Thunuguntla, P.; Duraiyan, D.; Sizemore, C.; Sulvaran-Guel, E.; Mishra, R.; Camacho, J.; Gonzales, S.; Daly, S.; Bagwill, K.; Colbert, D.; King, J.; Samuel, C.; David-Pennington, L.; Ki, A.; Anderson, S.; Bras Costa, C.; Zhang, J.; Vij, R.; Garcia, B. A.; DiPersio, J.; Silva-Fisher, J.

RNA modifications play critical roles in gene regulation. N6-methyladenosine (m6A) is the most abundant modification on mRNA and long noncoding RNA (lncRNA) and regulates RNA processing, stability, and translation. RNA modifications are not well characterized in multiple myeloma (MM), a plasma cell malignancy characterized by relapse and disease progression, and the contribution of m6A -modified lncRNAs to the disease remains unclear. Here, we define the RNA modification landscape of MM by combining mass spectrometry, Nanopore Direct RNA sequencing, and methylated RNA immunoprecipitation sequencing. We identify 20 RNA modification types and > 15,000 m6A sites, including sites on 2,398 lncRNAs. Among these, we validate m6A sites on the paraspeckle-associated lncRNA NEAT1. Functional studies reveal that NEAT1 expression is regulated by the methyltransferase METTL3 and site-specific demethylation of a NEAT1 m6A site reduces MM cell viability. Single-cell RNA sequencing shows consistent NEAT1 enrichment in malignant plasma cells but minimal expression in healthy cells. These findings identify m6A-modified lncRNAs as key regulators of MM biology and establish NEAT1 as an epitranscriptomically controlled driver of MM cell survival.