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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, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability. [ABSTRACT FROM AUTHOR]

We recently demonstrated that the major decapping activity in mammalian cells involves DcpS, a scavenger pyrophosphatase that hydrolyzes the residual cap structure following 3' to 5' decay of an mRNA. The association of DcpS with 3' to 5' exonuclease exosome components suggests that these two activities are linked and there is a coupled exonucleolytic decay-dependent decapping pathway. We purified DcpS from mammalian cells and identified the cDNA encoding a novel 40 kDa protein possessing DcpS activity. Consistent with purified DcpS, the recombinant protein specifically hydrolyzed methylated cap analog but did not hydrolyze unmethylated cap analog nor did it function on intact capped RNA. Sequence alignments of DcpS from different organisms revealed the presence of a conserved hexapeptide, containing a histidine triad (HIT) sequence with three histidines separated by hydrophobic residues. Mutagenesis analysis revealed that the central histidine within the DcpS HIT motif is critical for decapping activity and defines the HIT motif as a new mRNA decapping domain, making DcpS the first member of the HIT family of proteins with a defined biological function. [ABSTRACT FROM AUTHOR]

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 6 ,2'-O-dimethyladenosine (m 6 A m ), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m 6 A m we find that m 6 A m -initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m 6 A m -initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m 6 A m is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m 6 A m rather than N 6 -methyladenosine (m 6 A), and reduces the stability of m 6 A m mRNAs. Together, these findings show that the methylation status of m 6 A m in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

Neocortical development requires tightly controlled spatiotemporal gene expression. However, the mechanisms regulating ribosomal complexes and the timed specificity of neocortical mRNA translation are poorly understood. We show that active mRNA translation complexes (polysomes) contain ribosomal protein subsets that undergo dynamic spatiotemporal rearrangements during mouse neocortical development. Ribosomal protein specificity within polysome complexes is regulated by the arrival of in-growing thalamic axons, which secrete the morphogen Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3). Thalamic WNT3 release during midneurogenesis promotes a change in the levels of Ribosomal protein L7 in polysomes, thereby regulating neocortical translation machinery specificity. Furthermore, we present an RNA sequencing dataset analyzing mRNAs that dynamically associate with polysome complexes as neocortical development progresses, and thus may be regulated spatiotemporally at the level of translation. Thalamic WNT3 regulates neocortical translation of two such mRNAs, Foxp2 and Apc, to promote FOXP2 expression while inhibiting APC expression, thereby driving neocortical neuronal differentiation and suppressing oligodendrocyte maturation, respectively. This mechanism may enable targeted and rapid spatiotemporal control of ribosome composition and selective mRNA translation in complex developing systems like the neocortex., Significance Statement: The neocortex is a highly complex circuit generating the most evolutionarily advanced complex cognitive and sensorimotor functions. An intricate progression of molecular and cellular steps during neocortical development determines its structure and function. Our goal is to study the steps regulating spatiotemporal specificity of mRNA translation that govern neocortical development. In this work, we show that the timed secretion of Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3) by ingrowing axons from the thalamus regulates the combinatorial composition of ribosomal proteins in developing neocortex, which we term the "neocortical ribosome signature." Thalamic WNT3 further regulates the specificity of mRNA translation and development of neurons and oligodendrocytes in the neocortex. This study advances our overall understanding of WNT signaling and the spatiotemporal regulation of mRNA translation in highly complex developing systems., (Copyright © 2015 the authors 0270-6474/15/3510912-16$15.00/0.)

