pacman; Me31B; Oogenesis Abstract: Processing bodies (P-bodies) have emerged as important subcellular structures that are involved in mRNA metabolism. To date, a detailed description of P-bodies in Drosophila oogenesis is lacking. To this end, we first demonstrate that Drosophila decapping protein 2 (dDcp2) contains intrinsic decapping activity and its enzymatic activity was not detectably enhanced by Drosophila decapping protein 1 (dDcp1). dDcp1-containing bodies in the nurse cell cytoplasm can associate with the 5' to 3' exoribonuclease, Pacman in addition to dDcp2 and Me31B. The size and number of dDcp1 bodies are dynamic and dramatically increased in dDcp2 and pacman mutant backgrounds supporting the conclusion that dDcp1 bodies in nurse cell cytoplasm are Drosophila P-bodies. In stage 2-6 oocytes, dDcp1 bodies appear to be distinct from previously characterized P-bodies since they are insensitive to cycloheximide and RNase A treatments. Curiously, dDcp2 and Pacman do not colocalize with dDcp1 at the posterior end of the oocyte in stage 9-10 oocytes. This suggests that dDcp1 bodies are in a developmentally distinct state separate from the 5' end mRNA degradation enzymes at later stages in the oocyte. Interestingly, re-formation of maternally expressed dDcp1 with dDcp2 and Pacman was observed in early embryogenesis. With respect to developmental switching, the maternal dDcp1 is proposed to serve as a marker for the re-formation of P-bodies in early embryos. This also suggests that a regulated conversion occurs between maternal RNA granules and P-bodies from oogenesis to embryogenesis. Author Affiliation: (a) Institute of Molecular and Cellular Biology, College of Life Science, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan (b) Department of Cell Biology and Neuroscience, Rutgers University, 604 Allison Road, Piscataway, NJ 08854-8082, USA (c) Brighton and Sussex Medical School, University of Sussex, Falmer, Brighton, BN1 9QG, UK Article History: Received 27 February 2008; Revised 14 July 2008; Accepted 15 July 2008

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5 end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N[sup.6],2-O-dimethyladenosine (m[sup.6]A[sub.m]), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m[sup.6]A[sub.m] we find that m[sup.6]A[sub.m]-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m[sup.6]A[sub.m]-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m[sup.6]A[sub.m] is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m[sup.6]A[sub.m] rather than N[sup.6]-methyladenosine (m[sup.6]A), and reduces the stability of m[sup.6]A[sub.m] mRNAs. Together, these findings show that the methylation status of m[sup.6]A[sub.m] in the 5 cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability., Author(s): Jan Mauer [1]; Xiaobing Luo [2]; Alexandre Blanjoie [3]; Xinfu Jiao [2]; Anya V. Grozhik [1]; Deepak P. Patil [1]; Bastian Linder [1]; Brian F. Pickering [1]; Jean-Jacques Vasseur [...]

Decapping of mRNA is a critical step in eukaryotic mRNA turnover, yet the proteins involved in this activity remain elusive in mammals. We identified the human Dcp2 protein (hDcp2) as an enzyme containing intrinsic decapping activity, hDcp2 specifically hydrolyzed methylated capped RNA to release [m.sup.7]GDP; however, it did not function on the cap structure alone, hDcp2 is therefore functionally distinct from the recently identified mammalian scavenger decapping enzyme, DcpS. hDcp2-mediated decapping required a functional Nudix (nucleotide diphosphate linked to an X moiety) pyrophosphatase motif as mutations in conserved amino acids within this motif disrupted the decapping activity. hDcp2 is detected exclusively in the cytoplasm and predominantly cosediments with polysomes. Consistent with the localization of hDcp2, endogenous Dcp2-like decapping activity was detected in polysomal fractions prepared from mammalian cells. Similar to decapping in yeast, the presence of the poly(A) tail was inhibitory to the endogenous decapping activity, yet unlike yeast, competition of cap-binding proteins by cap analog did not influence the efficiency of decapping. Therefore the mammalian homologue of the yeast Dcp2 protein is an mRNA decapping enzyme demonstrated to contain intrinsic decapping activity.