Project Summary Regulation of RNA stability is an important determinant of the post-transcriptional control of eukaryotic gene expression. Minor alterations in mRNA stability can have profound consequences and may manifest as clinical phenotypes as illustrated by the ability of aberrantly expressed proto-oncogenes that can give rise to malignancies. Eukaryotic mRNAs are generally thought to possess an N7 methyl guanosine (m7G) cap at their 5¢ end to promote their stability and translation. However, our demonstration that mammalian mRNAs can also carry a 5´-end nicotinamide adenine dinucleotide (NAD+) cap that, in contrast to the m7G cap, does not support translation but instead promotes mRNA decay of exogenously introduced RNAs provides a new paradigm for mRNA 5´ end processing. Analogous to m7G cap decapping enzymes, cell also possess specific “deNADding” enzymes to hydrolyze the NAD+ cap. The mammalian and fungal noncanonical DXO/Rai1 decapping enzymes efficiently remove NAD+ caps and appear to function as deNADding enzymes in cells. Removal of DXO from cells increases NAD+-capped mRNA levels and enabled detection of NAD+-capped intronic small nucleolar RNAs (snoRNAs), suggesting NAD+ caps can be added to 5´- processed termini indicating the presence of a NAD+ capping mechanism. We will build on these novel findings throughout this proposal within three specific aims. The first will address the functional role of an NAD+ cap on endogenous mRNAs to determine whether it functions as a molecular tag to promote mRNA decay and the mechanism involved and assess potential interplay between cellular assimilation of NAD+ and RNA metabolism. The second aim will deduce the impact of NAD+ caps on small noncoding RNAs and the mechanism underlying the incorporation of a NAD+ cap onto an RNA, we refer to as NADding. It is apparent that in addition to DXO, mammalian cells harbor additional deNADding enzymes. In the last aim, we will identify and expand the spectrum of mammalian deNADding proteins and determine their potential to be regulated by novel NAD+ cap binding protein(s). Collectively, we have established NAD+ as an alternative mammalian RNA cap and the proposed studies will provide insight into a heretofore unknown fundamental post-transcriptional regulatory mechanism and will provide the framework for novel avenues to control gene expression in normal and disease states.