18S rRNA methyltransferases DIMT1 and BUD23 drive intergenerational hormesis.

Heritable non-genetic information can regulate a variety of complex phenotypes. However, what specific non-genetic cues are transmitted from parents to their descendants are poorly understood. Here, we perform metabolic methyl-labeling experiments to track the heritable transmission of methylation from ancestors to their descendants in the nematode Caenorhabditis elegans (C. elegans). We find heritable methylation in DNA, RNA, proteins, and lipids. We find that parental starvation elicits reduced fertility, increased heat stress resistance, and extended longevity in fed, naïve progeny. This intergenerational hormesis is accompanied by a heritable increase in N6'-dimethyl adenosine (m6,2A) on the 18S ribosomal RNA at adenosines 1735 and 1736. We identified DIMT-1/DIMT1 as the m6,2A and BUD-23/BUD23 as the m7G methyltransferases in C. elegans that are both required for intergenerational hormesis, while other rRNA methyltransferases are dispensable. This study labels and tracks heritable non-genetic material across generations and demonstrates the importance of rRNA methylation for regulating epigenetic inheritance.

N6-adenosine methylation of ribosomal RNA affects lipid oxidation and stress resistance.

During stress, global translation is reduced, but specific transcripts are actively translated. How stress-responsive mRNAs are selectively translated is unknown. We show that METL-5 methylates adenosine 1717 on 18S ribosomal RNA in C. elegans, enhancing selective ribosomal binding and translation of specific mRNAs. One of these mRNAs, CYP-29A3, oxidizes the omega-3 polyunsaturated fatty acid eicosapentaenoic acid to eicosanoids, key stress signaling molecules. While metl-5-deficient animals grow normally under homeostatic conditions, they are resistant to a variety of stresses. metl-5 mutant worms also show reduced bioactive lipid eicosanoids and dietary supplementation of eicosanoid products of CYP-29A3 restores stress sensitivity of metl-5 mutant worms. Thus, methylation of a specific residue of 18S rRNA by METL-5 selectively enhances translation of cyp-29A3 to increase production of eicosanoids, and blocking this pathway increases stress resistance. This study suggests that ribosome methylation can facilitate selective translation, providing another layer of regulation of the stress response.

Transgenerational epigenetic inheritance: from phenomena to molecular mechanisms.

Inherited information not encoded in the DNA sequence can regulate a variety of complex phenotypes. However, how this epigenetic information escapes the typical epigenetic erasure that occurs upon fertilization and how it regulates behavior is still unclear. Here we review recent examples of brain related transgenerational epigenetic inheritance and delineate potential molecular mechanisms that could regulate how non-genetic information could be transmitted.

Identification of the m6Am Methyltransferase PCIF1 Reveals the Location and Functions of m6Am in the Transcriptome.

mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2'-O-dimethyladenosine (m6Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m6Am and m6A. We find that m6A and m6Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m6Am that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Am's function.

DAP5 associates with eIF2ß and eIF4AI to promote Internal Ribosome Entry Site driven translation.

Initiation is a highly regulated rate-limiting step of mRNA translation. During cap-dependent translation, the cap-binding protein eIF4E recruits the mRNA to the ribosome. Specific elements in the 5'UTR of some mRNAs referred to as Internal Ribosome Entry Sites (IRESes) allow direct association of the mRNA with the ribosome without the requirement for eIF4E. Cap-independent initiation permits translation of a subset of cellular and viral mRNAs under conditions wherein cap-dependent translation is inhibited, such as stress, mitosis and viral infection. DAP5 is an eIF4G homolog that has been proposed to regulate both cap-dependent and cap-independent translation. Herein, we demonstrate that DAP5 associates with eIF2ß and eIF4AI to stimulate IRES-dependent translation of cellular mRNAs. In contrast, DAP5 is dispensable for cap-dependent translation. These findings provide the first mechanistic insights into the function of DAP5 as a selective regulator of cap-independent translation.

The translation initiation factor DAP5 is a regulator of cell survival during mitosis.

Initiation of protein translation is tightly regulated by various physiological signals and involves cap-dependent and independent mechanisms. DAP5 protein is an eIF4G family member previously implicated in mediating cap-independent IRES driven translation in response to various cellular stresses. Unexpectedly, we have recently found that DAP5 is also essential for continuous cell survival in non-stressed cells. We reported in this respect that the knock down of endogenous DAP5 by RNA-interference induces M-phase specific caspase-dependent cell death. Bcl-2 and CDK1 were identified as DAP5 mRNA targets, the translation of which was selectively reduced in the DAP5 knock down cells. They each possess a functional IRES element in their 5'UTR. Here we review the major results of this study and present new data on the link of DAP5 to additional Bcl-2 family members. In addition we discuss other possible cellular phenotypes resulting from the knock down of DAP5 in these cells.

The crystal structure of the C-terminal DAP5/p97 domain sheds light on the molecular basis for its processing by caspase cleavage.

DAP5/p97 (death-associated protein 5) is a member of the eukaryotic translation initiation factor 4G family. It functions as a scaffold protein promoting cap-independent translation of proteins. During apoptosis, DAP5/p97 is cleaved by caspases at position 792, yielding an 86-kDa C-terminal truncated isoform (DAP5/p86) that promotes translation of several mRNAs mediated by an internal ribosome entry site. In this study, we report the crystal structure of the C-terminal region of DAP5/p97 extending between amino acids 730 and 897. This structure consists of four HEAT-Repeats and is homologous to the C-terminal domain of eIF4GI, eIF5, and eIF2Bepsilon. Unlike the other proteins, DAP5/p97 lacks electron density in the loop connecting alpha3 and alpha4, which harbors the caspase cleavage site. Moreover, we observe fewer interactions between these two helices. Thus, previous mapping of this site by mutation analysis is confirmed here by the resolved structure of the DAP5/p97 C-terminus. In addition, we identified the position of two conserved aromatic and acidic boxes in the structure of the DAP5/p97 C-terminus. The acidic residues in the two aromatic and acidic boxes form a continuous negatively charged patch, which is suggested to make specific interactions with other proteins such as eIF2beta. The caspase cleavage of DAP5/p97 removes the subdomain carrying acidic residues in the AA-box motif, which may result in exposure of a hydrophobic surface. These intriguing structural differences between the two DAP5 isoforms suggest that they have different interaction partners and, subsequently, different functions.

DAP5 promotes cap-independent translation of Bcl-2 and CDK1 to facilitate cell survival during mitosis.

DAP5 is an eIF4G protein previously implicated in mediating cap-independent translation in response to cellular stresses. Here we report that DAP5 is crucial for continuous cell survival in nonstressed cells. The knockdown of endogenous DAP5 induced M phase-specific caspase-dependent apoptosis. Bcl-2 and CDK1 were identified by two independent screens as DAP5 translation targets. Notably, the activity of the Bcl-2 IRES was reduced in DAP5 knockdown cells and a selective shift of Bcl-2 mRNA toward light polysomal fractions was detected. Furthermore, a functional IRES was identified in the 5'UTR of CDK1. At the cellular level, attenuated translation of CDK1 by DAP5 knockdown decreased the phosphorylation of its M phase substrates. Ectopic expression of Bcl-2 or CDK1 proteins partially reduced the extent of caspase activation caused by DAP5 knockdown. Thus, DAP5 is necessary for maintaining cell survival during mitosis by promoting cap-independent translation of at least two prosurvival proteins.