Eric L. Greer, Ph.D. - US grants

N6-methyladenine (6mA) was recently identified as a new base modification in metazoan genomic DNA. Methylation on adenine was previously thought to be restricted to unicellular organisms. The identification of this mark in metazoans has raised questions about the role of 6mA in metazoan biology. Some studies suggest that 6mA may correlate with developmental regulation, active transcription start sites, or transposon expression, and potentially carry non-genetic information across generations. However, 6mA is a rare DNA modification in eukaryotes and its role in regulating biological processes is still unclear. Because of the low level of 6mA in eukaryotes, some researchers have questioned whether it plays a role in regulating biological processes or is an evolutionary holdover from bacteria. Studies of 6mA have been slowed by a lack of practical methods for accurately examining 6mA locations. Current epigenomic mapping techniques are either prohibitively expensive or do not provide sufficient resolution and accuracy to map the rare 6mA occurrences in metazoan genomes. Here we propose to develop a new technique for identifying sites of adenine methylation genome-wide at nucleotide resolution in a cost-effect manner. We will determine the utility of this technique first in bacteria and then apply it to examine 6mA distribution during development in zebrafish, where we have found that it changes dramatically. This technique will provide a new tool for scientists to define 6mA sites in metazoans to enable the study of its potential role in epigenetics, imprinting and development.

The long-term goal of this project is to define the function(s) of the cap-specific N6, 2'-O-dimethyladenosine (m6 Am) present at the transcription start site of eukaryotic mRNAs. We and others recently identified the cellular mRNA methyltransferase responsible for methylation of the cap-proximal adenosine at the N6 position as phosphorylated carboxy-terminal domain interacting factor 1 (PCIF1). PCIF1 binds the phosphorylated C- terminal domain of host RNA polymerase II to selectively modify the cap proximal A, following the sequential methylation of the cap-structure by the guanine-N7-methylase and ribose-2'-O methylase. The functional significance of the cap-proximal m6Am modification is uncertain with published literature reaching different conclusions regarding mRNA stability and translation. Viruses that replicate in the cytoplasm such as the negative-strand RNA virus vesicular stomatitis virus (VSV) also contain this cap-proximal m6Am modification on mRNA synthesized in infected cells despite the absence of a viral encoded N6, 2'-O-dimethyltransferase. In preliminary data we have found that PCIF1 is relocalized to the cytoplasm in VSV infected cells and methylates VSV mRNA. The 5 VSV mRNAs are well characterized and we have developed tools necessary to define how m6Am influences the function of each of those mRNAs. Our preliminary data shows that neither mRNA stability nor mRNA translation is impacted by the loss of m6Am, and that in 293T and Hela cells in culture, virus replication is unaffected under basal conditions. Pretreatment of cells with interferon, however, demonstrates that loss of PCIF1 results in the further translational suppression of viral mRNA and a more pronounced reduction in viral growth. This PCIF1 dependent phenotype suggests that one function of this cap-proximal m6Am is to discriminate self from non-self mRNA. The mRNA cap has been hypothesized to have emerged with eukaryotic evolution, when PCIF1 is first detected, to replace the Shine-Dalgarno sequence for directing ribosomes to mRNAs and to protect mRNAs from digestion by 5' exoribonucleases thus providing an early method for distinguishing self- versus foreign mRNAs. It is likely that extant viruses have evolved in the face of this RNA methylation to evade the eukaryotic self-defense system. To further probe the role of PCIF1 modification of viral RNA we generated a PCIF1 -/- mouse providing an additional unique reagent to mechanistically dissect the role of m6Am of viral mRNA in vivo. Capitalizing on this preliminary data we will use genetic, biochemical, cell biological and virological approaches both in cell-culture and in vivo to dissect the role of m6Am and PCIF1 mediated mRNA methylation. Our underlying hypothesis is that PCIF1 modification of mRNA contributes to distinguishing self from non-self mRNA, and that viruses have coopted PCIF1 to ensure efficient replication.