Abstract Disclosure: X. Tan: None. M.S. Stokes: None. T.S. Nandu: None. W.L. Kraus: None. ADP-ribosylation is a post-translational modification of proteins in which ADP-ribose units from NAD+ are covalently linked to various amino acids on substrates by ADP-ribosyltransferase (ART) enzymes. The NAD+ required for these reactions is synthesized by nicotinamide mononucleotide adenylyl transferases (NMNATs), which serve as the key NAD+ synthase enzymes in the salvage pathway. The three cellular NMNATs have different subcellular localizations (NMNAT-1 in the nucleus, NMNAT-2 in the cytoplasm, and NMNAT-3 in the mitochondria). We have shown previously that NMNAT-2 protein is rapidly and robustly induced during adipogenesis through a stabilization mechanism, causing rapid reduction in nuclear nicotinamide mononucleotide (NMN) levels and decreased PARP1 catalytic activity, leading to an increased expression of proadipogenic genes. We also recently demonstrated that NMNAT-2 support the catalytic activity of PARP16, an endoplasmic reticulum-localized enzyme, to maintain protein homeostasis in ovarian cancer cells by mono(ADP-ribosyl)ation (MARylation) of ribosomal proteins. The biological functions of increased cytoplasmic NAD+ synthesis by NMNAT-2 in adipocytes, however, is unknown. Our results demonstrate a reciprocal decrease of nuclear poly(ADP-ribose) (PAR) levels and increase of cytosolic MAR levels during the differentiation of 3T3-L1 preadipocytes, which is driven by the induction of NMNAT-2. The increase in cytosolic MAR levels leads to enhanced ribosome MARylation, and knockdown of NMNAT-2 or PARP16 results in reduced ribosome MARylation. Analysis of ribosomes isolated from differentiated 3T3-L1 cells revealed the enrichment of MARylation in fractions containing monosomes. Using TMT mass spectrometry, we identified numerous high confidence of ADP-ribosylation sites on ribosome proteins in both undifferentiated and differentiated 3T3-L1 cells, with over 30 sites showing significant increases in differentiated adipocytes. We are now examining the relationship between the MARylation of ribosome proteins and translational programming during adipogenesis to expand our understanding of MARylation in physiology and disease. Presentation: Friday, June 16, 2023