Posttranscriptional regulation is an important control mechanism governing gene expression in neurons. We recently demonstrated that VCX-A, a protein implicated in X-linked mental retardation, is an RNA-binding protein that specifically binds the 5' end of capped mRNAs to prevent their decapping and decay. Previously, expression of VCX-A was reported to be testes restricted. Consistent with a role in cognitive function, we demonstrate that VCX-A is ubiquitously expressed in human tissues including the brain. Moreover, retinoic acid-induced differentiation of human SH-SY5Y neuroblastoma cells promoted the accumulation of VCX-A in distinct cytoplasmic foci within neurites that colocalize with staufen1-containing RNA granules, suggesting a role in translational suppression and/or mRNA transport. Exogenous expression of VCX-A in rat primary hippocampal neurons, which normally do not express the primate-restricted VCX proteins, promoted neurite arborization, and shRNA-directed knockdown of the VCX genes in SH-SY5Y cells resulted in a reduction of both primary and secondary neurite projections upon differentiation. We propose that the cap-binding property of VCX-A reflects a role of this protein in mRNA translational regulation. In support of this hypothesized role, we demonstrate that VCX-A can specifically bind a subset of mRNAs involved in neuritogenesis and is also capable of promoting translational silencing. Thus, VCX-A contains the capacity to modulate the stability and translation of a subset of target mRNAs involved in neuronal differentiation and arborization. It is plausible that defects of these functions in the absence of the VCX genes could contribute to a mental retardation phenotype.

The 5'-->3' exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with crucial functions in RNA metabolism and RNA interference. XRN2, or Rat1 in yeast, functions primarily in the nucleus and also has an important role in transcription termination by RNA polymerase II (refs 7-14). Rat1 exoribonuclease activity is stimulated by the protein Rai1 (refs 15, 16). Here we report the crystal structure at 2.2 A resolution of Schizosaccharomyces pombe Rat1 in complex with Rai1, as well as the structures of Rai1 and its murine homologue Dom3Z alone at 2.0 A resolution. The structures reveal the molecular mechanism for the activation of Rat1 by Rai1 and for the exclusive exoribonuclease activity of Rat1. Biochemical studies confirm these observations, and show that Rai1 allows Rat1 to degrade RNAs with stable secondary structure more effectively. There are large differences in the active site landscape of Rat1 compared to related and PIN (PilT N terminus) domain-containing nucleases. Unexpectedly, we identified a large pocket in Rai1 and Dom3Z that contains highly conserved residues, including three acidic side chains that coordinate a divalent cation. Mutagenesis and biochemical studies demonstrate that Rai1 possesses pyrophosphohydrolase activity towards 5' triphosphorylated RNA. Such an activity is important for messenger RNA degradation in bacteria, but this is, to our knowledge, the first demonstration of this activity in eukaryotes and suggests that Rai1/Dom3Z may have additional important functions in RNA metabolism.

Hydrolysis of the mRNA cap plays a pivotal role in initiating and completing mRNA turnover. In nematodes, mRNA metabolism and cap-interacting proteins must deal with two populations of mRNAs, spliced leader trans-spliced mRNAs with a trimethylguanosine cap and non-trans-spliced mRNAs with a monomethylguanosine cap. We describe here the characterization of nematode Dcp1 and Dcp2 proteins. Dcp1 was inactive in vitro on both free cap and capped RNA and did not significantly enhance Dcp2 activity. Nematode Dcp2 is an RNA-decapping protein that does not bind cap and is not inhibited by cap analogs but is effectively inhibited by competing RNA irrespective of RNA sequence and cap. Nematode Dcp2 activity is influenced by both 5' end sequence and its context. The trans-spliced leader sequence on mRNAs reduces Dcp2 activity approximately 10-fold, suggesting that 5'-to-3' turnover of trans-spliced RNAs may be regulated. Nematode Dcp2 decaps both m(7)GpppG- and m(2,2,7)GpppG-capped RNAs. Surprisingly, both budding yeast and human Dcp2 are also active on m(2,2,7)GpppG-capped RNAs. Overall, the data suggest that Dcp2 activity can be influenced by both sequence and context and that Dcp2 may contribute to gene regulation in multiple RNA pathways, including monomethyl- and trimethylguanosine-capped RNAs.